Extracorporeal device and matrix for removing fibrinolytic proteins from biological fluids, and methods and uses thereof

Abstract

To provide, e.g., an extracorporeal device and matrix for removing fibrinolytic proteins from biological fluids, and methods and uses thereof.SOLUTION: The presently disclosed subject matter provides special compositions, conjugates, devices, kits and systems for depleting fibrinolytic agents from biological fluids. The presently disclosed subject matter further relates to the resulting biological fluid formulations that are devoid in fibrinolytic activity, and to therapeutic methods and uses thereof. The conjugates comprise particles, at least one linker, and at least one amino acid, derivative thereof or analog thereof being at least one of 4-(aminomethyl)cyclohexanecarboxylic acid (tranexamic acid), ε-aminocaproic acid, lysine, cyclohexanecarboxylic acid and 4-methylcyclohexanecarboxylic acid. Multiple different conjugates (e.g., differing in the particle size or the type of linker) can be used.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to the field of coagulation and transfusion medicine. More specifically, the present invention provides special devices and matrices for depleting fibrinolytic agents from biological fluids, the resulting biological fluid preparations lacking fibrinolytic activity, methods and uses thereof.

[0002] Background technology The following references are believed to be relevant background to the subject matter disclosed herein. Seligson U et al. Classification, Clinical Manifestations & Evaluation of Disorders of Hemostasis. In: Williams Hematology, 8 th ed, 2010, pp2322-2330 Abdel-Wahab OI et al. Effect of fresh-frozen plasma transfusion on prothrombin time and bleeding in patients with mild coagulation abnormalities. Transfusion 2006; 46: 1279-1285 Holland LL et al. Toward rational fresh frozen plasma transfusion: The effect of Plasma transfusion on coagulation test results. Am J Clin Pathol 2006; 126: 133-139 Meheux CJ et al. Efficacy of Intra-articular Platelet-Rich Plasma Injections in Knee Osteoarthritis: A Systematic Review. Arthroscopy, 2016, 32, 495-505 Pap G et al. Expression of stromelysin and urokinase type plasminogen activator protein in resection specimens and biopsies at different stages of osteoarthritis of the knee. Pathol. Res. Pract. 2000, 196: 219-226 U.S. Patent No. 7,125,569 U.S. Patent No. 3,998,946

[0003] The acknowledgment of the above references herein should not be inferred to mean that those references are in any way relevant to the patentability of the subject matter disclosed herein. [Background technology]

[0004] 2. Background of the Invention Normal hemostasis is a delicately balanced system. When it works as it should, blood is maintained in a fluid state within the vascular system, but clots rapidly when needed to seal an injury. In the 1960s, two groups proposed a model of clot formation, which postulated a series of sequential steps in which activation of one clotting (clotting) factor leads to activation of another factor, ultimately resulting in clot formation. As these clotting factors are sequentially activated to form a clot, a corresponding system called the fibrinolytic system is further activated and is responsible for the dissolution (lysis) of the clot. This fibrinolytic system contains anticoagulant proteins (plasminogen activator, plasminogen, and plasmin), which undergo sequential activation to lead to the dissolution of the clot (Selighson U et al.).

[0005] Hemostatic dysfunction due to deficiencies in clotting factors leads to impaired (or absent) clot formation. Similarly, excessive fibrinolytic activity results in rapid and unnecessary dissolution of formed clots. On the other hand, excessive stimulation of the coagulation cascade or inhibition of the fibrinolytic system can lead to the formation of pathological clots. Thus, the consequences of failure of each of the above systems can be bleeding or an increased tendency to clot.

[0006] Replacement therapy is effective in treating bleeding disorders; however, this treatment may not be sufficient. Patients who are bleeding or have prolonged coagulation tests are frequently transfused with fresh frozen plasma (FFP) under the assumption that it will improve hemostasis and correct and / or prevent bleeding. In a prospective audit conducted at Massachusetts General Hospital, the prothrombin time (PT) and The effect of FFP on coagulation parameters such as blood clotting time and international normalized ratio (INR) was investigated (Abdel-Wahab OI et al.). Data showed that FFP transfusion in this setting was effective in 99% of patients. showed that FFP failed to correct the PT and only 15% of patients corrected the INR to at least half of normal. Similarly, Holland et al. reported that FFP did not change the INR over time. These researchers hypothesized that the inability of FFP to correct the INR was due to dilution of the clotting factors present in the infused FFP by the recipient's plasma. FFP contains all the components (proteins) of the coagulation and fibrinolysis systems and is therefore theoretically suitable for treating bleeding in patients with inherited or acquired clotting factor deficiencies. In addition, this preparation should prevent bleeding before, during and after surgical procedures in subjects with coagulation disorders. However, these plasma-derived preparations contain fibrinolysis proteins in addition to clotting factors and may cause undesired dissolution (melting) of hemostatic clots formed during and after the preparation's clotting factor replacement.

[0007] Thus, to date, there appear to be limited solutions to the treatment of excessive bleeding. Over 192,000 patients die each year in the United States due to injury-related blood loss (according to the National Trauma Institute). Massive blood loss can occur during trauma surgery, childbirth, and disseminated intravascular coagulation. It is associated with hemorrhage, vascular infarction syndrome (DIG), gastrointestinal bleeding, etc. In all of these cases, efficient plasma transfusion is required to stop the bleeding. However, recent studies have revealed that plasma transfusion cannot stop hemorrhage, while other treatments show low efficacy and increased risk of death. The majority of treatment options focus on enhancing coagulation, however, such treatments for hemorrhage have poor outcomes and in some cases even death. In contrast, there are several treatments for hyperfibrinolysis, which also contributes to hemorrhage. Hyperfibrinolysis occurs when fibrinolytic activity potentially outpaces fibrin formation, threatening the integrity of the clot. Currently, 57% of trauma patients and 60% of cirrhosis patients exhibit hyperfibrinolysis, and therefore, although the outcomes of these patients with enhanced coagulation are poor, there are no alternative treatment options. The subject matter disclosed herein has been developed to create an innovative extracorporeal device that effectively and specifically extracts and removes plasma proteins that cause hyperfibrinolysis. Using the medical devices of the presently disclosed subject matter, physicians can improve transfusion plasma and shift hemostasis from hyperfibrinolysis to coagulation, thus preventing hemorrhage.

[0008] No. 3,998,946 discloses a method for treating plasma with fumed colloidal silica to remove fibrinogen without polymerization to fibrin, and to remove plasminogen and plasmin and other compounds, but to retain clotting factor II, or related preparations. Lacking fibrinogen, the resulting preparation cannot support clot formation and therefore cannot be used to treat bleeding and hemostatic disorders.

[0009] US Patent No. 7,125,569 and its corresponding applications and patents disclose a specific method using a very specific resin to remove only plasmin(ogen) from one or more protein mixtures. The resulting formulation was made with the aim of preparing plasmin(ogen)-free fibrinogen to be used as a bioadhesive. However, the resulting mixture still contains tissue plasminogen activator (tPA) and thus exhibits apparent fibrinolytic activity. More specifically, the tPA in this formulation activated plasminogen at the treatment site, which led to the severing of the newly formed fibrin network. Plasminogen is normally present in high concentrations in blood (approximately 2 μM), and therefore any blood leak during any surgical intervention would increase the plasminogen concentration at the extravascular site. Furthermore, when such adhesives are applied to injured blood vessels during surgical intervention, the tPA present in the bioadhesive may come into contact with plasminogen present in the blood, thereby activating the fibrinolytic cascade. Therefore, the plasminogen-free formulations disclosed in U.S. Pat. No. 7,125,569 may only be used for local application as a bioadhesive and are not suitable for systemic use in blood transfusions or for the treatment of bleeding associated with fibrinolytic or thrombolytic therapy. Summary of the Invention [Problem to be solved by the invention]

[0010] Thus, there is a need in the art for effective devices and conjugates that deplete fibrinolytic proteins from mammalian body fluids, specifically blood, plasma and any preparations thereof. [Means for solving the problem]

[0011] Summary of the Invention In a first aspect, the present disclosure provides a plurality of conjugates or a composition comprising a plurality of conjugates, each conjugate comprising a particle, at least one linker, and at least one amino acid, derivative or analog thereof, wherein the plurality of conjugates comprises at least two different conjugates. The amino acid or analog thereof may be at least one of 4-(aminomethyl)cyclohexanecarboxylic acid (tranexamic acid), ε-aminocaproic acid (also referred to herein as aminocaproic acid), and lysine, or any combination thereof, and wherein the amino acid derivative may be at least one of cyclohexanecarboxylic acid, 4-methylcyclohexanecarboxylic acid, or any combination thereof.

[0012] In some further aspects, the present disclosure provides a conjugate comprising at least one particle, at least one linker, and at least one amino acid, derivative or analog thereof. In some specific embodiments, the conjugate of the subject matter disclosed herein comprises: [ka] (In the formula, [ka] represents a particle).

[0013] In yet another aspect, the presently disclosed subject matter provides an apparatus for depleting at least one fibrinolytic protein from one or more mammalian bodily fluids, the apparatus comprising: - a housing having at least one fluid inlet port and at least one fluid outlet port; Includes; - the housing comprises at least one chamber defining a control volume in fluid communication with at least one fluid inlet port and at least one fluid outlet port; - said control volume comprises a plurality of conduits as defined by the subject matter disclosed herein; The composition may contain at least one of the conjugates or compositions comprising a plurality of conjugates and the conjugates as defined herein above.

[0014] According to another aspect of the subject matter disclosed herein, there is provided an apparatus for depleting at least one fibrinolytic protein from one or more mammalian body fluids, the apparatus comprising: - a housing having at least one fluid inlet port and at least one fluid outlet port; Includes; - the housing comprises at least one chamber defining a control volume in fluid communication with at least one fluid inlet port and at least one fluid outlet port; - the control volume contains a plurality of particle populations including at least a first population of first particles and a second population of second particles; - wherein said first particles differ in size from said second particles; wherein at least one of the first particle and the second particle is a conjugated particle conjugated with an amino acid, a derivative or an analogue thereof, wherein the amino acid or analogue thereof is at least one of 4-(aminomethyl)cyclohexanecarboxylic acid (tranexamic acid), ε-aminocaproic acid and lysine, and the amino acid derivative thereof is cyclohexanecarboxylic acid, 4-methylcyclohexanecarboxylic acid or any combination thereof.

[0015] For example, the first particle and the second particle are conjugated particles, each conjugated particle being conjugated to an amino acid, a derivative thereof, or an analog thereof, wherein each of the amino acids or analogs thereof is at least one of 4-(aminomethyl)cyclohexanecarboxylic acid (tranexamic acid), ε-aminocaproic acid, and lysine, and the amino acid derivative thereof is cyclohexanecarboxylic acid, 4-methylcyclohexanecarboxylic acid, or any combination thereof.

[0016] Additionally and / or alternatively, for example, the first particles and the second particles are TXA conjugate particles, specifically cyclohexane carboxylic acid conjugate particles, 4-methylcyclohexane carboxylic acid conjugate particles, or any combination thereof. Additionally, for example, the first particles and the second particles are TXA conjugate particles.

[0017] As used herein, the term conjugated particle refers to a particle conjugated to an amino acid, a derivative thereof, or an analog thereof via a linker.

[0018] Additionally or alternatively, for example, the conjugate particle is defined by a plurality of conjugates or compositions as defined herein above.

[0019] Additionally or alternatively, for example, the housing may include a longitudinal axis and may include a body portion and a pair of end caps including an inlet end cap having the at least one fluid inlet port, and an outlet end cap having the at least one fluid outlet port.

[0020] For example, the control volume may be defined by corresponding barrier members provided at opposite longitudinal ends of the body portion.

[0021] For example, the barrier member may be configured to prevent the particles from escaping from the control volume.

[0022] For example, the barrier member may be configured to simultaneously permit the flow of one or more mammalian bodily fluids through the control volume, or the barrier member may be configured to simultaneously permit the flow of one or more mammalian bodily fluids through the control volume, where, during use of the device, one or more mammalian bodily fluids enter the control volume through the inlet end cap and fluid inlet port and, after exiting the control volume, flow through the outlet end cap and fluid outlet port.

[0023] Additionally or alternatively, for example, the barrier member may include a plurality of openings each of which permits the flow of one or more mammalian bodily fluids therethrough, the openings being smaller in size than the particles.

[0024] In a further aspect, the subject matter disclosed herein relates to a battery for use in depleting at least one fibrinolytic protein from one or more mammalian body fluids, comprising a plurality of devices, each device being as defined by the subject matter disclosed herein, the devices of the plurality of devices being interconnected in a manner providing fluid communication between the control volumes of each of the plurality of devices.

[0025] Another aspect of the subject matter disclosed herein relates to a kit for depleting at least one fibrinolytic protein from one or more mammalian body fluids, the kit comprising: - at least one device as defined by the subject matter disclosed herein, - a saline reservoir in selective fluid communication with said at least one fluid inlet port; - a receiving plasma reservoir and a wash waste reservoir in selective and non-simultaneous fluid communication with said at least one fluid outlet port. Includes.

[0026] In yet another aspect, the presently disclosed subject matter provides a system for depleting at least one fibrinolytic protein from one or more mammalian body fluids, the system comprising: - at least one device as defined by the subject matter disclosed herein, - a saline reservoir and a donor reservoir in selective and non-simultaneous fluid communication with the at least one fluid inlet port; The system includes a receiving plasma reservoir and a wash waste reservoir in selective and non-simultaneous fluid communication with the at least one fluid outlet port.

[0027] Thus, in yet another aspect, the subject matter disclosed herein relates to a method for depleting at least one fibrinolytic protein from one or more mammalian body fluids or any preparation thereof. More specifically, the method comprises the steps of (i) subjecting said one or more body fluids to an affinity depletion procedure specific for at least one fibrinolytic protein; and (ii) recovering the at least one fibrinolytic protein-depleted body fluid obtained in step (i). It should be noted that the affinity depletion procedure may comprise contacting the body fluid with an effective amount of the conjugates or at least one composition comprising the conjugates. Alternatively, the body fluid may be applied to a device, a set, a kit or a system comprising the conjugates or any composition thereof. In some embodiments, each conjugate comprises at least one particle, at least one linker and at least one amino acid, derivative or analogue thereof. In some specific embodiments, the plurality of conjugates comprises at least two different conjugates, and wherein the amino acid, derivative or analog thereof is at least one of 4-(aminomethyl)cyclohexane carboxylic acid (tranexamic acid), ε-aminocaproic acid, lysine, cyclohexane carboxylic acid, and 4-methylcyclohexane carboxylic acid. In some further specific embodiments, the plurality of conjugates comprises at least two different conjugates, and wherein wherein the amino acid, derivative or analogue thereof is at least one of 4-(aminomethyl)cyclohexanecarboxylic acid (tranexamic acid), ε-aminocaproic acid and lysine.

[0028] In yet another aspect, the subject matter disclosed herein provides a method for treating, preventing, prophylaxis, ameliorating, inhibiting bleeding, hemostatic disorders, and any bleeding or pathological condition associated therewith, in a subject in need thereof. More specifically, the method may comprise administering to the subject under treatment a therapeutically effective amount of at least one blood and / or blood-derived product having reduced fibrinolytic activity. In some embodiments, the formulation may be prepared by a method as described herein.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS For a better understanding of the subject matter disclosed herein, and to illustrate how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0030] [Figure 1] FIG. 1: Conjugate 2. Schematic of the chemical reactions for the preparation of conjugate 2. [Diagram 2] Figure 2: Plasminogen (PLG) standard curve. A graph showing the standard curve for calculating PLG concentration. [Diagram 3] Figure 3: Plasminogen-depleted cryoprecipitate. Graph showing plasminogen concentration in unprocessed cryoprecipitate and in cryoprecipitate filtered using ClearPlasma. [Figure 4] Figure 4: Plasma filtration with ClearPlasma with conjugate 1 ("TXA conjugated to agarose 4% beads"). Schematic showing blood flow and separation of plasma and red blood cells. Additionally, the schematic shows the use of ClearPlasma to generate plasminogen-depleted plasma with reduced tPA levels. [Diagram 5] Figure 5: ClearPlasma reduces blood loss in porcine liver injury. Histogram shows blood loss as seen 30 min after liver laceration. Statistics were calculated using Student's t-test (two-tailed, equal variance). Data are expressed as mean ± SD. A value of P<0.05 was considered significant. [Figure 6] Figures 6A-6L: ClearPlasma with conjugate 1 ("TXA conjugated to agarose 4% Superflow beads") abolishes fibrinolytic activity. Pigs underwent plasma apheresis and the plasma was either treated with ClearPlasma or left untreated. The coagulation and fibrinolysis of the resulting porcine whole blood was determined by TEG. [Figure 6A]FIG. 6A: Clotting of blood obtained from a control pig before anesthesia. [Figure 6B] FIG. 6B: Blood from a control pig at 0.083 μM Wt-tPA before anesthesia. [Figure 6C] FIG. 6C: Blood from a control pig after anesthesia. [Figure 6D] FIG. 6D: Control pig blood at 0.083 μM Wt-tPA after anesthesia. [Figure 6E] FIG. 6E: Control pig blood before plasma apheresis and plasminogen depletion. [Figure 6F] FIG. 6F: Porcine blood before plasma apheresis and plasminogen depletion with 0.083 μM Wt-tPA. [Figure 6G] FIG. 6G: Pig blood before plasma apheresis. [Figure 6H] FIG. 6H: Porcine blood before plasma apheresis with 0.083 μM Wt-tPA. [Figure 6I] FIG. 6I: Control pig blood after plasma apheresis and plasminogen depletion. [Figure 6J] FIG. 6J: Pig blood after plasma apheresis and plasminogen depletion with 0.083 μM Wt-tPA. [Figure 6K] FIG. 6K: Pig blood after plasma apheresis control. [Figure 6L] FIG. 6L: Pig blood after plasma apheresis with 0.083 μM Wt-tPA. [Figure 7] 7A-7F: TEG Analysis - ClearPlasma Eliminates Fibrinolytic Activity in Human Plasma The clotting and fibrinolysis of fresh frozen plasma (referred to as PDP) flowing through ClearPlasma was compared to the clotting and fibrinolysis of unprocessed plasma (referred to as FFP) by thromboelastography (TEG). [Figure 7A] FIG. 7A: FFP demonstrates clot formation without further processing. [Figure 7B] FIG. 7B: Addition of tissue plasminogen activator (tPA-1.85 nM) results in clot disassembly. [Figure 7C]Figure 7C: Overlay of Figures 7A and 7B. [Figure 7D] FIG. 7D: Plasminogen-depleted plasma (PDP) demonstrates clot formation without further treatment. [Figure 7E] FIG. 7E: Addition of tissue plasminogen activator (tPA-1.85 nM) did not result in clot disassembly. [Figure 7F] Figure 7F: Overlay of Figures 7D and 7E. The data show a representative experiment (out of three independent experiments). [Figure 8] Figure 8: Bleeding times following tail cut in mice. Graph showing bleeding times for individual mice (numbered 1-8) following tail cut treated with either ClearPlasma, plasma or saline. [Figure 9] Figure 9: Pellet size results for individual mice (at 24 hours). Graph showing pellet size for individual mice (numbered 1-8) following tail amputation treated with either ClearPlasma, plasma or saline. [Figure 10] Figure 10: Bleeding test results after tail amputation in mice: Blood cell pellets from the bleeding test were centrifuged and the supernatant aspirated. Pellet size was then measured using a ruler. Statistical analysis of pellet size measurements was performed using one-way ANOVA followed by post hoc LSD / SCHELF (p<0.05 considered significant). [Figure 11] Figures 11A-11B: ClearPlasma reduces both plasminogen and tPA protein levels in pigs. Sows underwent plasma apheresis using the Haemonetics mcs+ system under anesthesia. Both procedures were performed similarly (blood filtration volume, anticoagulant administered, plasma collection time and volume). [Figure 11A] FIG. 11A: Graph depicting plasminogen depletion in plasma samples from pigs filtered with ClearPlasma (referred to as PDP) in comparison to unfiltered control plasma. [Figure 11B]Figure 11B: Graph showing tPA depletion in plasma samples from pigs filtered with ClearPlasma (designated PDP) in comparison to unfiltered control plasma. Representative results from four independent experiments. [Figure 12] FIG. 12: The Device A cross-sectional side view of a device according to a first example of the subject matter disclosed herein is shown. [Figure 12A] FIG. 12A: An exploded isometric view of an apparatus according to an alternative variation of the example of FIG. [Figure 12B] FIG. 12B: A cross-sectional detailed side view of a portion of the device according to an alternative variation of the example of FIG. [Figure 13] FIG. 13A to FIG. 13D: This device [Figure 13A] FIG. 13A: A top view of the main body part of the device according to the example of FIG. [Figure 13B] FIG. 13B: A side view of the main body portion of the device according to the example of FIG. 13A. [Figure 13C] FIG. 13C: A cross-sectional side view of the main body portion of the device according to the example of FIG. 13B along AA. [Figure 13D] FIG. 13D: A cross-sectional detailed side view of a portion of the device at "G" according to the example of FIG. 13C. [Figure 14] FIG. 14: FIG. 14A-FIG. 14D: End caps of the device [Figure 14A] FIG. 14A: The end cap of the device according to the example of FIG. 12 is shown in top view. [Figure 14B] FIG. 14B: A side view of the end cap of the device according to the example of FIG. 14A. [Figure 14C] FIG. 14C: An end cap of the device according to the example of FIG. 14B is shown in cross-sectional side view along AA. [Figure 14D] FIG. 14D: A cross-sectional detailed side view of a portion of the device at "G" according to the example of FIG. 14C. [Figure 15] FIG. 15: The System Schematically illustrates a system according to an example of the subject matter disclosed herein, the system in a cleaning configuration. [Figure 16] FIG. 16: The present system illustrates a schematic diagram of a system according to the example of FIG. 15, the system being in a treatment configuration. [Figure 17] FIG. 17: Present Kit A kit corresponding to the system according to the example of FIG. 15 is illustrated diagrammatically. [Figure 18] FIG. 18: The System Schematically illustrates a system according to another example of the subject matter disclosed herein, the system including a suite of devices. [Figure 19] FIG. 19: The System A system according to another example of the subject matter disclosed herein is illustrated generally by way of example, the system including a suite of devices. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Detailed Description of the Invention According to its broadest aspect, the disclosure provides a plurality of conjugates or a composition comprising a plurality of conjugates, each conjugate having the general formula (I): XYZ(I) The During the ceremony: X is a solid support moiety, e.g., a particle; Y is a chemically reactive moiety that links moieties X and Z; Z is a moiety comprising at least one of an amino acid, a derivative thereof, or an analog thereof; and In the formula, each "-" denotes an interaction / association, e.g., a chemical bond optionally including one or more intervening atoms that act as a spacer or as a selectivity-inducing moiety.

[0032] The term amino acid, as used herein, refers to a compound (e.g., an organic compound) containing an amine (-NH2) group and a carboxyl (-COOH) group, including any derivatives thereof or any analogs thereof as detailed herein. The term "moiety" in the context of this disclosure may refer to any functional fragment of an atom, group of atoms, or molecule that functions as described herein. A moiety may also be in the form of a physical element (i.e., a single atom or multiple atoms) of at least one material that functions as described herein, such as a capsule, a sphere, a nanoparticle, a liposome, etc.

[0033] An "amino acid analog" is a compound (also referred to herein as a structural analog) that has the same chemical structure, i.e., carboxyl and amino groups, or R groups, as a naturally occurring amino acid. Examples include homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. In still some further embodiments, the term amino acid analog also encompasses functional analogs, specifically molecules that perform the same biological function. A non-limiting example of such a functional analog of an amino acid, e.g., lysine, is tranexamic acid (TXA), which acts as a functional analog to lysine and thus functions as an antifibrinolytic agent by reversibly binding to the 4-5 lysine receptor sites on plasminogen.

[0034] "Amino acid derivative" as used herein refers to an amino acid or any analogue thereof (e.g., An analog compound is at least a fragment (part, moiety) of an amino acid (structural and / or functional), e.g., a fragment lacking at least an amino group. In a specific example, an amino acid derivative includes a cyclohexane carboxylic acid fragment of an amino acid or an analog thereof, i.e., a fragment lacking an amino group.

[0035] The amino acid, derivative or analog thereof, in some embodiments, may be at least one of 4-(aminomethyl)cyclohexanecarboxylic acid (tranexamic acid), ε-aminocaproic acid, lysine, cyclohexanecarboxylic acid, 4-methylcyclohexanecarboxylic acid, or any combination thereof.

[0036] In a first aspect, the present disclosure provides a plurality of conjugates or a composition comprising a plurality of conjugates, each conjugate comprising a particle, at least one linker, and at least one amino acid, derivative or analog thereof. In some embodiments, the plurality of conjugates comprises at least two different conjugates. Still further, in some embodiments, the amino acid, derivative or analog thereof may be at least one of 4-(aminomethyl)cyclohexanecarboxylic acid (tranexamic acid), ε-aminocaproic acid, lysine, cyclohexanecarboxylic acid, and 4-methylcyclohexanecarboxylic acid, or any combination thereof. Still further, in some embodiments, the amino acid, derivative or analog thereof may be at least one of 4-(aminomethyl)cyclohexanecarboxylic acid (tranexamic acid), ε-aminocaproic acid, and lysine, or any combination thereof. Still in some embodiments, the amino acid, derivative or analog thereof may be tranexamic acid (TXA).

[0037] A conjugate, as used herein, refers to a compound that is composed of several elements (components), including at least one particle, at least one linker, and at least one amino acid, derivative or analog thereof, all of which are associated therewith. Although the term "at least one particle" is used in this application, it should be noted that any solid support applicable to the claimed conjugates is encompassed herein.

[0038] Any one of the subject conjugates disclosed herein or any composition thereof may also be referred to as a composition of matter. In terms, the term "composition of matter", like "conjugate" (both used interchangeably), refers to an association of at least one particle, at least one linker and at least one amino acid, derivative or analogue thereof, which produces properties that can be attributed to the composition of matter (or conjugate) as a whole and that can not be attributed to any one of the components of the conjugate in its individual state, as detailed below.

[0039] In some embodiments, any one of the conjugates of the subject matter disclosed herein comprises an association of at least one particle with at least one chemically reactive moiety that is a linker, and an association of the at least one linker with at least one amino acid, derivative or analog thereof, wherein the linker is an assocaition between the particle and the amino acid, derivative or analog thereof. and an analog thereof, and thus associated with the particle at one end (at one arm) and associated with the amino acid, derivative or analog thereof at the other end (at a second, different arm).

[0040] As used herein, the term "association" or any grammatical variation thereof refers to a chemical or physical force that holds two entities (e.g., a particle and a linker) together. Such forces may be any type of chemical or physical binding interaction known to those of skill in the art. Non-limiting examples of such association interactions are covalent bonds, ionic bonds, coordinate bonds, complexation, hydrogen bonds, van der Waals bonds, hydrophobic-hydrophilic interactions, etc. Thus, the linker and at least The association / conjugation of both the particle and the linker with the amino acid may be by any chemical bond including covalent bonds, electrostatic interactions, acid-base interactions, van der Waals interactions, etc. As will be appreciated, the association of the particle and the linker and the association of the linker with the amino acid, derivatives or analogs thereof may be the same or may be different as further detailed below.

[0041] For example, and as discussed in more detail below, for an amino acid derivative that is cyclohexanecarboxylic acid or 4-methylcyclohexanecarboxylic acid (i.e., lacking at least the NH2 group (amino group) of the amino acid analog), the linker may include an amino group (either alone or attached to a methylene group, i.e., -NH2-CH2- (where "-" is a covalent bond)). In an alternative example, for an amino acid or an analog thereof (i.e., including the NH2 group (amino group) of the amino acid), the linker may not include an amino group (either alone or attached to a methylene group, i.e., -NH2-CH2- (where "-" is a covalent bond)).

[0042] As pointed out above, a plurality of conjugates may be provided in a composition by the subject matter disclosed herein. A composition as used herein includes a plurality of conjugates, including at least two different conjugates. Different conjugates should be understood to be different in at least one parameter, more specifically, different conjugates that differ in at least one parameter or characteristic of at least one of the conjugate components, such as particles, linkers, amino acids, derivatives or analogs thereof, sometimes at least two of the conjugate components, and sometimes at least three of the conjugate components. Thus, at least two different conjugates of a plurality of conjugates or compositions thereof may include different particles and / or different linkers and / or different amino acids, derivatives or analogs thereof. In other words, conjugates referred to herein as "different conjugates" include conjugate components, such as particles, linkers, amino acids, where at least one, at least two, or all three components are not identical (e.g., different).

[0043] In some embodiments, at least two different conjugates comprise different particles.

[0044] In some embodiments, at least two different conjugates comprise different linkers.

[0045] In some embodiments, at least two different conjugates comprise different amino acids, derivatives or analogs thereof.

[0046] In some embodiments, at least two different conjugates comprise the same particle, the same linker, and different amino acids, derivatives or analogs thereof.

[0047] In some other embodiments, at least two different conjugates comprise the same particle, a different linker, and the same amino acid, derivative, or analog thereof.

[0048] In some other embodiments, the at least two different conjugates may comprise different particles, the same linker, and the same amino acid, derivative, or analog thereof.

[0049] In some embodiments, the at least two different conjugates comprise different particles and different linkers. In some embodiments, the at least two different conjugates comprise different particles, different linkers and the same amino acid, derivative or analog thereof.

[0050] In some embodiments, the at least two different conjugates comprise different particles and different amino acids, derivatives or analogs thereof. In some embodiments, the at least two different conjugates comprise different particles, different amino acids, derivatives or analogs thereof and the same linker.

[0051] In some embodiments, the at least two different conjugates comprise different linkers and different amino acids, derivatives or analogs thereof. In some embodiments, the at least two different conjugates comprise different linkers, different amino acids, derivatives or analogs thereof and the same particle.

[0052] In some embodiments, the at least two different conjugates comprise different particles, different linkers, and different amino acids, derivatives or analogs thereof.

[0053] The term different linkers should be understood such that the differences can be in the linker properties, such as the chemical formula of the linker.

[0054] As described above, the plurality of conjugates or any composition thereof as provided by the presently disclosed subject matter may comprise at least two different conjugates, at least three different conjugates, at least four, at least five, at least six, at least seven, or at least two different conjugates. It may comprise at least 8, at least 10 different conjugates, at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more different conjugates.

[0055] Still further, when referring to different conjugates, it should be understood that the difference can be in at least one characteristic (property, parameter) of the conjugate or any of its components (e.g., particles, linkers and amino acids), such as size, chemical composition, shape, structure, density, conductivity, solubility, material, etc. For example, at least one of the particles, linkers or amino acids that differ in at least one of size and / or different composition and / or different shape and / or different structure are considered to be different particles.

[0056] Surprisingly, it has been found that a plurality of conjugates comprising at least two different conjugates as described herein or a composition comprising at least two different conjugates is effective in depleting fibrinolytic proteins such as plasminogen, plasmin and / or tPA from body fluids, in particular blood, plasma and any blood product.

[0057] In some embodiments, the conjugates include particles having an average particle size between about 10 μm or less and about 500 μm or more, such as μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm or more. In some specific embodiments, the conjugates comprise particles having an average particle size of at least 70 μm, sometimes at least 80 μm, sometimes at least 90 μm, sometimes at least 100 μm, sometimes at least 110 μm, sometimes at least 120 μm, sometimes at least 130 μm, sometimes at least 140 μm, sometimes at least 150 μm, In some embodiments, the conjugates have an average particle size of between about 90 μm and about 150 μm or more.

[0058] The terms "average size" or "average diameter" or "average diameter" "Mean size" refers to the arithmetic mean of diameter measurements, where the diameter is within ±25% of the mean. The average particle size can be measured by any method known in the art.

[0059] In some embodiments, the plurality of conjugates comprises at least two different particles with different average diameters.Without being bound by theory, it should be noted that using two or more different sizes of particles (e.g., beads) has the advantage of maximizing surface area, so that bodily fluids, e.g., plasma, flow mainly on the surface of the beads, and there is little dead space.The more beads of different sizes there are, the less free space in the resin bed.

[0060] On the other hand, if the resin is packed tightly, there will be little space for the plasma to flow, which means lower flow rates.

[0061] More specifically, the inventors have unexpectedly found that a mixture of particles of different sizes can improve the microfluidic flow rate and expose more plasma to the conjugate (resin) while maintaining the same flow rate. Furthermore, mixing at least two particle sizes of conjugates can reduce the amount of beads and therefore minimize the cost of the conjugate used. In some embodiments, the conjugates of the subject matter disclosed herein can form a resin containing the conjugate using at least two particle populations of different sizes. In some detailed embodiments, the particles can be present in a ratio of about 0.001:1 to about 1:10000, more specifically, about 0.01:1 to 1:10000, about 1:1 to 1:1000. In still some further embodiments, the ratio may be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000. More specifically, particles of a plurality of conjugates or compositions thereof according to the presently disclosed subject matter may be of at least two different sizes exhibiting a ratio of about 1:1 to 1:10, specifically 1:4, in some embodiments. In some embodiments, the conjugates of the presently disclosed subject matter, also referred to herein as resins of the presently disclosed subject matter, can include a mixture of different conjugates of particles having an average diameter of about 90 μm or less and particles having an average diameter of 150 μm or more. In still some further specific embodiments, the conjugates or compositions of the presently disclosed subject matter can include a 4:1 ratio of conjugates of particles having an average diameter of 90 μm and particles having an average diameter of 150 μm.

[0062] The term particle, as used herein, refers to a portion of a substance having a surface that can be attached to chemical or biological compounds, small or large molecules, which may be attached by either covalent or non-covalent bonds. The particle may include a porous material. The particle may be "spherical" (generally referring to a substantially (nearly) round ball geometric shape) or "non-spherical", e.g., ("elongated" shape, with defined long and short axes). Non-limiting examples of particles include beads such as at least one of polysaccharide beads, glass beads, cotton beads, plastic beads, nylon beads, latex beads, magnetic beads, paramagnetic beads, superparamagnetic beads, starch beads, etc., silicon beads, PTFE beads, polystyrene beads, gallium arsenide beads, gold beads, or silver beads. In some embodiments, the particle is a bead, including agarose beads, optionally with different cross-linking degrees, different material (agarose) percentages.

[0063] Thus, agarose beads include beads containing agarose with various degrees of cross-linking, such as beads called sepharose beads. In some embodiments, the beads include agarose beads. In some embodiments, the beads include sepharose beads. In some embodiments, the multiple conjugates include a combination of particles including agarose beads and sepharose beads. It should be noted that in the present disclosure, particles that are either agarose beads or sepharose beads are considered to be two different conjugates with different particles.

[0064] Sepharose is the trade name for a cross-linked, beaded form of agarose, a polysaccharide polymer material extracted from seaweed. The trade name is derived from Separation-Pharmacia-Agarose. Sepharose is manufactured by GE Healthcare (formerly Pharmacia, Pharmacia LKB Biotechnology, Pharmacia Biotech, Amersham Sepharose is a registered trademark of Pharmacia Biotech, Inc. and Amersham Biosciences. Sepharose is available in a variety of grades and chemistries.

[0065] As described herein, the plurality of conjugates may include at least two different conjugates, optionally having particles with different properties, such as different sizes, different shapes, different compositions, and different materials. In some embodiments, the plurality of conjugates may include agarose beads with an average diameter of about 90 μm. In some embodiments, the plurality of conjugates may include agarose beads with an average diameter of about 150 μm. In some embodiments, the plurality of conjugates may include sepharose beads with an average diameter of about 90 μm. In some embodiments, the plurality of conjugates may include sepharose beads with an average diameter of about 150 μm. It should be noted that in the present disclosure, particles that are either sepharose beads with an average diameter of about 90 μm or sepharose beads with an average diameter of about 150 μm are considered to be two different conjugates with different particles.

[0066] Particles, particularly beads, as described herein may be associated with chemically reactive moieties, referred to herein as linkers. Linkers, as used herein, may be any chemical entity composed of any collection of atoms, including oligomeric and polymeric chains of any length, which, according to some embodiments, have the ability to bind at one end to the particle and at the other end to at least one amino acid, derivative or analog thereof. Furthermore, the present inventors have found that the coverage of the linker on the particle surface may range from about 9 to about 23 μmol beads / ml of drained medium. More specifically, it should be noted that in some embodiments, drained medium refers herein to dry beads.

[0067] As described herein, the linker has one end capable of binding to the particle and the second end capable of binding to an amino acid, its derivative or its analog, thus the linker has functional ends on both sides. In other words, the linker can be a bifunctional linker. In some embodiments, the linker is a bifunctional crosslinker and the particle is a bead that binds to the bifunctional crosslinker. As used herein, the term "crosslinker" refers to a reagent that contains two or more reactive ends capable of chemically linking with specific functional groups (e.g., primary amines, carboxyls, sulfhydryls, etc.) on amino acids, peptides, proteins, or other molecules.

[0068] In some embodiments, the linker (or cross-linker) may be a bifunctional linker or may include a portion / fragment of a bifunctional linker.

[0069] As will be appreciated, the linker can have different lengths depending on various experimental requirements. Length refers to the molecular span of the crosslinker, i.e., the distance between the conjugated components, e.g., between the particle and the amino acid. In some embodiments, the crosslinker is cleavable (i.e., whether the linkage can be reversed or broken as needed, e.g., EDC). In some embodiments In the above, the crosslinker is a zero-length crosslinker. In some embodiments, the crosslinker causes direct conjugation without becoming part of the final crosslinked covalent bond. The crosslinker may be a homobifunctional or heterobifunctional crosslinker. A homobifunctional crosslinker is a reagent that has the same type of reactive group at both ends. An amine crosslinker (i.e., binds to an amine reactive group) may be selected from, for example, glutaraldehyde, bis(imidoesters) or bis(succinimidyl esters) (also known as NHS esters). According to some embodiments, a homobifunctional crosslinker such as, but not limited to, dimethyl pimelimidate (DMP) or glutaraldehyde can bind to the NH2 group (the main group) on the magnetic beads and the NH2 group of tranexamic acid. A sulfhydryl crosslinker may be selected from, for example, maleimides or pyridyldithiols.

[0070] In some embodiments, the linker is a heterobifunctional crosslinker, which is a reagent that has different types of reactive groups at both ends, such as, but not limited to, amine to sulfhydryl or amine to carboxyl.

[0071] Amine to sulfhydryl crosslinkers can have NHS esters and maleimides at each end, or NHS esters and pyridyldithiols at each end. Examples of heterobifunctional crosslinkers capable of linking amine and sulfhydryl groups are selected from, but not limited to, N-succinimidyl 3-[2-pyridyldithio]-propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), or succinimidyl-4-(p-maleimidophenyl)butyrate (SMPB).

[0072] Amine to carboxyl crosslinkers may have carbodiimide crosslinkers that activate carboxyl groups so that they react spontaneously with primary amines. These crosslinkers may conjugate carboxyl groups (glutamate, aspartate, C-terminus) to primary amines (lysine, N-terminus) and N-hydroxysuccinimide (NHS). Examples of heterobifunctional crosslinkers capable of linking amine and carboxyl groups are selected from, but not limited to, dicyclohexylcarbodiimide (DCC) and (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride (EDAC). These crosslinkers are used to conjugate carboxyl groups (glutamate, aspartate, C-terminus) to primary amines (lysine, N-terminus) and N-hydroxysuccinimide (NHS) for stable activation of carboxylates for amine conjugation.

[0073] In some embodiments, the linker is aromatic. Non-limiting examples include benzoic acid or substituted benzoic acids, benzenesulfonyl chloride, benzaldehyde, chloromethylbenzene.

[0074] In some specific embodiments, the linker used is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC).

[0075] As detailed herein above, for conjugates comprising an amino acid derivative, such as cyclohexanecarboxylic acid or 4-methylcyclohexanecarboxylic acid (i.e. lacking the NH2 group (amino group) of the amino acid), the linker may comprise an amino group (either alone or attached to a methylene group, i.e. -NH2-CH2- (where "-" is a covalent bond). It should therefore be noted that in some embodiments, the linkers described herein may comprise at least an additional amino group or an amino group attached to a methylene group. Thus, in such embodiments, the linker terminus attached to the amino acid derivative may be The end is an amino group of the linker or a methylene group of the linker. In other words, the association between one end of the linker and the amino acid derivative comprises a covalent bond between the cyclohexane ring and the amino group or the methylene group. Such association can be by any known synthetic method known to those skilled in the art.

[0076] In some embodiments, beads may be associated with a linker via a spacer or coating present on the beads. Thus, the beads are first activated by association with a spacer / coating ("activated beads") and then react with a linker. It should be noted that sometimes, when the spacer / coating is directly bonded to at least one amino acid, a linker may not be necessary any more. Sometimes, the beads may not have functional groups capable of binding to a linker, and a spacer or coating may be used.

[0077] Activated beads are obtained by pre-coating beads with a suitable material having an active moiety that allows binding to the beads and to the linker and / or amino acid, in other words, the beads are pre-coated to contain reactive groups that allow covalent binding to either the linker or the amino acid.

[0078] In some embodiments, the beads may be activated, for example, by pre-coating with any coating material. Non-limiting examples of such materials include, for example, amino acids, proteins, epoxies, tosyls, carboxylic acids, carboxylated polyvinyl alcohol. When referring to "pre-coating", it should be understood as a preliminary step that results in the beads being coated with an active material, which in turn allows the beads to be covalently bonded to tranexamic acid (i.e. directly) or via at least one linker. In some embodiments, the beads are pre-coated with amino acids, peptides or any derivatives thereof. Pre-coated magnetic beads may contain, for example, primary amines (-NH2), carboxyls (-COOH), sulfhydryls (-SH) or carbonyls (-CHO) as active groups. In some embodiments, the beads are pre-coated to contain moieties that can react with primary or secondary amino groups. In some other embodiments, the magnetic beads are coated with polylysine.

[0079] As used herein, the term "linker" includes any spacer or pre-coating present on the beads.

[0080] In some embodiments, the linker comprises or is a chain of atoms, e.g., a linear chain. In some embodiments, the linker comprises at least 1 atom, at least 4 atoms, sometimes 5 atoms, sometimes 10 atoms, sometimes 20 atoms, sometimes 30 atoms, sometimes 40 atoms. In some embodiments, the linker is or comprises a linear chain of 1-40 atoms. In some embodiments, the linker is or comprises a linear chain of 1 atom. In some embodiments, the linker is a linear chain comprising 5 atoms. In some embodiments, the linker is a linear chain comprising 15 atoms.

[0081] In some embodiments, the linker is a straight chain containing 31 atoms. In some embodiments, the linker is a fragment of 26-(2-hydroxy-3-methoxypropylamino)-hexacosanoic acid 2,5-dioxopyrrolidin-1-yl ester. In some embodiments, the linker is a fragment of 4-oxopentanoic acid methyl ester. In some embodiments, the linker is methylene.

[0082] As described herein, for amino acid derivatives, the linker contains an amino or methylene (-CH2-) group at one end (the end that is reactive with the cyclohexane ring).

[0083] As will be appreciated, when a linker is associated with an amino acid, derivative or analog thereof, the linker is modified due to the association, and in some embodiments includes a fragment of the linker.

[0084] The linker properties depend on the nature of the atoms in the linker. Linkers have been found that contain at least one atom with at least one lone pair of electrons, such as oxygen, nitrogen, or sulfur. In some embodiments, the linker contains at least one oxygen atom.

[0085] In some other embodiments, the association between the linker to the at least one amino acid, derivative or analogue thereof may be a covalent bond. In some further embodiments, the association between the linker to the at least one amino acid may be by a bond between the nitrogen (N) atom of the amino acid and the carbon (C) atom of the linker. As will be appreciated, upon association and chemical bond formation, the amino group (-NH2-) loses one hydrogen atom to become -NH-. In some embodiments, the covalent association is via an amine, imine or amide bond. In some embodiments, the amino acid is tranexamic acid (TXA). As detailed herein, in some embodiments, the amino acid derivative is cyclohexane carboxylic acid and the association / interaction between the cyclohexane carboxylic acid and the linker is via the cyclohexane ring and the methylene group of the linker. As detailed in Example 1.1 below, in at least some of the multiple conjugates, the beads may be pre-coated with 26-(2-hydroxy-3-methoxypropylamino)-hexacosanoic acid 2,5-dioxopyrrolidin-1-yl ester. Thus, the agarose beads associated with 26-(2-hydroxy-3-methoxypropylamino)-hexacosanoic acid 2,5-dioxopyrrolidin-1-yl ester are subjected to association with at least one amino acid or analog thereof. The linker in the conjugate is a fragment of 26-(2-hydroxy-3-methoxypropylamino)-hexacosanoic acid 2,5-dioxopyrrolidin-1-yl ester, specifically 26-(2-hydroxy-3-methoxypropylamino)-hexacosanal. As can be seen, due to the association between 26-(2-hydroxy-3-methoxypropylamino)-hexacosanoic acid 2,5-dioxopyrrolidin-1-yl ester and an amino acid or analog thereof, the linker is a fragment of 26-(2-hydroxy-3-methoxypropylamino)-hexacosanoic acid 2,5-dioxopyrrolidin-1-yl ester, specifically 26-(2-hydroxy-3-methoxypropylamino)-hexacosanal.

[0086] As detailed in Example 1.2 below, in at least some of the conjugates, the beads may be pre-coated with hydroxide, reacted with succinic anhydride and pyridine, and subsequently reacted with N-Hydroxylsuccinamide (NHS) and pyridine. and EDC. Thus, the agarose beads associated with 4-(2,5-dioxopyrrolidin-1-yl)-4-oxobutyric acid methyl ester are subjected to association with at least one amino acid or analog thereof. The linker in the conjugate is a fragment of 4-(2,5-dioxopyrrolidin-1-yl)-4-oxobutyric acid methyl ester, specifically 4-oxobutyric acid methyl ester.

[0087] In some embodiments, the conjugate comprises agarose beads having an average diameter of 90 μm. In some embodiments, the linker comprises 26-(2-hydroxy-3-methoxypropylamino)-hexacosanal. In some embodiments, the conjugate has the structure (referred to herein as "conjugate 1"): [ka] (In the ceremony [ka] may comprise particles, such as agarose beads, specifically 4% agarose beads having an average diameter of 90 μm.

[0088] In some embodiments, the amino acid is TXA, the particle is an agarose bead having an average diameter of 90 μm, and the linker comprises 26-(2-hydroxy-3-methoxypropylamino)-hexacosanoic acid methylamide, and the conjugate has a structure referred to herein as Conjugate 1. In some embodiments, the amino acid derivative is cyclohexane carboxylic acid, the particle is an agarose bead having an average diameter of 90 μm, and the linker comprises 26-(2-hydroxy-3-methoxypropylamino)-hexacosanoic acid methylamide, and the conjugate has a structure referred to herein as Conjugate 1.

[0089] In some embodiments, the conjugate of the presently disclosed subject matter comprises a sepharose bead having an average diameter of 150 μm. In some embodiments, the linker comprises 4-oxobutyric acid methyl ester. In some embodiments, the conjugate has the structure (referred to herein as "conjugate 2"): [ka] (In the ceremony [ka] represents particles, e.g., sepharose beads having an average diameter of 150 μm).

[0090] In some embodiments, the amino acid is TXA, the particles are sepharose beads having an average diameter of 150 μm, and the linker comprises 4-oxobutyric acid methyl ester, and The conjugate has a structure referred to herein as conjugate 2.

[0091] In some embodiments where the amino acid derivative is cyclohexane carboxylic acid, the particles are sepharose beads with an average diameter of 150 μm, and the linker comprises N-methyl-succinamic acid methyl ester, and the conjugate has the structure referred to herein as Conjugate 2.

[0092] In some embodiments, the conjugate comprises agarose beads having an average diameter of 150 μm. In some embodiments, the linker comprises methylene. In some embodiments, the conjugate comprises the structure (referred to herein as "Conjugate 3"): [ka] (In the ceremony [ka] represents particles, e.g., agarose beads with an average diameter of 150 μm).

[0093] In some embodiments, the amino acid is TXA, the particles are agarose beads with an average diameter of 150 μm, and the linker comprises methylene, and the conjugate has a structure referred to herein as Conjugate 3.

[0094] In some embodiments where the amino acid derivative is cyclohexane carboxylic acid, the particles are agarose beads with an average diameter of 150 μm, and the linker comprises dimethylamine, and the conjugate has the structure referred to herein as Conjugate 3.

[0095] In some embodiments, the conjugate comprises an agarose bead. In some embodiments, the linker comprises a 15-atom carbon chain. In some embodiments, the linker is hexadecanal. In some embodiments, the conjugate has the structure (Conjugate 4): [ka] (In the ceremony [ka] represents a particle).

[0096] In some embodiments, the conjugate comprises an agarose bead. In some embodiments, the linker comprises a 15-atom carbon chain. In some embodiments, the linker is hexadecanoic acid methylamide. In some embodiments, the conjugate may have the structure (Conjugate 4):

[0097] In some further aspects, the present disclosure provides at least one conjugate comprising at least one particle, at least one linker, and at least one amino acid, derivative or analog thereof. In some embodiments, the conjugate of the presently disclosed subject matter may be any one of the following conjugates or any combination thereof. More specifically, [ka] (In the ceremony [ka] represents a particle).

[0098] In some embodiments, the particles are agarose beads or sepharose beads. In some other embodiments, the particles have an average particle size, specifically as defined above, between about 10 μm or less and about 500 μm or more. More specifically, between about 90 μm and about 150 μm. In some embodiments, the conjugates of the subject matter disclosed herein may bind to at least one fibrinolytic protein, more specifically, the fibrinolytic protein may be tissue plasminogen activator (tPA) and, optionally, plasminogen.

[0099] 12-19, an apparatus for processing mammalian bodily fluids, particularly plasma or whole blood, according to a first example of the subject matter disclosed herein, is generally designated by reference numeral 100 and includes a housing 300 and multiple collections of particles, particularly beads, including at least a first collection 110 of first particles or beads 210 and a second collection 120 of second particles or beads 220.

[0100] Although the device according to this and other examples finds particular use with human blood plasma (also used synonymously herein with "human plasma") and / or human whole blood (also used synonymously herein with "human blood"), and / or any formulations thereof, it may also be used for the processing of other mammalian but non-human plasma or mammalian but non-human whole blood, or indeed at least some types of animal plasma or animal whole blood, including, for example, at least some types of non-mammalian plasma or non-mammalian whole blood.

[0101] As will become apparent herein, the device is configured to deplete at least one fibrinolytic protein (e.g., tPA and / or plasminogen) from a mammalian bodily fluid (e.g., human plasma and / or human whole blood and / or other mammalian plasma and / or other mammalian whole blood) as an example of processing a mammalian bodily fluid.

[0102] Housing 300 defines a longitudinal axis AA and includes a body portion 350 and a pair of end caps including an inlet end cap 310 having a fluid inlet port 330 and an outlet end cap 320 having a fluid outlet port 340. In alternative variations of this example, the housing can include two or more body portions, and / or two or more inlet ports per body portion, and / or one or more inlet ports, and / or two or more outlet ports per body portion, and / or one or more outlet ports.

[0103] Each of the inlet end cap 310 and the outlet end cap 320 has a generally frustoconical base 360 ​​having a respective frustoconical inner surface 370 and a respective frustoconical outer surface 380. In this example and at least some other examples, the frustoconical inner surface 370 and the frustoconical outer surface 380 are highly polished. In an alternative variation of this example, the frustoconical inner surface 370 and / or the frustoconical outer surface 380 are not polished.

[0104] For example, fluid inlet port 330 and fluid outlet port 340 may each project distally from a respective frusto-conical base 360 ​​and each be fitted with a suitable respective leak-proof connector portion 335. For example, such leak-proof connector portion 335 may be in the form of a luer lock to facilitate connection of a conduit (e.g., medical grade tubing) thereto. Alternatively, fluid inlet port 330 and fluid outlet port 340 may each be fitted with any other type of suitable leak-proof connector portion 335 that facilitates connection of conduits (e.g., medical grade tubing) thereto.

[0105] 14A-14D, each frusto-conical base 360 ​​has a cross-sectional area (orthogonal to longitudinal axis AA) that increases from its respective small end 312, 322 to its respective large end 314, 324. In at least this example, each frusto-conical base 360 ​​includes a plurality of web members 301 that project radially and axially inwardly from an inner surface of the respective inlet end cap 310 or outlet end cap 320. However, in at least some alternative variations of this example, as well as other examples, the web members may be omitted. Without being bound by theory, the web members 301 may increase the overall stiffness of the device 100 and / or help regulate the flow of untreated bodily fluid into and out of the device 100, particularly into the chamber 400 generally along the longitudinal axis AA.

[0106] Further, without being bound by theory, it is believed that the web member 301 serves to hold or otherwise maintain the barrier members 352, 354 in position at their respective longitudinal ends E1, E2, and more particularly, may serve to clamp the barrier members 352, 354 between the respective inlet end caps 310 or outlet end caps 320 and the body portion 350 in the housing 300, as will become more clear below.

[0107] The web members 301 each have a free end 306 that faces a respective barrier member 352, 354 in the assembled device 100. As best seen in Figures 14B and 14C, the free ends 306 project beyond the respective larger ends 314, 324 in a longitudinal direction parallel to the longitudinal axis AA, away from the respective frusto-conical inner surfaces 370.

[0108] In at least this example, fluid inlet port 330 is mated to small end 312 of inlet end cap 310 and fluid outlet port 340 is mated to small end 322 of outlet end cap 320. In alternative variations of this example, fluid inlet port 330 may be connected to inlet end cap 310 at any other suitable location and / or fluid outlet port 340 may be connected to outlet end cap 320 at any other suitable location.

[0109] In this example, and referring also to Figures 13A-13D, body portion 350 includes a generally cylindrical wall 355 (ie, having a circular cross-section transverse to the longitudinal axis AA of the device) and has longitudinally opposed ends 356, 358.

[0110] In this example, housing 300, and in particular body portion 350 including cylindrical wall 355, is relatively rigid. By relatively rigid, we mean that body portion 350 maintains its shape without significant deformation (e.g., without deformation visible to the naked eye of an untrained observer) under its own weight or when containing a plurality of particle populations as well as the flow of plasma or other bodily fluid through the device during operation of the device.

[0111] In alternative variations of this example, the body portion may be semi-flexible or fully flexible (e.g., in the form of a flexible bag) and / or the body portion may have a non-cylindrical cross-section, e.g., elliptical, polygonal, etc.

[0112] In this and other examples, the cross-section of body portion 350 is generally uniform along the longitudinal axis AA of the device. In alternative variations of this example, the cross-section of body portion 350 may instead be non-uniform along the longitudinal axis AA of the device - e.g., the cross-section may vary in shape and / or size along the longitudinal axis AA. For example, body portion The portion may be frusto-conical, with the circular cross-section of the body portion increasing in size or alternatively decreasing in size along the longitudinal axis AA; for example, such a frusto-conical shape may be approximately cylindrical, for example having a half apex angle of about 0.5° or about 1° (see FIG. 13C ).

[0113] The housing 300, and in particular the body portion 350 and the inlet and outlet end caps 310 and 320, may each be made of any suitable medically compatible material. For example, such materials include medical grade plastics, such as those available from Covestro, USA. Supply PC (Makrolon 2458), Apec® 1745 polycarbonate; polypropylene The material may include any one of the following: polyethylene; polysulfone; polyetheretherketone (PEEK).

[0114] In at least this example, housing 300, and in particular body portion 350, inlet end cap 310, and outlet end cap 320, are transparent, i.e., made of a transparent material. However, in alternative variations of this example, housing 300, and in particular body portion 350 and / or inlet end cap 310 and / or outlet end cap 320, are not transparent, e.g., translucent or opaque, or any combination of transparent, translucent, or opaque portions.

[0115] The housing 300, and more particularly the body portion 350, includes an interior chamber 400 that defines a control volume V. In this example, the control volume V is defined by its transverse perimeter P, which is defined by the inner surface of the body portion 350, which in this example is a cylindrical inner surface, and by longitudinal ends E1 and E2 at opposite longitudinal ends of the body portion 350.

[0116] The longitudinal ends E1 and E2 of the control volume V are defined by corresponding barrier members 352, 354 provided at opposite longitudinal ends of the body portion 350. As will become clearer herein, the barrier members 352, 354 are configured to prevent the first particles 210 and the second particles 220 from exiting the body portion 350, and in particular from the control volume V, in particular through the inlet end cap 310 and the fluid inlet port 330, or through the outlet end cap and the fluid outlet port 340. The barrier members 352, 354 are also configured to simultaneously allow the flow of body fluids, in particular liquids, more particularly plasma, and more particularly human plasma, through the body portion 350, and in particular through the control volume V, in particular one or more mammalian body fluids entering the control volume V through the inlet end cap 310 and the fluid inlet port 330, and flowing through the outlet end cap and the fluid outlet port 340 after exiting the control volume V.

[0117] In at least this example, barrier members 352, 354 are similar or identical to one another and are in the form of filter discs 352A, 354A, respectively, each having a respective upstream surface and a respective downstream surface separated by the thickness of the respective filter disc 352A, 354A. Barrier members 352, 354, and more particularly filter discs 352A, 354A, include a plurality of pores or other openings to permit the flow of fluid, more particularly liquid, more particularly bodily fluid, and more particularly human plasma or human whole blood (or alternatively, any mammalian or non-mammalian plasma or whole blood) therethrough, the pores being sized less than the smaller of (a) the mean or median diameter of first particles 210 or (b) the mean or median diameter of second particles 220. For example, in an example where the smaller of the first particles 210 or second particles 220 has an effective (average or median) diameter of about 90 μm (e.g., the larger particle has an effective (average or median) diameter of about 150 μm), the pores have an effective (average or median) diameter of less than 90 μm, e.g., any one of 80 μm; 70 μm; 60 μm; 50 μm; 40 μm, or less than 40 μm. In such example particle diameters, the pores can have an effective (average or median) diameter within the range 45 μm to 50 μm, e.g., or alternatively, the pores can have an effective (average or median) diameter within the following ranges: 20 μm to 80 μm; 30 μm to 80 μm; It may have an effective (average or median) diameter within any one of the following: 40 μm to 70 μm; 40 μm to 55 μm; 40 μm to 50 μm; 40 μm to 60 μm; 40 μm to 70 μm; 40 μm to 80 μm.

[0118] An advantage of at least some examples where the barrier members 352, 354 are in the form of filter discs 352A, 354A is that the filter discs 352A, 354A can be rapidly manufactured.

[0119] Another feature of at least some examples of the device is the ability to control the flow through the device 100. For example, selecting a large amount of particles (i.e., the first particles 210 and the second particles 220) in the control volume V will tend to reduce the volumetric flow rate of bodily fluid through the device 100, while selecting a reduced amount of particles in the control volume V will reduce the ability and effectiveness of the particles to deplete fibrinolytic proteins from the bodily fluid. Thus, in effect, the volumetric flow rate of the device may depend on the ratio of the amount of particles (i.e., the first particles 210 and the second particles 220) in the control volume V to the volume of bodily fluid that can be accommodated in the control volume V when this amount of particles is accommodated in the control volume V.

[0120] In alternative variations of this and other examples, the barrier member may comprise, for example, a filter including a fiber or plastic matrix to which the ligand is conjugated, or for example a suitable membrane, for example a unidirectional membrane that allows the flow of bodily fluid therethrough in one direction but not in the opposite direction through the membrane.

[0121] In this and other examples, the filter disks 352A, 354A are Spectra Mesh® woven filters (e.g., nylon, PEEK) supplied by Spectrum, USA. , polypropylene, polyester, stainless steel), or MS® nylon mesh filters supplied by Yair Technologies, Israel. It is possible.

[0122] In at least this example, the barrier members 352, 354, particularly the filter discs 352A, 354A, are attached to the housing 300, particularly the body portion 350, and optionally also to the inlet end cap 310 and the outlet end cap 320, in a manner that maintains the integrity of the control volume V. In other words, the connection between the barrier members 352, 354, particularly the filter discs 352A, 354A, and the housing 300, particularly the body portion 350, is such that it minimizes or avoids any leakage of the first particles 210 or the second particles 220 from the connection, while maximizing the exposed area of ​​the barrier members 352, 354, particularly the filter discs 352A, 354A, to the bodily fluid passing through the device 100. Further, the barrier members 352, 354, and particularly the filter discs 352A, 354A, are mounted to the housing 300, and particularly to the body portion 350, and optionally also to the inlet end cap 310 and the outlet end cap 320, such that their respective similar upstream surfaces of the two barrier members 352, 354, and particularly to the two filter discs 352A, 354A, face in the upstream direction, while their respective downstream surfaces face in the downstream direction.

[0123] 13D and 14D, each of ends 356, 358 of body portion 350 includes a raised annular shoulder 359 that is radially offset from inner edge 344 (see, e.g., FIGS. 12B, 13C) of each end 356, 358 and defines respective inner ledge 357 and outer ledge 353. Each of barrier members 352, 354, and particularly filter discs 352A, 354A, has an outer diameter that is greater than the outer diameter of its respective inner edge 344 and less than the inner diameter of its respective annular shoulder 359, and thus each The inlet end cap 310 and the outlet end cap 320 are seated on an inner ledge 357. The respective inlet end cap 310 and the outlet end cap 320 each have a respective annular rim 351 at a respective large end 314, 324 of the frusto-conical base 360 ​​that sealingly secures the respective inlet end cap 310 and the outlet end cap 320 to the respective ends 356, 358. For example, the respective annular rims 351 may be glued, heat welded, ultrasonically welded, or otherwise sealingly secured to the respective outer ledge 353.

[0124] In this example, each annular shoulder 359 on each end 356, 358 includes a protruding lip 359A having a generally triangular cross-section with the apex of the triangle pointing away from the respective end 356, 358 and converging toward the apex in a direction generally parallel to the longitudinal axis AA away from the body portion 350. Each annular edge 351 includes an annular recess 351A that mates with the protruding lip 359A when the inlet end cap 310, body portion 350 and outlet end cap 320 are simultaneously aligned relative to the longitudinal axis AA.

[0125] It should be noted that web member 301 has a dimension D (FIG. 14C) parallel to longitudinal axis AA such that when the respective inlet end cap 310 or outlet end cap 320 is sealingly secured to body portion 350, the respective barrier members 352, 354, particularly filter discs 352A, 354A, are immobilized relative to housing 300 and provide an abutting, and optionally clamped, relationship between the respective web member 301 and the respective inner ledge 357.

[0126] During the thermal or ultrasonic welding process, for example, respective lip 359 A deforms and forms a sealing connection with respective annular recess 351 A, thereby sealingly securing respective inlet end cap 310 or outlet end cap 320 to body portion 350 .

[0127] 13A-14D, and referring to FIG. 12B as one such example, each annular shoulder 359 of each end 356, 358 has an inclined surface 359B (instead of protruding lip 359A) that is generally parallel to the frustoconical inner surface 370. On the other hand, in this example, each annular edge 351 includes (instead of annular recess 351A) a protruding lip 351B that mates with outer ledge 353 when inlet end cap 310, body portion 350 and outlet end cap 320 are simultaneously aligned with longitudinal axis AA. Protruding lip 351B has a generally triangular cross section with the apex of the triangle pointing towards each end 356, 358 and the generally triangular cross section converging towards the apex in a direction parallel to longitudinal axis AA, i.e., towards body portion 350. During the thermal or ultrasonic welding process, each lip 351B deforms and forms a sealing connection with a respective annular outer ledge 353.

[0128] 13A-13D provide dimensional data (in mm) for various portions of body portion 350 according to one embodiment of this example (e.g., tolerance ±0.05). In alternative embodiments of this example, the dimensional data may be changed proportionally, e.g., all annotated dimensions may be increased or decreased by the same percentage, e.g., increased by 50%, 100%, 150%, 200%, etc., or decreased by 10%, 25%, 50%, etc.

[0129] 14A-14D provide dimensional data (in mm) (e.g., with tolerances of ±0.05) for various portions of inlet end cap 310 according to one embodiment of this example, and similar dimensional data, modified accordingly, apply to outlet end cap 320. In alternative embodiments of this example, the dimensional data may be changed proportionally, e.g., all annotated dimensions may be increased or decreased by the same percentage, e.g., by 50%, 100%, 150%, 200%, etc., or by 10%, 25%, 50%, etc. 12 and 13A-14D, it should be noted that body portion 350 has a longitudinal dimension (parallel to longitudinal axis AA) that is approximately the same size as the inner diameter of body portion 350. In alternative variations of this example, referring for example to FIG. 12A, the longitudinal dimension may be greater than the inner diameter of body portion 350. In yet other alternative variations of this example, the longitudinal dimension may be less than the inner diameter of body portion 350.

[0130] In an alternative variation of this example, the housing 300 may be integrally formed and provided with transverse slots at each of the longitudinal ends E1 and E2 to allow the respective barrier members 352, 354, particularly the filter discs 352A, 354A, to be inserted transversely and sealed against the housing 300.

[0131] In at least this example, the housing 300, and more particularly the body portion 350, includes a particle inlet port 390 provided in the cylindrical wall 355 and is configured to allow the plurality of particle collections, including at least a first collection 110 of first particles 210 and a second collection 120 of second particles 220, to be loaded into the control volume V through the particle inlet port 390. The particle inlet port 390 includes a sealing cap 392 for reversibly or permanently sealing the particle inlet port 390 after loading of the plurality of particle collections into the control volume V. For example, such sealing cap 392 has a tapered highly polished stem portion 393 that abuts a complementary tapered inner wall 394 of the particle inlet port 390 and a head portion 395 that deformably engages an annular flange 399 provided at the mouth of the particle inlet port 390.

[0132] Alternatively, particle inlet port 390 may be reversibly or permanently sealed in another manner after filling of control volume V with the plurality of particle populations.

[0133] The device 100 is assembled by first positioning the barrier members 352, 354, specifically filter discs 352A, 354A, into alignment with the ends 356, 358 of the housing 300, and then sealingly fastening the inlet end cap 310 and the outlet end cap 320 to the body portion 350, thereby engaging the barrier members 352, 354, specifically filter discs 352A, 354A, to the housing 300. The integrity of the assembly may be tested by, for example, connecting the particle inlet port 390 to a pressure source (e.g., set at 1 bar gauge pressure) while reversibly sealing the inlet port 330 and outlet port 340, and submerging the housing 300 in a liquid, e.g., water, to see if there are any leaks, which may be detected by observing any air bubbles escaping from the housing 300. For example, the housing 300 may be considered adequately sealed if a leak is observed with no more than one air bubble escaping from the housing 300 every 30 seconds.

[0134] In at least some applications of the above example device 100, the device 100 is pre-loaded with the plurality of particle populations prior to delivery to an end user. In such cases, a preservative may be used in the plurality of particle populations. For example, such a preservative may be 20% ethanol.

[0135] In another alternative application of the above example of device 100, device 100 is filled with the plurality of particle populations by an end user. At least in this example, the size of control volume V is between 20 ml and about 35 ml.

[0136] In alternative variations of this example, and in other examples, the size of the control volume V may vary, e.g., less than 35 ml or more than 35 ml. For example, the size of the device, and therefore the size of the control volume V, may be scaled, e.g., by a linear scale factor. For example, the linear scale factor may be any one of the following values: 2, 3, 4, 5, 10, 20, 30, 40, 50, or more than 50. Or, for example, the linear scale factor may be any one of the following values: 2, 3, 4, 5, 10, 20, 30, 40, 50, or more than 50. is "n" times larger than in the current example, where n is any one of the following values: 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 150 or more than 150; correspondingly or alternatively, the control volume V may be enlarged to 70 ml, 100 ml, 150 ml, 175 ml, 200 ml, 250 ml, 500 ml, 1 liter, 1.5 liters, 2 liters, 2.5 liters, 3 liters, 4 liters, 5 liters or more than 5 liters.

[0137] In this example, first particle 210 and / or second particle 220 may be conjugate particles (referred to herein as "conjugates"), each of which may optionally comprise any one of the conjugate particles, e.g., as disclosed herein, or compositions thereof which may include at least two or more different conjugates.

[0138] In at least some alternative variations of these examples, only one of the first population 110 and the second population 120 includes conjugated particles, while the other of the first population 110 and the second population 120 includes non-conjugated particles. In still other alternative variations of these examples, the first population 110 and the second population 120 both include the same conjugated particles, such that there is effectively only a single, combined population of conjugated particles.

[0139] In at least this example, the first particle 210 and / or the second particle 220 are conjugate particles, where the first particle differs in size from the second particle. In particular, the first conjugate particle 210 and the second conjugate particle 220 differ in size from the second particle. In at least some such examples, the first conjugate particle has an average diameter that differs from the average diameter of the second particle. In still some further embodiments, the first particle, specifically the conjugate, may be chemically distinct from the second conjugate.

[0140] Without being bound by theory, the inventors believe that including at least two particle populations in which the first conjugate particles have a different average diameter than the average diameter of the second particles may provide spacing between the particles, resulting in an arrangement that is substantially less dense than a similar arrangement in which all particles are the same or similar size. This characteristic of low density in turn provides each of the particles with a larger exposed area than a similar arrangement of particles of the same or similar size. The increased exposed area in turn allows for a higher interaction between the conjugate particles and the bodily fluid (as such fluid is flowed through the device 100), thereby increasing the effectiveness of the particles in depleting at least one fibrinolytic protein from the bodily fluid. Thus, a relatively small total amount of first and second conjugate particles of different dimensions can be as effective in depleting at least one fibrinolytic protein from the bodily fluid as a relatively large total amount of first and second conjugate particles of similar dimensions, which may have economical effects.

[0141] One way to calculate how much plasminogen can be absorbed using a single conjugate particle population is as follows: Resin capacity calculation -Start with standard fresh frozen plasma units- Each unit contains 250mL plasma Plasma plasminogen concentration is 160 μg / mL Therefore, the total amount of plasminogen in one unit of FFP is 40 mg. The molecular mass of plasminogen is 92 kDa. For purposes of calculation, 1 Dalton unit is considered to be numerically equivalent to 1 g / mol. Therefore, 92000 Da is 92000 g / mol or 92000 μg / μmol or 9 2mg / μmol Calculate the number of moles per 40 mg FFP unit: 40mg÷92mg / μmol=0.435μmol - Sepharose 4 Fast Flow with 25 atom linker acting on the resin after 16-23 μmol beads / mL depletion was able to reduce 1.317 g of plasminogen. This is 33 times more than found in one plasma unit.

[0142] Referring to Figures 15 and 16, a system for depleting at least one fibrinolytic protein from one or more mammalian bodily fluids, generally indicated by reference numeral 1000, according to one example of the subject matter disclosed herein, includes a device 100, a saline reservoir 1100, a donor reservoir 1200, a recipient plasma reservoir 1300 and a wash waste reservoir 1400.

[0143] The saline reservoir 1100 and the donor reservoir 1200 are in selective and non-simultaneous fluid communication with the fluid inlet port 330 via the first three-way valve 70 .

[0144] The receiving plasma reservoir 1300 and the wash waste reservoir 1400 are in selective and non-simultaneous fluid communication with the fluid outlet port 340 via the second three-way valve 90 .

[0145] Conduits 82 , 84 connect the saline reservoir 1100 and the donor reservoir 1200 to respective ports 72 , 74 of the first three-way valve 70 , and another conduit 76 connects the third port 76 of the first three-way valve 70 to the inlet port 330 of the device 100 .

[0146] Conduits 62, 64 connect the receiving plasma reservoir 1300 and the wash waste reservoir 1400 to respective ports 92, 94 of the second three-way valve 90, and another conduit 66 connects a third port 96 of the second three-way valve 90 to an outlet port 340 of the device 100.

[0147] In at least this example, the apparatus 100 is as disclosed in that example or alternative variations of those examples herein, in either case, a control volume V contains the plurality of particle populations including at least a first population of first particles and a second population of second particles.

[0148] Saline reservoir 1100 may include any suitable reservoir containing a suitable volume of saline, which is typically several times the size of control volume V. In this example, saline reservoir 1100 is in the form of a syringe, however, in alternative variations of this example, saline reservoir 1100 may take other forms, such as a non-rigid bag that can be squeezed to force saline out of the bag through conduit 82.

[0149] Donor reservoir 1200 may include a bag or other reservoir containing a mammalian bodily fluid to be processed by device 100. For example, donor reservoir 1200 may contain human plasma and / or human whole blood and / or other mammalian plasma and / or other mammalian whole blood. Alternatively, donor reservoir 1200 may include a living donor, e.g., a human, whose blood vessels have blood catheters inserted in such a way that blood can flow directly to device 100 through a catheter (connected to or part of conduit 84) or indirectly through a suitable blood product separation device that separates plasma from the whole blood prior to delivery to device 100.

[0150] The receiving plasma reservoir 1300 may include a bag or other reservoir for receiving the treated mammalian bodily fluid after treatment by the device 100. For example, the plasma reservoir 1300 may contain human plasma that has been fully or partially depleted of at least one fibrinolytic protein and / or It may contain human whole blood and / or other mammalian plasma and / or other mammalian whole blood, or alternatively, the receiving plasma reservoir 1300 may comprise a living patient, e.g., a human, having a blood catheter inserted into its vasculature in such a manner as to allow flow of treated mammalian bodily fluid from the device 100 through the catheter (which is connected to or part of the conduit 62).

[0151] The wash waste reservoir 1400 may comprise a bag or other reservoir capable of receiving saline from the saline reservoir 1100 after it has passed through the device.

[0152] 17, it should be noted that the device 100 as disclosed herein may be provided as a kit 800, along with a saline reservoir 1100, a receiving plasma reservoir 1300 and a wash waste reservoir 1400. The kit 800 may further include a first three-way valve 70, and a conduit 82 that connects to the saline reservoir 1100 and connects to the first three-way valve 70 via port 72, and a conduit 86 that connects to the first three-way valve 70 and connects to the inlet port 330 of the device 100. The kit 800 may further include a second three-way valve 90, and conduits 62, 64 that connect to the receiving plasma reservoir 1300 and the wash waste reservoir 1400, respectively, and connect to the second three-way valve 90 via respective ports 92, 94, and a conduit 66 that connects to a third port 96 of the second three-way valve 90 and connects to the outlet port 340 of the device 100. The kit 800 may further include a conduit 84 that connects to the first three-way valve 70 via port 74, where the other end of the conduit 84 is connectable to the donor reservoir 1200.

[0153] The system 1000 can be used, for example, as follows.

[0154] The kit 800 may be opened from its sterile packaging and the various components inspected to ensure the integrity of all components.

[0155] 15, the cleaning configuration is set up by ensuring that the first three-way valve 70 is set to provide fluid communication between ports 72 and 76 while blocking port 74, and the second three-way valve 90 is set to provide fluid communication between ports 94 and 96 while blocking port 92. The device 100 is then primed (cleaned with saline) by passing saline from the saline reservoir 1100 through the device 100, through the first three-way valve 70, and through the second three-way valve 90 to the cleaning waste reservoir 1400.

[0156] A donor reservoir 1200 is connected to the kit 800 to provide the system 1000. In instances where the donor reservoir is in the form of a bag or other reservoir containing a mammalian bodily fluid to be processed by the device 100, the donor reservoir 1200 may be fused to the conduit 84. The donor reservoir 1200 may then be suspended from, for example, an IV pole or connected to a peristaltic pump, which may allow the mammalian bodily fluid to be processed by the device 100 to selectively flow therethrough, as will become more clear below.

[0157] In the example where donor reservoir 1200 is a live donor, such as a human, a blood catheter is inserted into the live donor's vein and the catheter is connected to or is part of conduit 84 .

[0158] 16, after the cleaning configuration is completed and the device 100 is primed with saline (using the configuration of FIG. 15), the processing configuration is set up. The processing configuration is configured such that the first three-way valve 70 provides fluid communication between ports 74 and 76 while blocking port 72, and the second three-way valve 90 allows flow between ports 92 and 96 while blocking port 94. The apparatus 100 is then set up to provide bodily communication with the donor reservoir 1200. The unprocessed mammalian bodily fluid is then processed, specifically by passing the unprocessed mammalian bodily fluid from the donor reservoir 1200 through the first three-way valve 70, through the apparatus 100, and through the second three-way valve 90 to the recipient plasma reservoir 1300 to deplete the mammalian bodily fluid of at least one fibrinolytic protein.

[0159] 18, another example of a system for depleting at least one fibrinolytic protein from one or more mammalian bodily fluids in accordance with the subject matter disclosed herein, generally designated by reference numeral 2000, includes a suite 2500 including a plurality of devices 100. In the example shown in FIG. 18, suite 2500 includes four devices 100, although in alternative variations of this example suite 2500 may instead include two, three or more than four devices 100, with appropriate modifications.

[0160] The system 2500 also includes a saline reservoir 1100, a donor reservoir 1200, a receiver plasma reservoir 1300 and a wash waste reservoir 1400, as well as a first 70 and a second 90 three-way valve, with appropriate modifications, as disclosed herein for the system 1000 shown in Figures 15 and 16. In this example, the apparatus set 2500 includes a plurality of apparatus 100 interconnected to provide fluid communication between the respective control volumes V of the plurality of apparatus 100.

[0161] In at least this example, each device 100 (of the suite of devices 2500) is as disclosed in that example or alternative variations of those examples herein. In any case, the control volume V of each device 100 of the suite of devices 2500 contains a respective one of the plurality of particle populations including at least a first population of first particles and a second population of second particles.

[0162] In at least this example, the devices 100 of the suite 2500 are interconnected in series, where for each pair of serially (adjacently) interconnected devices 100, a respective fluid inlet port 330 of one device 100 of the pair is connected to and in fluid communication with a respective fluid outlet port 340 of the other device 100 of the pair. The most upstream device 100 of the suite 2500 (also designated device 100A in FIG. 18) connects to a saline reservoir 1100 and a donor reservoir 1200 via a first three-way valve 70 in a manner similar to the devices 100 of the system 1000 shown in FIGs. 15 and 16, with appropriate modifications. The most downstream device 100 of the set of devices 2500 (also designated device 100B in FIG. 18) connects to the receiving plasma reservoir 1300 and the wash waste reservoir 1400 via a second three-way valve 90 in a manner similar to the device 100 of the system 1000 shown in FIGS. 15 and 16, with appropriate modifications.

[0163] In this example, the saline reservoir 1100 contains enough saline to wash and prime all of the devices 100 in the suite 2500 .

[0164] It should be noted that the complete set 2500 of the device 100 as disclosed herein, together with the saline reservoir 1100, the receiver plasma reservoir 1300 and the wash waste reservoir 1400 (and excluding the respective donor reservoirs 1200) may be provided as a kit 2800. The kit 2800 may further include a first three-way valve 70, and a conduit 82 connecting to the saline reservoir 1100 and connecting to the first three-way valve 70 via port 72, and a conduit 86 connecting to the first three-way valve 70 and connecting to the inlet port 330 of the most upstream device 100A. The kit 2800 includes a second three-way valve 90, conduits 62, 64 which connect to the receiving plasma reservoir 1300 and the wash waste reservoir 1400, respectively, and connect to the second three-way valve 90 via respective ports 92, 94, and a conduit 66 which connects to a third port 96 of the second three-way valve 90 and connects to the outlet port 340 of the most downstream device 100B. 66. The kit 2800 may further include a conduit 84 that connects to the first three-way valve 70 via port 74, where the other end of the conduit 84 is connectable to the donor reservoir 1200.

[0165] System 2000 can be used in the same cleaning and processing configurations as disclosed herein for system 1000, particularly in a manner similar to system 1000 starting with the respective cleaning configurations followed by the respective processing configurations, with appropriate modifications.

[0166] A feature of the system 2000 is that it allows for the continuous flow of mammalian body fluid from the donor reservoir 1200 to each device 100 within the set of devices 2500, thereby providing high quality processed mammalian body fluid, i.e., as the processed and increasingly depleted mammalian body fluid is sent from one device 100 to the next device 100 within the set of devices 2500, the levels of at least one fibrinolytic protein in the mammalian body fluid become successively more and more depleted.

[0167] On the other hand, multiple devices 100 connected in series may be able to set up backpressure in the system 2000, which may result in a lower flow rate of mammalian body fluids through the system 2000 compared to, for example, in the system 1000.

[0168] 19, another example of a system for depleting at least one fibrinolytic protein from one or more mammalian bodily fluids in accordance with the subject matter disclosed herein, generally designated by reference numeral 3000, includes a device suite 3500 including a plurality of devices 100. In the illustrated example of FIG. 19, the device suite 3500 includes four devices 100, although in alternative variations of this example, the device suite 3500 may instead include two, three or more than four devices 100, with appropriate modifications.

[0169] The system 3500 also includes a saline reservoir 1100, a donor reservoir 1200, a receiver plasma reservoir 1300 and a wash waste reservoir 1400, as well as a first three-way valve 70 and a second three-way valve 90, with appropriate modifications, as disclosed herein for the system 1000 shown in Figures 15 and 16. In this example, the apparatus 3500 includes a plurality of apparatus 100 interconnected to provide fluid communication between the respective control volumes V of the plurality of apparatus 100.

[0170] In at least this example, each device 100 (of the suite of devices 3500) is as disclosed in that example or alternative variations of those examples herein. In any case, the control volume V of each device 100 of the suite of devices 3500 contains a respective one of the plurality of particle populations including at least a first population of first particles and a second population of second particles.

[0171] In at least this example, the devices 100 of the complete set 3500 are interconnected in parallel, with the fluid inlet ports 330 of the devices 100 interconnected and in fluid communication with one another via an inlet manifold 3330. Additionally, the fluid outlet ports 340 of the devices 100 interconnected and in fluid communication with one another via an outlet manifold 3340. The inlet manifold 3330 of the complete set 3500 connects to the saline reservoir 1100 and the donor reservoir 1200 via a first three-way valve 70, similar to the device 100 of the system 1000 shown in Figures 15 and 16, with appropriate modifications. The outlet manifold 3340 of the complete set 3500 connects to the receiver plasma reservoir 1300 and the wash waste reservoir 1400 via a second three-way valve 90, similar to the device 100 of the system 1000 shown in Figures 15 and 16, with appropriate modifications.

[0172] In this example, the saline reservoir 1100 contains enough saline to wash and prime all of the devices 100 in the device suite 3500 .

[0173] It should be noted that the complete set 3500 of the device 100 as disclosed herein, together with the saline reservoir 1100, the receiving plasma reservoir 1300 and the wash waste reservoir 1400, the inlet manifold 3330 and the outlet manifold 3340 (and excluding the respective donor reservoirs 1200), may be provided as a kit 3800. The kit 3800 may further include a first three-way valve 70, and a conduit 82 connecting to the saline reservoir 1100 and connecting to the first three-way valve 70 via port 72, and a conduit 86 connecting to the first three-way valve 70 and connecting to the inlet manifold 3330. The kit 3800 may further include a second three-way valve 90, and conduits 62, 64 that connect to the receiving plasma reservoir 1300 and the wash waste reservoir 1400, respectively, and connect to the second three-way valve 90 via respective ports 92, 94, and a conduit 66 that connects to a third port 96 of the second three-way valve 90 and connects to an outlet manifold 3340. The kit 3800 may further include a conduit 84 that connects to the first three-way valve 70 via port 74, where the other end of the conduit 84 is connectable to the donor reservoir 1200. The system 3000 may be used in the same manner as the washing and processing configurations as disclosed herein for the system 1000, specifically starting from the respective washing configurations followed by the respective processing configurations, with appropriate modifications.

[0174] A feature of the system 3000 is that it can process relatively high volumetric flows and / or volumetric flow rates of mammalian bodily fluid from the donor reservoir 1200 by dividing the mammalian bodily fluid among multiple devices 100 in the set of devices 3500 and simultaneously processing the mammalian bodily fluid in the devices 100.

[0175] On the other hand, multiple devices may set up a backpressure in system 2000 that may result in a lower flow rate of mammalian bodily fluids through system 2000 compared to within system 1000, for example.

[0176] In alternative variations of the system 3000, the fluid inlet ports 330 of multiple devices 100 in the set of devices 3500 may be directly connected to and in fluid communication with a respective donor plasma reservoir, and / or each of the fluid outlet ports 340 may be directly connected to and selectively in fluid communication with a respective recipient plasma reservoir.

[0177] In another alternative variation of system 3000, each device 100 may be replaced with a set of serially connected devices 100 corresponding, for example, to set 2500, with appropriate modifications.

[0178] The proposed physiological cell-based model of hemostasis is initiated when activated factor VII (VIIa) binds to tissue factor-bearing cells, leading to further activation of factors IX and X, which in turn cleaves (activates) factor II (prothrombin) to form thrombin (IIa). Thrombin activates factor XI, which in turn activates other factors to generate more thrombin. Thrombin then further cleaves fibrinogen to form a preliminary fibrin clot, which is then stabilized by the cross-linking action of factor XIII to form a firm hemostatic clot.

[0179] The above-mentioned activation of the coagulation system in response to vascular trauma leads to the deposition of cross-linked fibrin in tissues and blood vessels, thus compromising blood flow. Therefore, an additional system is needed that can adequately melt the fibrin clot, thereby preventing the clot from growing further beyond physiological needs, and triggering clot lysis once the clot is no longer needed. This system involves fibrinolytic proteins, which are then activated and convert fibrin into its soluble degradation products through the action of the serine protease, plasmin. Under physiological conditions, fibrinolysis is achieved by a precisely measured combination of activators, inhibitors and cofactors. It is tightly regulated by engagement.

[0180] Plasminogen, the main component of the fibrinolytic system, is synthesized primarily in the liver. Its cleavage (activation) at a single Arg-Val peptide bond at positions 560-561 generates the active serine protease, plasmin, which then dissolves fibrin clots. Plasminogen cleavage is mediated by plasminogen activators.

[0181] The major endogenous plasminogen activator is tissue plasminogen activator (tPA). Functionally, t-PA itself is a weak activator of plasminogen. However, in the presence of fibrin, the catalytic efficiency of tPA-dependent plasminogen activation increases 500-fold. Excess plasmin activity can also cleave clotting factors, thereby preventing the formation of new clots.

[0182] The second endogenous plasminogen activator is single-chain u-PA or pro-urokinase. u-PA has a much lower affinity for fibrin than tPA. uPA is an effective plasminogen activator in the presence or absence of fibrin, but its plasminogen activating activity is greatly stimulated by fibrin. u-PA is expressed on several cells, including activated endothelial cells, macrophages, renal epithelial cells, and some tumor cells.

[0183] The fibrinolytic system is indeed balanced by the action of fibrinolytic protein activators (as detailed above) and inhibitors. The major inhibitor of plasmin is α2-antiplasmin - a single-chain glycoprotein that is synthesized primarily in the liver and circulates in relatively high concentrations (2 μM) in plasma. Plasmin released into the bloodstream or in the vicinity of a clot is immediately neutralized following the formation of an irreversible 1:1 stoichiometric complex with α2-antiplasmin.

[0184] Among the plasminogen activator inhibitors, plasminogen activator inhibitor-1 (PAI-1) is the most ubiquitous. It is released by endothelial cells, monocytes, macrophages, hepatocytes, adipocytes, and platelets. PAI-1 is the most important and rapidly acting physiological inhibitor of both tPA and u-PA.

[0185] Plasminogen activator inhibitor 2 (PAI 2) is synthesized by the human placenta. High levels of PAI 2 are found in human plasma, primarily during pregnancy.

[0186] Finally, thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma carboxypeptidase that acts as a potent fibrinolysis inhibitor with specificity for carboxy-terminal arginine and lysine residues.

[0187] Treatment of patients with various coagulation disorders is essential during spontaneous bleeding episodes, trauma, and throughout surgical procedures. In many such situations, blood / plasma-derived products (e.g., standard plasma or fresh frozen plasma-FFP) are used. These products contain coagulation factors and fibrinolytic proteins, and are therefore said to stop bleeding by inducing the formation of hemostatic clots and correct defective or impaired coagulation disorders. In general, the absence or abnormality of any coagulation factor may lead to a tendency to hemorrhage due to an insufficient ability to create a stable hemostatic clot. Without being bound by any theory, the inventors hypothesized that the presence of fibrinolytic proteins may be involved in the dissolution of hemostatic clots, leading to the melting of the clots and the worsening of the bleeding phenomenon.

[0188] Thus, after the formation of a hemostatic clot following replacement of the missing coagulation factor(s) with one or more available blood / plasma derived products, clot lysis is achieved by the fibrinolytic system, if necessary. However, there are cases where lysis of the clot is not desired, e.g. to maintain a hemostatic clot in bleeding situations, or to improve the functioning of the blood. If indeed the reverse activity is required for the generation of many clots, treatment with bodily fluids composed of clotting factors but depleted of fibrinolytic factors, specifically blood or plasma and any derived products, may be a desirable solution.

[0189] As noted herein, the subject matter disclosed herein provides conjugates, conjugates or any compositions thereof, as well as devices, kits and systems for depleting fibrinolytic proteins from bodily fluids, particularly blood, plasma and preparations thereof, and therefore also provides methods of using these preparations.

[0190] Thus, in yet another aspect, the subject matter disclosed herein relates to a method for depleting at least one fibrinolytic protein from one or more mammalian body fluids or any preparation thereof. More specifically, the method comprises in a first step (i) subjecting one or more body fluids or any preparation thereof to an affinity depletion procedure specific for at least one fibrinolytic protein. The next step (ii) involves recovering the body fluid depleted of at least one fibrinolytic protein obtained in step (i).

[0191] It should be noted that the affinity depletion procedure, as disclosed by the presently disclosed subject matter, comprises contacting the body fluid with an effective amount of the conjugates according to the presently disclosed subject matter or at least one composition comprising the conjugates. Alternatively, the affinity depletion procedure may be performed in the presently disclosed subject matter by applying the body fluid to a device, a set of devices, a kit or a system comprising the conjugate, any of the conjugates or any composition comprising the conjugates of the presently disclosed subject matter.

[0192] It should be noted that in some embodiments, each conjugate comprises at least one particle, at least one linker, and at least one amino acid, derivative or analog thereof. In some specific embodiments, the plurality of conjugates comprises at least two different conjugates, wherein the amino acid, derivative or analog thereof is at least one of 4-(aminomethyl)cyclohexanecarboxylic acid (tranexamic acid), ε-aminocaproic acid, lysine, cyclohexanecarboxylic acid, and 4-methylcyclohexanecarboxylic acid. In some further specific embodiments, the plurality of conjugates comprises at least two different conjugates, wherein the amino acid, derivative or analog thereof is at least one of 4-(aminomethyl)cyclohexanecarboxylic acid (tranexamic acid), ε-aminocaproic acid, and lysine.

[0193] It should be noted that in some embodiments, the conjugate particles useful in the methods of the presently disclosed subject matter may be as defined herein above by the presently disclosed subject matter. In still some further embodiments, the methods of the presently disclosed subject matter may use any of the devices, apparatus, kits or systems provided by the presently disclosed subject matter as defined herein above.

[0194] In some embodiments, the methods of the subject matter disclosed herein may be applicable to depleting fibrinolytic proteins from a bodily fluid, which may be at least one of whole blood, plasma, or a blood-derived product containing at least one coagulation factor.

[0195] In some specific embodiments, such blood-derived products may be at least one of whole blood, plasma, fresh frozen plasma (FFP), platelet-rich plasma (PRP), and cryoprecipitate.

[0196] It should be understood that in some embodiments, the methods of the subject matter disclosed herein may be performed ex vivo or in vitro, more specifically, in bodily fluids that are no longer part of the human body.

[0197] Blood transfusion remains a vital part of saving lives. In modern bank blood therapy, blood components are transfused rather than whole blood.

[0198] Blood component therapy refers to the separation of blood into its components, allowing only the specific desired components to be transfused to a patient, thus avoiding the use of unnecessary components. The use of blood components allows several patients to be treated with blood from a single donor.

[0199] The term "fresh frozen plasma" (FFP), as used herein, refers to the major blood component, the cell-free liquid fraction of human blood, that has been frozen and stored after donation and that is to be used for transfusion. After donation, a unit of human blood is centrifuged to separate the cellular content of the blood, and the remaining plasma is frozen at -18C (0F) or below within 8 hours of collection.

[0200] FFP contains all components (factors / proteins) of the coagulation, fibrinolysis and complement systems. There are clearly defined indications for its use in single or multiple coagulation deficiencies, and in existing or anticipated bleeding, as occurs in trauma or surgery.

[0201] "Cryoprecipitate", as used herein, relates to the precipitated proteins of plasma obtained from a unit of fresh plasma by rapid freezing (as is done for FFP) within 6-8 hours of collection and rapid thawing at 4°C. Cryoprecipitate is rich in factors VIII, XIII, von Willebrand factor and fibrinogen. This component is therefore suitable for the treatment or prevention of bleeding in inherited or acquired pathologies associated with a deficiency or disorder of the abovementioned coagulation proteins.

[0202] "Platelet-rich plasma" (PRP) blood component is prepared from a unit of fresh (donated) blood by centrifugation or an apheresis procedure.

[0203] In addition to being prepared from standard units of whole blood, blood components may also be obtained by apheresis procedures. Apheresis is performed using a pheresis machine, a semi-automated blood separation instrument. In this procedure, if plasma is planned to be used for the donation, the donor's anticoagulated whole blood is passed through the machine, which separates the blood into red blood cells, plasma, and white blood cell / platelet fractions, which are then returned to the subject. Only the separated plasma is not returned to the subject, but is used for further donations.

[0204] Several semi-automated blood cell separator instruments are available for collection of platelets, granulocytes, blood stem cells, mononuclear cells, and plasma. All of these instruments use centrifugation to separate blood components. Some apheresis procedures involve two venipunctures while a continuous flow of blood from the donor is passed through the blood cell separator; other procedures can be accomplished with a single venipuncture and intermittent blood withdrawal and return.

[0205] In still some further specific embodiments, the methods of the presently disclosed subject matter may be used to deplete fibrinolytic proteins, which may be at least one of plasminogen and tissue plasminogen activator (tPA).

[0206] More specifically, plasminogen (also known as PEG, enzyme entry EC: 3.4.21.7), as used herein, is a major component of the fibrinolytic system and is primarily synthesized in the liver. There are two major glycoforms of plasminogen in humans - two glycosylated forms. Type I plasminogen contains cosylated moieties (N-linked to N289 and O-linked to T346), whereas type II plasminogen contains only a single O-linked sugar (O-linked to T346). Type II plasminogen is preferentially recruited to cell surfaces compared to type I glycoforms. Conversely, type I plasminogen appears to be more readily recruited to clots. In the circulation, plasminogen adopts a closed, activation-resistant conformation. Upon binding to a clot or to a cell surface, plasminogen adopts an open form that can be converted to active plasmin by a variety of enzymes, including tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and factor XII (Hagemann factor). More specifically, cleavage (activation) of plasminogen at the single Arg-Val peptide bond at positions 560-561 generates the active serine protease, plasmin, which in turn dissolves the fibrin clot.

[0207] Full-length plasminogen contains seven domains. In addition to the C-terminal chymotrypsin-like serine protease domain, plasminogen contains five N-terminal Pan Apple domains (PAps). The Pan-Apple domain is a protein that binds to the Kringle domain (KR1-5) of the plasmid. It contains determinants important for maintaining the endothelin in a closed conformation, and the kringle domain is involved in binding to lysine residues present in receptors and substrates.

[0208] In some embodiments, the plasminogen referred to in the presently disclosed subject matter may be human plasminogen. In such embodiments, the plasminogen gene (GenBank: AY192161.1, mapped to chr6q26) is located at about 52.5 It spans 10 kb of DNA and contains 19 exons (OMIM number 173350). It should be noted that in some embodiments, plasminogen as used herein refers to human plasminogen comprising an amino acid sequence encoded by a nucleic acid sequence comprising a sequence as set forth by SEQ ID NO:1. In some further embodiments, human plasminogen may comprise an amino acid sequence encoded by a nucleic acid sequence comprising a sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to a sequence as set forth by SEQ ID NO:1. In yet some further embodiments, such human plasminogen molecule may comprise an amino acid sequence as set forth by SEQ ID NO:2. In yet some other embodiments, such human plasminogen molecules may comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to the sequence as set forth by SEQ ID NO:2.

[0209] "Plasminogen deficient body fluid" or "plasminogen free body fluid", as used herein, means that a formulation of the subject matter disclosed herein (which, according to some embodiments, is prepared by treating a body fluid, such as blood, plasma or a blood product, with a fibrinolytic protein binding agent, specifically a plasminogen binding agent) exhibits about 100% to 50% reduction, decrease, or attenuation in the amount of plasminogen when compared to untreated blood or blood products. More specifically, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the plasminogen normally present in the body fluid, specifically blood or blood products, is removed from the formulation of the subject matter disclosed herein, specifically when compared to untreated blood or blood products. In other words, the formulations of the subject matter disclosed herein may contain plasminogen in an amount that is about 0.01% to about 50% of the amount of plasminogen in other formulations or unprocessed blood or blood products, specifically, less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or ...5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or less than about 0.01%, 0.05%, 0.5%, 1%, 2%, 3%, 4 50%, 60%, 70% or less.

[0210] Plasminogen, when activated to form the active plasmin enzyme, exhibits proteolytic activity, specifically the cleavage or degradation of proteins into small polypeptides or amino acids. In this regard, the bodily fluids treated by the methods of the presently disclosed subject matter lack plasminogen or plasmin proteolytic activity. In some specific embodiments, the proteolytic activity of plasmin and plasminogen involves the cleavage of fibrin, thus dissolving the fibrin clot. The term "lacking plasmin and plasminogen activity" means that the body fluid treated by the methods of the subject matter disclosed herein is completely devoid of plasmin and plasminogen proteolytic activity, or at least, is devoid of any one of about 1% to 99.9% of the proteolytic activity of active plasmin or plasminogen when compared to the proteolytic activity of active plasmin or plasminogen in a body fluid such as blood, plasma or blood products, specifically untreated blood or blood products, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85%. It should be understood to mean exhibiting a "reduced," "diminished," "mitigated," "inhibited" or "attenuated" proteolytic activity by about 85%-90%, about 90%-95%, about 95%-99%, or about 99%-99.9% or 100%.

[0211] Still further, in some embodiments, the bodily fluids treated by the methods of the presently disclosed subject matter are devoid of tPA. The depletion of tPA using the methods, conjugates and devices of the presently disclosed subject matter is clearly demonstrated by FIG.

[0212] The term tPA, as used herein with respect to tissue plasminogen activator (also known as PLAT; enzyme entry EC 3.4.21.68.), should be understood to refer to a secreted serine protease that converts and activates the proenzyme plasminogen to the potent fibrinolytic enzyme plasmin. tPA is synthesized in vascular endothelial cells as a single polypeptide chain that undergoes proteolytic cleavage by plasmin or trypsin at a centrally located arginine-isoleucine bond to a two-chain disulfide-linked form consisting of an N-terminally derived heavy chain and a C-terminal light chain. The tPA gene (DNA accession number NT_167187.1, mapped to 8p11.21) contains 14 exons that code for a heavy chain domain that contains two kringle regions (K1 and K2) and a region similar to growth factors, and a light chain domain that contains the serine protease catalytic site. Alternative splicing of the tPA gene results in multiple transcript variants encoding different isoforms that are involved in multiple biological processes, such as cell migration and tissue remodeling, apart from fibrinolysis. Increased tPA activity leads to hyperfibrinolysis, which manifests as excessive bleeding; decreased tPA activity leads to hypofibrinolysis, which may lead to thrombosis or embolism. Phenotypes associated with tPA include familial hyperfibrinolysis (due to increased tPA release) and familial thrombophilia (due to decreased tPA release (OMIM number 612348)). It should be noted that in some embodiments, tPA as used herein refers to human tPA comprising an amino acid sequence encoded by a nucleic acid sequence comprising the sequence as set forth by SEQ ID NO:3. In some further embodiments, human tPA may comprise an amino acid sequence encoded by a nucleic acid sequence comprising a sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to a sequence as set forth in SEQ ID NO: 3. In still some further embodiments, such a human tPA molecule may comprise an amino acid sequence as set forth in SEQ ID NO: 4.In yet some other embodiments, the human tPA is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identical to the sequence as set forth by SEQ ID NO:4. , 99% or 100% homology thereto.

[0213] "tPA-deficient body fluid" or "tPA-free body fluid," as used herein, means that a body fluid treated by the methods of the presently disclosed subject matter (which, according to some embodiments, is prepared by treating a body fluid, such as blood, plasma, or a blood product, with a conjugate of the presently disclosed subject matter) exhibits about 100% to 50% of the amount of tPA normally present when compared to untreated blood or blood products. More specifically, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the tPA is removed from the body fluid, particularly when compared to untreated blood, plasma, or blood products. In other words, a body fluid treated by the methods of the presently disclosed subject matter may contain tPA in an amount that is about 0.01% to about 50% of the amount of tPA in other formulations or in untreated blood or blood products. Specifically, about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or even 60% or 70% of the amount of tPA compared to an untreated body fluid, such as blood, plasma or a blood product.

[0214] In still some further embodiments, the body fluids or specifically blood, plasma or blood-derived products prepared by the methods of the presently disclosed subject matter may be devoid of plasminogen and tPA, and therefore may be devoid of any fibrinolytic activity as specified above. In still some further embodiments, the body fluids or products prepared by the methods of the presently disclosed subject matter may also be devoid of any other fibrinolytic agents, such as urokinase (uPA). Still further, it should be understood that in some embodiments, additional antifibrinolytic agents may be added to the body fluid formulations prepared by the methods of the presently disclosed subject matter.

[0215] In some optional embodiments, the method may further comprise the step of measuring the amount of plasminogen in the fibrinolytic protein-depleted body fluid recovered in step (ii) by determining at least one of the clotting time and the total clot lysis time in said fibrinolytic protein-depleted body fluid.

[0216] In some embodiments, the methods of the presently disclosed subject matter may be used in the preparation of at least one blood and / or blood derived product having reduced fibrinolytic activity.

[0217] Fibrinolytic activity, as used herein, refers to the ability of some proteolytic enzymes in blood and blood-derived products to dissolve fibrin and clots. The main proteolytic enzyme that cleaves fibrin is plasmin. Plasmin is formed by the activation of plasminogen by tPA and / or uPA. When plasmin breaks down fibrin, fibrin degradation products (FDPs) are formed. FDPs compete with thrombin, thus slowing down clot formation by preventing the conversion of fibrinogen to fibrin. This effect can be seen in the thrombin clotting time (TCT) test, which is prolonged in people with active fibrinolysis. FDP, and the specific FDP, D-dimer, can be measured using antibody-antigen techniques. This is more specific than TCT and confirms that fibrinolysis is occurring. It is therefore used to obtain an indication of the effectiveness of treatment in deep vein thrombosis, pulmonary embolism, DIC and acute myocardial infarction. Alternatively, thromboelastometry (TEM) in whole blood allows for a more rapid detection of fibrinolytic activity, especially hyperfibrinolysis, even in heparinized patients. In this assay, increased fibrinolysis is assessed by comparing TEM profiles in the absence or presence of the fibrinolysis inhibitor aprotinin. Still further, a test of overall fibrinolysis can be measured by the euglobulin lysis time (ELT) assay. ELT is the extraction of the euglobulin fraction (mainly the important fibrinolytic factor FRET) from plasma. Fibrinolysis is measured by clotting blood with a number of different proteins (fibrinogen, PAI-1, tPA, α2-antiplasmin, and plasminogen) and then observing the time required for the clot to dissolve. A shorter lysis time indicates a hyperfibrinolytic state and risk of bleeding.

[0218] As noted above, bodily fluids, blood, plasma or blood products treated by the methods of the presently disclosed subject matter exhibit reduced or decreased fibrinolytic activity. The terms "reduced," "reduced," "mitigated," "suppressed," or "attenuated," as referred to herein, refer to a process, a decrease in fibrinolytic activity, or a decrease in fibrinolytic activity, as compared to a bodily fluid, such as blood, plasma or blood products, that contains tPA and / or plasminogen, as compared to blood or blood products that have not been treated with the conjugates of the presently disclosed subject matter, as compared to normal blood or blood products, or as compared to commercially available blood products. Specifically, it should be understood to refer to a delay, restriction, decrease or reduction of any one of about 1% to 99.9% of fibrinolytic activity, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85%, about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, or even 100%. In other words, these preparations exhibit no fibrinolytic activity or at most a minimally reduced fibrinolytic activity, specifically about 0.01% or less, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or less of fibrinolytic activity when compared to the fibrinolytic activity of unprocessed blood or blood products. In some embodiments, the body fluids, blood, plasma or blood products processed by the methods, conjugates, compositions, and devices, complete devices, kits or systems provided by the presently disclosed subject matter exhibiting reduced or no fibrinolytic activity as defined above may be used in any therapeutic application disclosed by the presently disclosed subject matter, as discussed later herein.

[0219] In yet some other specific embodiments, the methods of the subject matter disclosed herein may be used in vivo / ex vivo to deplete at least one fibrinolytic protein fluid in and / or in a subject in need thereof.

[0220] Thus, in another aspect thereof, the presently disclosed subject matter provides a method for extracorporeal depletion of at least one fibrinolytic protein from one or more body fluids of a subject in need thereof. More specifically, the method comprises the steps of: In a first step (i), transferring the subject's bodily fluid to an extracorporeal device, in some embodiments, a device, a set of devices, a kit or a system provided by the subject matter disclosed herein may be considered as such an extracorporeal device.

[0221] The second step (ii) involves subjecting the body fluid to an affinity depletion procedure specific for at least one fibrinolytic protein. It should be noted that such depletion may be carried out before, during or after the transfer of blood to and from said device. In this way, an extracorporeal body fluid of interest is obtained, which is depleted of at least one fibrinolytic protein.

[0222] The next step (iii) involves returning or reintroducing the bodily fluid obtained in step (ii), which has been depleted in at least one fibrinolytic protein, back into the subject.

[0223] It should be noted that the affinity depletion procedure involves contacting the subject's bodily fluid with an effective amount of a plurality of conjugates or any composition thereof, specifically ex vivo. Still further, the method can be performed in an extracorporeal device or in a tissue culture system as defined by the subject matter disclosed herein. , the conjugate of the subject matter disclosed herein or any composition thereof may be included in a device, a set, a kit, or a system connected to such an extracorporeal device. As mentioned above, each conjugate discussed herein may, in some embodiments, include at least one particle, at least one linker, and at least one amino acid, derivative, or analog thereof. In still some further embodiments, the plurality of conjugates includes at least two different conjugates. In some further embodiments, the amino acid, derivative, or analog thereof may be at least one of 4-(aminomethyl)cyclohexanecarboxylic acid (tranexamic acid), ε-aminocaproic acid, lysine, cyclohexanecarboxylic acid, and 4-methylcyclohexanecarboxylic acid. In still some further embodiments, the amino acid, derivative, or analog thereof is at least one of 4-(aminomethyl)cyclohexanecarboxylic acid (tranexamic acid), ε-aminocaproic acid, and lysine. In still some embodiments, the amino acid, derivative, or analog thereof may be tranexamic acid (TXA).

[0224] In some embodiments, the extracorporeal device is a cardiopulmonary bypass machine (CPB), and wherein the extracorporeal device is a plasmapheresis machine.

[0225] The term "extracorporeal" refers to a medical procedure that is performed outside the body. For example, such an extracorporeal procedure may refer to a circulation procedure, i.e., a procedure in which blood is removed from the patient's circulation, a treatment is applied to it, and then it is returned to the circulation. Any device that transports blood outside the body is called an extracorporeal circulation. Such circulation procedures include, for example, but are not limited to, apheresis, autologous blood transfusion, hemodialysis, hemofiltration, plasmapheresis, extracorporeal carbon dioxide removal, extracorporeal cardiopulmonary resuscitation, extracorporeal membrane oxygenation (ECMO), and cardiopulmonary bypass during open heart surgery.

[0226] Cardiopulmonary bypass (CPB) is a technique that temporarily takes over the heart and lung functions during surgery, maintaining blood circulation and oxygen content in the patient's body. The CPB pump itself is often referred to as the cardiopulmonary bypass or "pump". The cardiopulmonary bypass pump is operated by a perfusionist. CPB is a form of extracorporeal circulation. Extracorporeal membrane oxygenation is generally used for long-term treatment.

[0227] An apheresis machine is a device that takes blood that has been removed from a patient's or donor's body and separates it into its various components: plasma, platelets, white blood cells, and red blood cells.

[0228] It should be noted that in some embodiments, the conjugate particles useful in the methods of the presently disclosed subject matter may be as defined by the presently disclosed subject matter. In still some further embodiments, the methods of the presently disclosed subject matter may use an apparatus, a device, a kit, or a system as defined herein above.

[0229] In some embodiments, the methods of the subject matter disclosed herein may be applicable to depleting at least one fibrinolytic protein from a bodily fluid, which may be at least one of whole blood, plasma, or a blood-derived product containing at least one coagulation factor.

[0230] In some specific embodiments, such blood-derived products may be at least one of whole blood, plasma, fresh frozen plasma (FFP), platelet-rich plasma (PRP), and cryoprecipitate.

[0231] In still some further specific embodiments, the methods of the presently disclosed subject matter may be used to deplete fibrinolytic proteins, which may be at least one of plasminogen and tPA. In some specific embodiments, the methods of the presently disclosed subject matter may be used to deplete tPA. In still some further embodiments, the methods of the presently disclosed subject matter may be used to deplete fibrinolytic proteins, which may be at least one of plasminogen and tPA. The methods of the presently disclosed subject matter may be used to deplete plasminogen. Still further, in certain embodiments, the methods of the presently disclosed subject matter may be used to deplete plasminogen and tPA.

[0232] In still some further embodiments, the method may further comprise recovering at least one of plasminogen and tPA from the conjugate, conjugates or any composition thereof, or specifically TXA conjugate particles, of the subject matter disclosed herein. It should be understood that the depleted fibrinolytic protein removed from the body fluid by the method of the subject matter disclosed herein may be used for other purposes. In some specific embodiments, recovering plasminogen and / or tPA from the device or conjugate of the subject matter disclosed herein may be performed by applying an effective amount of the conjugate, composition thereof, or specifically TXA, its derivative or any analog thereof, to the composition, conjugate or specifically TXA conjugate particles, bound to the plasminogen and / or tPA, on the device. In still some further embodiments, the recovered plasminogen may be used to treat the subject when fibrinolysis is required.

[0233] Still further, it should be understood that the present disclosure further provides an extracorporeal device for use in a method of depleting at least one fibrinolytic protein from one or more body fluids of a subject in need thereof by an extracorporeal procedure. More specifically, the method comprises the following steps: in a first step (i), transferring the body fluid of said subject into. In some embodiments, a device, a complete device, a kit or a system provided by the subject matter disclosed herein may be considered as such an extracorporeal device. A second step (ii) comprises subjecting the body fluid to an affinity depletion procedure specific for at least one fibrinolytic protein. It should be noted that such depletion may be performed before, during or after the transfer of blood to and from said device. In this way, an extracorporeal body fluid of the subject is obtained. This extracorporeal body fluid is depleted of at least one fibrinolytic protein. A next step (iii) comprises returning or reintroducing the body fluid obtained in step (ii), depleted of at least one fibrinolytic protein, to the subject.

[0234] In yet another aspect, the subject matter disclosed herein provides a method for treating, preventing, prophylaxis, ameliorating, inhibiting bleeding, hemostatic disorders and any bleeding or pathological condition associated therewith in a subject in need thereof. More specifically, the method may comprise administering to the subject under treatment a therapeutically effective amount of at least one blood and / or blood-derived product having reduced fibrinolytic activity. In some embodiments, the formulation may be prepared by a method as described herein.

[0235] In some embodiments, the methods of the presently disclosed subject matter may be applicable to hemostatic disorders, which may be inherited or acquired bleeding disorders.

[0236] Hemostatic disorders are bleeding disorders classified as either inherited or acquired. Acquired bleeding disorders are disorders in which bleeding is caused by external factors (acquired causes), such as trauma, surgery or fibrinolytic therapy, as discussed herein below.

[0237] Bleeding disorders caused by genetic deficiencies of one or more clotting factors are rare disorders distributed worldwide. Homozygotes or compound heterozygotes for the mutated genes responsible for these deficiencies present with bleeding manifestations of varying severity, usually related to the degree of reduced activity of a particular clotting factor.

[0238] In yet another embodiment, the method of the presently disclosed subject matter relates to an inherited hemostatic disorder. The present invention is applicable to the treatment, prevention, amelioration, suppression or delay of associated bleeding and undetermined bleeding tendency.

[0239] "Inherited hemostatic disorders" as used herein refers to a genetic deficiency of at least one coagulation factor. More specifically, a number of mutations have been identified in the genes encoding coagulation factors I, II, V, VII, X and XI that lead to a deficiency or reduced activity of at least one of said factors. Homozygotes for these mutations exhibit bleeding tendency, either idiopathic or after trauma / surgery. Heterozygotes for the various deficiencies rarely exhibit bleeding tendency.

[0240] Undetermined bleeding tendency, as used herein, relates to a condition in which there is a tendency to bleed, although a precise diagnosis of the condition cannot be established.

[0241] Some patients referred for evaluation of minor bleeding symptoms have an undiagnosed bleeding tendency that may not have been recognized until a challenging event occurs that causes bleeding, such as surgery or delivery. Clinical variability is common among such individuals with regard to bleeding manifestations, suggesting that environmental and other genetic factors may modify bleeding risk. Although minor bleeding problems may not become apparent until they face a significant hemostatic challenge (surgery, tooth extraction, major trauma, menarche, or delivery), a predictive postoperative bleeding risk has not been established for such individuals. Gender influences bleeding manifestations. Women are more likely to be referred for evaluation due to bothersome bleeding associated with menstruation and / or delivery. In addition, bleeding that persists or becomes problematic for more than 24 hours after tooth extraction raises the possibility of a bleeding disorder. Inability to establish a diagnosis in patients with minor mucocutaneous bleeding is a common problem in practice.

[0242] The diagnosis of an undetermined bleeding tendency is evidenced by routine laboratory tests. The inability to establish a diagnosis can be problematic for patients who need to undergo surgery or give birth.

[0243] Fibrinolytic inhibitor therapy with ε-aminocaproic acid or tranexamic acid may be used for mild bleeding episodes in patients with undetermined bleeding disorders for dental and oral surgery, which may reduce bleeding associated with other surgical procedures. However, when severe bleeding occurs, for example during surgery or childbirth, blood or blood-derived components are required.

[0244] In more specific embodiments, the inherited hemostatic disorder can be a disorder resulting from at least one deficiency of at least one clotting factor and / or an undetermined bleeding tendency.

[0245] In still some further embodiments, the deficiency of at least one coagulation factor can be a deficiency of at least one of Factor XI, Factor X, Factor V, Factor VII, Factor II (prothrombin), and Factor I (fibrinogen). Thus, in some embodiments, the methods described by the presently disclosed subject matter can be applicable to any form of bleeding associated with an inherited hemostatic disorder caused by a deficiency of at least one of Factor XI, Factor X, Factor V, Factor VII, Factor II (prothrombin), and Factor I (fibrinogen) as disclosed herein.

[0246] In still some further embodiments, the methods of the subject matter disclosed herein may be applicable to the treatment of disorders characterized by genetic deficiencies of coagulation factors I, II, V, VII, X and XI, including at least one or any of the associated bleeding tendencies. Genetic deficiencies of coagulation factors I, II, V, VII, X and XI are autosomal recessive bleeding disorders that have been described in the majority of populations. Their relative frequency varies between populations, in part as a result of the high frequency of certain mutant genes in inbred populations. Several population studies have shown that deficiencies of factors XI and VII are common in these bleeding disorders, while less common disorders are characterized by deficiencies of factors V and X. and afibrinogenemia, and the rarest disorder is deficiency of factor II (prothrombin) and factor XIII. The severity of bleeding manifestations in affected patients who are homozygous or compound heterozygous for the mutated genes varies and is usually related to the degree of deficiency. Some patients have only light bruising or present with excessive bleeding only after trauma. Other patients, who usually have less than one percent of normal factor VII, XIII, or X activity, may present with intracranial hemorrhages and hemarthrosis similar to those of severe hemophilia patients.

[0247] In some specific embodiments, the methods of the subject matter disclosed herein may be applicable to the treatment, prevention, reduction, attenuation, or inhibition of bleeding associated with inherited factor XI deficiency, or any acquired bleeding or hemostatic condition in patients suffering from factor IX deficiency.

[0248] Hereditary factor XI deficiency is inherited as an autosomal recessive trait. The disorder manifests in homozygotes or compound heterozygotes as a mild to moderate bleeding tendency that is primarily injury-related. Although affected subjects have been described in many populations, the disorder is more prevalent in Jews, particularly Ashkenazi Jews.

[0249] Factor XI deficiency as a result of a dysfunctional protein is rare, and the majority of patients have reduced levels of factor XI protein. Over 150 mutations have been reported in non-Jewish and Jewish patients of various ancestries, most of which are missense mutations.

[0250] Many bleeding manifestations in homozygotes and compound heterozygotes are injury-related. Excessive bleeding may occur at the time of injury or begin hours or days after trauma. Bleeding tendency varies according to the hemostatic challenge and the various injury sites. Surgical procedures involving tissues with high fibrinolytic activity (urinary tract, tonsils, nose, alveolus) are frequently associated with excessive bleeding in patients with severe factor XI deficiency, regardless of genotype. Site-related bleeding tendency can now be understood in light of the documented function of factor XI in preventing clot dissolution. Factor XI deficiency is itself associated with increased fibrinolysis, and therefore the bleeding tendency may be increased even further by the additional bleeding risk of surgery at fibrinolysis-rich sites in these patients.

[0251] Current treatment of bleeding patients with factor XI deficiency is based on FFP. Patients with severe factor XI deficiency who must undergo surgical procedures must be carefully evaluated and meticulously prepared for surgery. In patients undergoing surgery at sites of high local fibrinolytic activity, the use of antifibrinolytic agents must be considered. Thus, in some embodiments, the tPA and / or plasminogen-deficient FFP provided by the subject matter disclosed herein may be particularly relevant for the treatment of patients with factor XI deficiency, more specifically, subjects suffering from any of the conditions discussed above.

[0252] In other embodiments, the methods of the subject matter disclosed herein may be applicable to the treatment, prevention, reduction, attenuation, inhibition of bleeding associated with inherited factor VII deficiency, or any acquired bleeding or hemostatic condition in patients suffering from factor VII deficiency.

[0253] Genetic deficiency of factor VII is a rare autosomal recessive disorder observed in many populations. Presumptive diagnosis is easy to make because factor VII deficiency is the only blood clotting disorder that results in a prolongation of the clotting time test, prothrombin time (PT). Most mutations causing factor VII deficiency are missense mutations.

[0254] Hemorrhagic manifestations occur in homozygotes and compound heterozygotes for factor VII deficiency. Patients with factor VII activity below 1 percent of normal frequently develop severe bleeding manifestations, including hemarthrosis leading to severe arthropathy and fatal intracerebral hemorrhage.

[0255] Patients with somewhat elevated levels of factor VII (factor VII activity 5 percent or greater than normal) have a much milder disease characterized by nosebleeds, bleeding gums, menorrhagia, and easy bruising. Some surgical procedures, such as tooth extraction, tonsillectomy, and procedures involving the genitourinary tract, are frequently associated with bleeding if pretreatment is not administered prior to the procedure. In contrast, surgical procedures such as laparotomy, herniorrhaphy, appendectomy, and hysterectomy are performed without incident. This apparent difference can be explained by the different degrees of local fibrinolysis exhibited by the respective traumatic tissues.

[0256] Replacement therapy with FFP is essential for patients with severe bleeding, such as hemarthrosis or intracerebral hemorrhage. Tooth extraction, tonsillectomy, nasal surgery, and urological interventions are likely to be accompanied by bleeding due to local fibrinolysis, and the surgical site must be considered when surgery is required. Thus, in some embodiments, the tPA and plasminogen deficient FFP provided by the subject matter disclosed herein may be particularly relevant for treating patients with factor VII deficiency, particularly any of the conditions discussed above.

[0257] In yet another embodiment, the methods of the subject matter disclosed herein may be applicable to the treatment, prevention, reduction, attenuation, inhibition of bleeding associated with inherited factor X deficiency, or any acquired bleeding or hemostatic condition in patients suffering from factor X deficiency.

[0258] Hereditary factor X deficiency, a moderate to severe bleeding tendency, is an autosomal recessive disorder. The 95 mutations causing factor X deficiency currently described include large deletions, small frameshift deletions, nonsense mutations, and missense mutations. Clinical signs of factor X deficiency are related to the functional level of factor X. Individuals with severe factor X deficiency and functional factor X levels less than 1 percent of normal bleeding levels bleed spontaneously and after trauma. Bleeding occurs primarily in joints and soft tissues, however bleeding from mucous membranes, such as menorrhagia, can be a particular problem in women. More unusual bleeding is intracerebral bleeding, intramural bleeding in the intestine (which can produce symptoms like acute abdomen), urinary tract bleeding, and soft tissue bleeding with the development of hemorrhagic pseudocysts or pseudotumors. In individuals with mild factor X deficiency, bleeding is less common and usually occurs only after trauma or during or after surgery. Patients with factor X deficiency are treated with fresh frozen plasma. Thus, in some embodiments, tPA and / or plasminogen-deficient FFP (or any other blood product) provided by the subject matter disclosed herein may be of particular relevance to the treatment of patients with factor X deficiency, particularly those suffering from any of the conditions discussed above.

[0259] In still some other embodiments, the subject matter disclosed herein may be applicable to the treatment, prevention, reduction, attenuation, and inhibition of bleeding associated with inherited factor V deficiency, or any acquired bleeding or hemostatic condition in patients suffering from factor V deficiency.

[0260] Hereditary factor V deficiency is one of the less common inherited bleeding disorders and manifests as a moderate bleeding tendency in homozygotes or compound heterozygotes. Factor V deficiency is inherited as an autosomal recessive trait. Heterozygotes with plasma factor V activity in the range of 25 to 60 percent of normal are usually asymptomatic. Factor V protein assays indicate that many homozygotes and compound heterozygotes have a true deficiency rather than a dysfunctional protein. Overall, more than 80 separate mutations have been identified, one-quarter of which are missense. Homozygous or compound heterozygous patients with factor V levels in the range of less than 1 to 10 percent of normal exhibit a lifelong tendency to bleed. Common symptoms include ecchymosis, nosebleeds, bleeding gums, bleeding after minor lacerations, and menorrhagia. Postpartum bleeding occurs in more than 50 percent of pregnancies in patients with severe factor V deficiency. Bleeding from other sites is uncommon. Trauma, tooth extraction, and surgery are associated with bleeding. , resulting in a high risk of excessive bleeding. If severe spontaneous bleeding occurs or surgery is performed, fresh frozen plasma replacement must be performed. When planning plasma replacement therapy, it is important to consider surgical procedures in areas with high local fibrinolytic activity, such as the genitourinary tract, oral cavity, and nose, as surgery in these areas will result in excessive bleeding and postpartum bleeding is common. Thus, in some embodiments, the tPA and plasminogen depleting formulations provided by the subject matter disclosed herein may be particularly relevant for treating patients with factor V deficiency, specifically any of the conditions discussed above.

[0261] In certain embodiments, the methods of the subject matter disclosed herein may be particularly applicable to the treatment, prevention, reduction, attenuation, inhibition of bleeding associated with inherited factor II deficiency, or any acquired bleeding or hemostatic condition in patients suffering from factor II deficiency.

[0262] Hereditary factor II (prothrombin) deficiency is one of the rarest clotting factor deficiencies. It presents in two forms: type I (hypoprothrombinemia), which is a true deficiency, and type II (dysprothrombinemia), which is the production of dysfunctional prothrombin. These autosomal recessive disorders are genetically heterogeneous and are characterized by a mild to moderate bleeding tendency.

[0263] Prothrombin defects are inherited in an autosomal recessive manner. Among individuals with type I deficiency, heterozygotes exhibit prothrombin levels that are approximately 50 percent of normal, while homozygotes typically exhibit levels that are less than 10 percent of normal. More than 50 mutations causing prothrombin deficiency have been identified, most of which are missense mutations.

[0264] Hereditary types I and II deficiency are characterized by mild to moderate mucocutaneous and soft tissue bleeding that usually correlates with the degree of deficiency of functional prothrombin. If prothrombin levels are about 1 percent of normal, bleeding may occur spontaneously or after trauma. Surgery bleeding may be severe. Menorrhagia, nosebleeds, bleeding gums, easy bruising, and subcutaneous hematomas may occur.

[0265] Replacement therapy for patients with congenital prothrombin deficiency consists of administration of FFP. Thus, in some embodiments, the tPA and plasminogen deficient FFP provided by the presently disclosed subject matter may be of particular relevance to the treatment of patients with factor II deficiency, particularly any of the conditions discussed above.

[0266] In still some other embodiments, the subject matter disclosed herein may be applicable to the treatment, prevention, reduction, attenuation, inhibition of bleeding associated with hereditary fibrinogen deficiency or any acquired bleeding or hemostatic condition in patients suffering from hereditary fibrinogen deficiency.

[0267] "Fibrinogen (Factor I) deficiency" as used herein relates to inherited fibrinogen abnormalities, including afibrinogenemia (total absence of fibrinogen), dysfibrinogenemia and hypodysfibrinogenemia. Inherited fibrinogen disorders are rare and can be subdivided into type I and type II disorders. Type I disorders (afibrinogenemia and hypofibrinogenemia) affect the amount of fibrinogen in circulation. Type II disorders (dysfibrinogenemia and hypodysfibrinogenemia) affect the quality of circulating fibrinogen. Afibrinogenemia, the most severe form of fibrinogen deficiency, is characterized by autosomal recessive inheritance and a total absence of fibrinogen in plasma.

[0268] Dysfibrinogenemia is the presence of functionally abnormal plasma fibrinogen at normal levels. Fibrinogenemia is defined by the presence of a deficiency in the fibrinogen chain. Hypodysfibrinogenemia is defined by low levels of dysfunctional protein. These are heterogeneous disorders caused by many different mutations in the three fibrinogen-encoding genes. Dysfibrinogenemia and hypodysfibrinogenemia are autosomal dominant disorders. Most susceptible patients are heterozygous for a missense mutation in the coding region of one of these three fibrinogen genes. Because the secreted fibrinogen hexamer contains two copies of each of these three fibrinogen chains and the resulting fibrin web contains multiple copies of this molecule, heterozygosity for one mutant allele is sufficient to impair the structure and function of the fibrin clot.

[0269] Bleeding due to afibrinogenemia usually presents in the neonatal period, with umbilical cord bleeding in 85 percent of cases, but later age of onset is not uncommon. Bleeding may occur in the skin, gastrointestinal tract, genitourinary tract, or central nervous system, with intracranial bleeding being the main cause of death. Patients with afibrinogenemia have an interesting susceptibility to idiopathic splenic rupture. Menstruating women may experience dysfunctional uterine bleeding. In addition, first trimester miscarriage is common in afibrinogenemic women. These patients may also have antepartum and postpartum bleeding. Intraperitoneal bleeding after rupture of the corpus luteum has also been observed.

[0270] Replacement therapy with commercially available preparations containing fibrinogen is the only treatment option for patients with congenital fibrinogen deficiency. Thus, in some embodiments, the methods of the subject matter disclosed herein may be particularly relevant for treating patients with fibrinogen deficiency, particularly any of the conditions discussed above. In particular, in some embodiments, fibrinogen is supplemented in fibrinolytic protein-depleted body fluids prepared by the conjugates and methods of the subject matter disclosed herein.

[0271] In contrast to commercially available preparations of blood-derived products used to treat inherited coagulation factor deficiencies, fibrinolytic protein-depleted body fluids prepared by the conjugates, compositions and methods of the subject matter disclosed herein have a substantial advantage since, in addition to providing the missing factors (procoagulant qualities), the removal of t-PA and plasminogen from the product provides antifibrinolytic qualities that are essential in preventing further clot lysis in cases of hemorrhage.

[0272] In some embodiments, the method of the subject matter disclosed herein may be applicable to the treatment of acquired hemostatic disorders. The acquired hemostatic disorders may be at least one of bleeding caused by surgery, bleeding caused by trauma, acute gastrointestinal bleeding, bleeding associated with burns, hemorrhagic stroke, lung injury associated with emphysema and chronic obstructive pulmonary disease (COPD), bleeding associated with childbirth, disseminated intravascular coagulation (DIC), and bleeding caused by fibrinolytic or thrombolytic therapy. In some specific embodiments, the method of the subject matter disclosed herein may be applicable to the treatment, prevention, reduction, attenuation, and inhibition of bleeding associated with surgical procedures, particularly minor or major surgical procedures.

[0273] Surgical procedures are a major challenge to the hemostatic system, especially when surgery is performed in locations (e.g., tissues, organs) that are rich in fibrinolytic proteins. Even patients with no or mild to moderate bleeding disorders may bleed excessively after surgery. In addition to the degree of surgical trauma, the magnitude of fibrinolytic activity at the surgical site must be considered.

[0274] It should be understood that if the surgical procedure is elective, scheduled, or non-urgent (e.g., Caesarean section, or any other major surgery that allows sufficient time for pre-operative preparation), the formulations of the subject matter disclosed herein may be used in pre-operative treatment to help prevent or reduce excessive bleeding during surgical intervention. Thus, in some embodiments, the subject matter disclosed herein is used in patients with genetic disorders, patients suffering from hyperfibrinolysis, and / or patients undergoing surgery. This may provide a particularly useful prophylactic method for patients scheduled for surgery.

[0275] In some further specific embodiments, the methods of the presently disclosed subject matter are suitable for treating bleeding caused by trauma (traumatic hemorrhage).

[0276] Traumatic hemorrhage can be caused by any type of injury, such as any injury caused by industrial accidents and automobile accidents, combat, or falls. There are various types of traumatic wounds that can cause hemorrhage. Generally, trauma causes damage to blood vessels, so that blood flows outwardly to the outside of the body, or inwardly to vital organs such as the brain, lungs, liver, kidneys, spleen, or vital cavities such as the chest and abdomen.

[0277] In addition to physical measures to stop the bleeding, blood and blood-derived components are usually administered to induce blood clotting, which will ultimately result in the cessation of bleeding.

[0278] The tPA and / or plasminogen deficient blood and blood derived products of the subject matter disclosed herein offer advantages over commercially available blood derived products as they provide additional antifibrinolytic qualities, thereby preventing the dissolution of formed blood clots, which may be essential for rapid cessation of bleeding.

[0279] In some specific embodiments, fibrinolytic protein-depleted body fluids prepared by the conjugates and methods of the presently disclosed subject matter may be suitable for the treatment of acute or chronic gastrointestinal bleeding.

[0280] "Gastrointestinal (GI) bleeding", also known as gastrointestinal bleeding, as used herein, relates to any form of bleeding in the digestive tract from the mouth to the rectum. "Acute GI bleeding" means there is a large amount of blood loss in a short period of time, leading to acute blood loss and hemorrhagic shock. Symptoms may include vomiting (hemathemesis), either red or black blood (due to digested blood, also known as "coffee grounds"), bloody stools, or melena (digested blood, called melena). In contrast, chronic GI bleeding is the bleeding of small amounts of blood over a long period of time. In this case, the symptom is iron deficiency anemia. GI bleeding is typically divided into two main types: upper GI bleeding and lower GI bleeding. Causes of upper GI bleeding include peptic ulcer disease, esophageal varices (which in some embodiments may result from cirrhosis and cancer), among others. Causes of lower GI bleeding include hemorrhoids, cancer, and inflammatory bowel disease, among others. Endoscopy of the upper and lower GI tract may identify the site of bleeding. If not clear, medical imaging may be useful.

[0281] Acute upper GI bleeding is more common than lower GI bleeding. Upper GI bleeding occurs in 50-150 adults per 100,000 each year. Lower GI bleeding is estimated to occur in 20-30 adults per 100,000 each year, resulting in approximately 300,000 hospitalizations per year in the United States. Mortality risk from GI bleeding ranges from 5% to 30%. Bleeding risk is more common in men and increases with age.

[0282] The most common source of upper GI bleeding is peptic ulcer disease. Esophagitis and erosive disease are the next most common causes. In patients with cirrhosis, 50-60% of bleeding is due to esophageal varices. Nearly half of patients with peptic ulcer have Helicobacter pylori infection. Other causes include gastric or duodenal ulcers, Mallory-Weiss tears, cancer, and angiodysplasia. Several medications are known to cause upper GI bleeding: NSAIDs, COX-2 inhibitors, SSRIs, corticosteroids, and anticoagulants.

[0283] Lower gastrointestinal bleeding is typically from the colon, rectum, or anus. Causes seen include hemorrhoids, cancer, angiodysplasia, ulcerative colitis, Crohn's disease, and aortoenteric fistula.

[0284] The initial focus of treatment for acute gastrointestinal bleeding is on resuscitation, beginning with airway management and fluid resuscitation with intravenous fluids and blood.

[0285] Colonoscopy is useful in the diagnosis and treatment of lower GI bleeding. A number of techniques may be used, including clipping, cauterization, and sclerotherapy. Surgery is rarely used to treat upper GI bleeding, but is still commonly used to manage lower GI bleeding by removing the part of the intestine that is causing the problem. Angiographic embolization can be used for both upper and lower GI bleeding.

[0286] Still further, plasminogen and / or tPA depleted blood products prepared by the methods of the presently disclosed subject matter using the conjugates and devices of the presently disclosed subject matter may be used in the treatment of hemorrhagic stroke.

[0287] "Hemorrhagic stroke" as used herein refers to bleeding occurring directly into the brain parenchyma. The usual mechanism is believed to be leakage from small intracerebral arteries damaged by chronic hypertension. Patients with intracerebral hemorrhage are more likely to have headaches, altered mental status, seizures, nausea and vomiting, and / or significantly elevated blood pressure compared to patients with ischemic stroke. None of these findings, however, reliably distinguishes hemorrhagic stroke from ischemic stroke. Specific symptoms may result from focal neurological impairment. This type of impairment depends on the site of brain lesion. In the case of lesion in the dominant hemisphere (usually the left hemisphere), a syndrome consisting of the following may result: right hemiparesis, right hemisensation, left gaze preference, right visual field defect, and aphasia. In the case of lesion in the non-dominant hemisphere (usually the right hemisphere), a syndrome consisting of the following may result: left hemiparesis, left hemisensation, right gaze preference, and left visual field defect.

[0288] Brain imaging is a critical step in determining a suspected hemorrhagic stroke and must be obtained in an emergency setting. Brain imaging helps diagnose hemorrhage and may identify complications such as intraventricular hemorrhage, cerebral edema, or hydrocephalus. Either non-contrast computed tomography (NCCT) scanning or magnetic resonance imaging (MRI) are the modalities of choice.

[0289] When blood product therapy is indicated for hemorrhagic stroke, blood, FFP and platelets obtained from a blood bank are used. Prothromboyic products of fibrinolytic protein-depleted body fluids prepared by the conjugates and methods of the subject matter disclosed herein. Considering the quality and fibrinolytic quality, this appears to be a more suitable bleeding cessation than conventional blood products in patients suffering from hemorrhagic stroke.

[0290] In some specific embodiments, the methods of the subject matter disclosed herein may be suitable for treating lung injury associated with emphysema and COPD. In more specific embodiments, the methods of the subject matter disclosed herein may include administering to a subject under treatment a therapeutically effective amount of fibrinolytic protein-depleted body fluid prepared by the conjugates and methods of the subject matter disclosed herein, which, due to its high concentration of alpha 1-antitrypsin, is more suitable for use in treating subjects with emphysema and COPD. In these diseases, leukocyte proteases destroy the elasticity of the lungs, resulting in lung fuller, which in turn leads to emphysema and chronic lung disease. Thus, α1-antitrypsin in fibrinolytic protein-depleted body fluids prepared by the conjugates and methods of the presently disclosed subject matter can inhibit the activity of leukocyte proteases, thereby restoring lung tissue elasticity.

[0291] Emphysema is a form of chronic (long-term) lung disease. People with emphysema can only exhale air in a limited way. Emphysema can occur in many different ways, but the most common is smoking.

[0292] Emphysema is one of the main types of chronic obstructive pulmonary disease (COPD). It is called "obstructive" because people with emphysema breathe out as if something is blocking the airflow. The other form of COPD is chronic bronchitis, which can also be caused by smoking.

[0293] Emphysema occurs when the delicate lining of the air sacs in the lungs is irreparably damaged. Most commonly, toxins in cigarette smoke cause the damage. Emphysematous lung changes develop slowly over many years as the fragile tissue between the air sacs is destroyed and air pockets develop in the lungs. Air becomes trapped in those spaces in the damaged lung tissue. The lungs slowly expand, making breathing more difficult.

[0294] This problem with emphysema is called airflow limitation. During a pulmonary function test, people with emphysema take much longer to empty their lungs than people without emphysema.

[0295] In addition to smoking, another known major cause of emphysema is α1-antitrypsin deficiency. However, this is a less important cause than smoking. α1-antitrypsin is a natural protein that circulates in human blood. Its main function is to prevent white blood cells from damaging normal tissue. Thus, treatment of subjects with emphysema with fibrinolytic protein-depleted body fluids enriched in α1-antitrypsin and prepared by the subject conjugates and methods disclosed herein may inhibit the activity of proteases, thereby restoring the elasticity of lung tissue.

[0296] Chronic obstructive pulmonary disease (COPD) is a type of obstructive lung disease characterized by long-term reduced airflow. Primary symptoms include shortness of breath and coughing with phlegm production. COPD typically worsens over time. Eventually, patients have difficulty climbing stairs or carrying objects. Chronic bronchitis and emphysema are older terms used for different types of COPD. The term "chronic bronchitis" is still used to define a productive cough that is present for at least 3 months each year for 2 years.

[0297] COPD is a type of obstructive pulmonary disease in which there is chronic, incompletely reversible reduced airflow (airflow limitation) and inability to fully exhale (air trapping). The reduced airflow is the result of lung tissue destruction (known as emphysema) and small airway disease (known as bronchiolitis obliterans). The relative contribution of these two factors varies from person to person. Severe destruction of the small airways can lead to the formation of large air pockets known as bullae that replace lung tissue. This form of the disease is called bullous emphysema.

[0298] Smoking is the most common cause of COPD, with several other factors, such as air pollution and genetics, playing smaller roles. In the developing world, one of the common sources of air pollution is heating and cooking fires with inadequate ventilation. Prolonged exposure to these irritants induces an inflammatory response in the lungs, leading to narrowing of the small airways and destruction of lung tissue. Diagnosis is based on reduced airflow as measured by pulmonary function tests. In contrast to asthma, reduced airflow does not improve significantly with the use of bronchodilators.

[0299] The most common symptoms of COPD are sputum production, shortness of breath, and productive cough. These symptoms are present for a long time and typically worsen over time. It is unclear whether there are different types of COPD. Although previously separated into emphysema and chronic bronchitis, emphysema describes the lung changes rather than the disease itself, while chronic bronchitis simply refers to the It describes symptoms that may or may not be associated with COPD.

[0300] Advanced COPD leads to high pulmonary artery pressure, which distorts the right ventricle and causes right heart failure. This condition is called cor pulmonale and leads to symptoms of leg swelling and jugular vein distention. COPD is more common as a cause of cor pulmonale than any other lung disease. Since the use of supplemental oxygen, cor pulmonale has become less common.

[0301] Consequently, fibrinolytic protein-depleted body fluids prepared by the conjugates and methods of the subject matter disclosed herein may be beneficial as a complementary treatment for COPD, as its concentrated alpha-1 antiplasmin inhibits leukocyte proteases, thereby restoring the elastic qualities of damaged lung tissue.

[0302] Still further, the plasminogen and / or tPA depleted blood products prepared by the methods of the presently disclosed subject matter using the conjugates and devices of the presently disclosed subject matter may be used to treat burns and any associated bleeding. The term "burn" as used herein refers to tissue injury involving damage to the skin and possibly the tissues underneath the skin. Burns can be caused by contact with heat, flame, chemicals, electricity, or radiation.

[0303] Burns are mainly caused by accidents and can be classified as high temperature burns, electrical burns, chemical burns, and radiation burns depending on the cause. The severity of burns is classified as first, second, third, and fourth degree burns depending on the width and depth of the burn, the time of contact with the temperature of the object causing the burn, and the condition of the skin. Burns of second degree or higher may leave scars and require treatment in a hospital.

[0304] A first-degree burn causes redness of the skin and pain accompanied by itching. It causes damage to the epidermis, the outermost layer of the skin, and swelling accompanied by pain and redness. The symptoms disappear after a few days, but scaling and pigmentation may remain. No scarring (burn marks) remains after recovery. Sunburn is the most common example of a first-degree burn.

[0305] Second-degree burns affect the epidermis and dermis and cause redness, pain, swelling, and blisters 24 hours after the incident. Second-degree burns may affect sweat glands or pores. Severe burning and extreme pain occur. Blisters burst, leaving eroded areas and releasing copious secretions. Special care must be taken if the burn area covers more than about 15 percent of the body surface area. Second-degree burns heal in a few weeks, but often leave behind pigmentation or depigmentation. If a secondary infection occurs, some symptoms become more severe and healing takes longer.

[0306] Third-degree burns affect the epidermis, dermis, and even subcutaneous fat, causing darkening or lightening of the skin and coagulation of blood vessels just below the skin surface. The burned area may become numb, but the patient feels extreme pain, there is death of skin tissue and structures, and healing takes a long time and leaves a scar. Two weeks after the incident, the eschar falls off and an ulcerated surface appears. A large amount of fluid is secreted and bleeding is likely, but gradually new tissue is formed, leading to regeneration of the epidermis and the third-degree burn heals, leaving a scar behind. If deep skin necrosis occurs or secondary infection occurs, healing is delayed and an uneven scar surface may develop, resulting in keloid formation or deformation, or impaired mobility. Special care is required if the burn area is more than 10 percent of the body surface area.

[0307] Fourth-degree burns involve charred and blackened tissue in the burn area, spreading through the skin layer and damaging the fat layer, ligaments, fascia, muscles, and even bone tissue. Fourth-degree burns mainly include high-voltage electrical burns, and in some cases, deep dermal second- and third-degree burns may occur when viral infection occurs. If burns are more than 20 percent in extent, a systemic reaction may occur; hypotension, shock, acute kidney failure due to excessive fluid loss, and later wound infection or pneumonia, sepsis, and multiple organ dysfunction syndrome.

[0308] At temperatures above 44°C (111°F), proteins begin to lose their three-dimensional shape and begin to degrade. This results in cell and tissue damage. Many of the direct health effects of burns are secondary to the disruption of the skin's normal functions. This includes the disruption of skin's sensation, ability to prevent water loss through evaporation, and ability to regulate body temperature. Disruption of cell membranes results in the loss of potassium from the cell into the extracellular space, and the uptake of water and sodium.

[0309] In extensive burns (greater than 30% of the total body surface area), there is a significant inflammatory response. This leads to increased fluid leakage from capillaries and subsequent tissue edema. This causes a global blood volume loss, and the remaining blood undergoes significant plasma loss, causing it to become further concentrated. Renal failure and gastric ulcers can result from reduced blood flow to organs such as the kidneys and digestive tract.

[0310] In still some further embodiments, the bleeding caused by surgery can be bleeding caused by minor surgery or major surgery. Major surgery is defined as any surgical procedure involving anesthesia or respiratory support. In the case of bleeding during major surgery, treatment includes replacement of missing or non-functional clotting factors with commercially available FP, FFP or cryoprecipitate.

[0311] As detailed herein above, in contrast to major surgery, which refers to any surgical procedure involving anesthesia or respiratory assistance, minor surgery is a medical procedure involving incision with an instrument, performed to repair damage or stop disease in a living body. Because minor surgery involves incision or resection, which is the act of piercing or opening any part of the human body with a sharp blade, in subjects prone to bleeding, the procedure can cause significant bleeding.

[0312] It should be noted that in some embodiments, the method of the subject matter disclosed herein may be applicable to minor surgery, which may include any dental treatment or procedure. The term "dental treatment" refers to any treatment aimed at preventing and / or curing diseases, disorders and conditions of the soft and hard tissues of the jaw (mandible), oral cavity, maxillofacial region and adjacent and associated structures of the human body. Such treatments obviously relate to dentistry, orthodontics, periodontology, oral medicine and oral surgery, but may also relate to other fields of dentistry and medical practice that may be generally involved in oral health.

[0313] Still further, the major surgery may be open heart surgery or a liver transplant.

[0314] Major surgery is defined as any surgical procedure involving anesthesia or respiratory support. In the case of bleeding during major surgery, treatment includes replacement of missing or non-functional coagulation factors with commercially available FP, FFP, or cryoprecipitate. However, this replacement may not be sufficient because the presence of tPA and plasminogen in the above preparations may mediate fibrinolytic activity at the site of surgical injury, thereby promoting the dissolution of clots formed to stop or prevent bleeding. Thus, administration of the t-PA and plasminogen deficient preparations of the presently disclosed subject matter, which exhibit significantly reduced fibrinolytic activity, is a better and more efficient treatment option compared to commercially available preparations, because in addition to the supplementation of coagulation factors by the presently disclosed subject matter preparations, their antifibrinolytic activity (as demonstrated by the examples) may reduce or prevent bleeding contributed by clot lysis caused by fibrinolytic proteins present in the commercially available preparations.

[0315] In particular embodiments, the methods of the presently disclosed subject matter are applicable to open heart surgery. Some surgical procedures are predicted to cause severe bleeding, such as open heart surgery. In such procedures, extracorporeal circulation (cardiopulmonary bypass - CPB) is used.

[0316] Cardiovascular surgery (open heart surgery) is surgery on the heart or great vessels performed by a cardiac surgeon. Frequently it is performed to treat complications of ischemic heart disease (e.g., coronary artery bypass graft surgery), to correct congenital heart disease, or to treat valvular heart disease from a variety of causes, including endocarditis, rheumatic heart disease, and atherosclerosis. It also includes heart transplants.

[0317] During open heart surgery, the heart is temporarily stopped. Patients undergoing open heart surgery are placed on cardiopulmonary bypass, meaning that a machine pumps their blood and oxygen for them. The machine never performs the same functions as a normal heart and lungs, and therefore, as with many surgical procedures, time on the machine is kept to a minimum. This artificial method provides a bypass, temporarily eliminating the patient's need for cardiopulmonary function.

[0318] The bleeding phenomena occurring during these operations are due to the anticoagulants used during surgery, which intentionally create a clotting deficiency, and in addition, platelet dysfunction caused by blood passing through the extracorporeal circuit also contributes to the bleeding tendency.

[0319] It should be understood that the methods of the presently disclosed subject matter may be particularly applicable to subjects undergoing open heart surgery with CPB. The formulations, compositions and methods of the presently disclosed subject matter may confer a dual beneficial effect on such patients, as follows:

[0320] 1. During open heart surgery, blood will flow through tubing attached to a cardiopulmonary bypass (CPB) machine while the tubing is coated with tranexamic acid to deplete t-PA and / or plasminogen from the blood (as detailed in the experimental procedures). A pump will send blood from the CPB machine to the patient. During the flow through this tubing, blood depleted of t-PA and plasminogen is returned to the patient's circulation.

[0321] The blood returned in this way has poor fibrinolytic activity and a high concentration of antifibrinolytic activity, thus providing protection against bleeding tendencies.

[0322] 2. If bleeding occurs due to the above reasons, then the patient may be treated to stop the bleeding with fibrinolytic protein-depleted body fluid prepared by the conjugates and methods of the subject matter disclosed herein, which is expected to be more effective in stopping bleeding due to its antifibrinolytic qualities.

[0323] In further embodiments, the methods of the presently disclosed subject matter are suitable for practice in the treatment of bleeding associated with liver transplantation.

[0324] The liver plays a central role in hemostasis and thrombosis. Hepatocytes are the site of synthesis of many clotting factors, physiological coagulation inhibitors, and essential components of the fibrinolytic system. The liver also regulates hemostasis and fibrinolysis by clearing activated clotting factors and enzyme inhibitor complexes from the circulation. Thus, when liver dysfunction occurs in patients with liver disease, a complex hemostatic disturbance follows, which can lead to bleeding.

[0325] During the first phase of liver transplantation, removal of the diseased liver (extrahepatic phase), significant hemostatic changes can occur. As activated clotting factors are not removed from the circulation, their consumption can increase along with platelet consumption and secondary hyperfibrinolysis. In addition, primary hyperfibrinolysis also occurs as a result of incomplete tPA clearance. The most significant hemostatic changes during liver transplantation occur after reperfusion of the donor liver. Platelet incorporation into the graft leads to increased thrombocytopenia and endothelial cell apoptosis. Induction of fibrinolysis causes injury to the graft. Release of tissue factor and tPA from the reperfused graft further induces fibrinolysis. Thus, hyperfibrinolysis is considered to be a major contributor to hemostatic disorders during the extrahepatic and reperfusion phases. Furthermore, the graft releases heparin-like substances that may inhibit coagulation. In addition, other factors such as hypothermia, metabolic acidosis, and hemodilution adversely affect hemostasis during this phase. Liver transplantation is a lengthy procedure with extensive wound surfaces, including potential collateral venous access. With improved surgical techniques and anesthesiological care, blood loss during liver transplantation has been significantly reduced. In the event of uncontrolled bleeding, packed red blood cells, platelets, and fresh frozen plasma can be transfused. The use of synthetic antifibrinolytic agents such as tranexamic acid (a lysine analogue) and aprotinin (a serine protease inhibitor) is a common approach.

[0326] It should therefore be appreciated that the formulations of the subject matter disclosed herein, the conjugates, compositions, and methods described by the subject matter disclosed herein, by virtue of their antifibrinolytic qualities, may be particularly applicable to the cessation of bleeding associated with the hyperfibrinolytic state caused by liver transplantation.

[0327] It should be understood that the subject methods disclosed herein may be applicable to any surgery involving any organ or tissue transplant, e.g., liver, kidney, lung, heart, pancreas, skin, blood vessels, etc.

[0328] In still some further embodiments, the methods of the presently disclosed subject matter may be applicable to acquired hemostatic disorders, which may be bleeding resulting from fibrinolytic or thrombolytic therapy.

[0329] Fibrinolytic / thrombolytic therapy is administered primarily in patients with acute myocardial infarction (acute coronary thrombosis) or acute stroke (acute cerebral artery thrombosis). The goal of fibrinolytic / thrombolytic therapy is rapid restoration of blood flow in occluded vessels, achieved by accelerating fibrinolytic proteolysis of the clot. Fibrinolytic therapy typically results in a fibrinolytic state, as plasminogen activation is not restricted to the clot. These effects are multiple and include reduced fibrinogen levels, increased fibrinogen degradation products, and reduced clotting factors. A complication of fibrinolytic therapy is bleeding. Hemorrhagic complications are more frequent with fibrinolytic therapy than with anticoagulant therapy, and require prompt diagnosis and management. Two problems contribute to excessive bleeding. First, the fibrinolytic effect is not restricted to the site of thrombosis, but is usually systemic. Thus, any hemostatic clots required to prevent bleeding at the site of vascular trauma, either caused by catheters required for treatment or in lesions in the brain, gastrointestinal tract, or elsewhere, are also susceptible to lysis. The most severe complication is intracranial bleeding, which occurs in about 1% of patients and is associated with high mortality and severe disability in survivors. The most common bleeding complications are related to invasive vascular procedures, such as the placement of arterial and intravenous catheters. Any bleeding at these sites is frequent and should not be a reason to discontinue therapy if it can be managed with local compression or other simple measures. This problem can be minimized by limiting venous and arterial punctures and early introduction of local measures. Heavy bleeding can also result from pre-existing lesions, such as gastrointestinal ulcers or genitourinary lesions.

[0330] Treatment of bleeding complications following fibrinolysis / thrombolysis involves targeted treatment of the local site, and correction of the systemic hypocoagulable state involves replacement therapy to correct the hemostatic abnormalities caused by systemic plasminemia. Fibrinogen replacement is often required, which can be achieved by administration of cryoprecipitate, and fresh frozen plasma can be used to replace other hemostatic proteins.

[0331] It should be noted that fibrinolytic / thrombolytic therapy involves the use of anticoagulants or anticoagulants. As used herein, the term "anticoagulant" is intended to mean any agent that prevents blood from clotting. Some anticoagulants, such as the coumarin derivative bishydroxycoumarin (dicumarol) and warfarin (Coumadin), inhibit the synthesis of the clot-forming substance prothrombin and other clotting factors. Anticoagulants can include, but are not limited to, beta2 adrenergic receptor antagonists, neuropeptide V2 antagonists, prostacyclin analogs, thromboxane synthase inhibitors, calcium agonists, coumarin derivatives, elastase inhibitors, nonsteroidal anti-inflammatory drugs, thrombin inhibitors, lipoxygenase inhibitors, factor Vila inhibitors, factor Xa inhibitors, phosphodiesterase III inhibitors, heparin, and compounds that act as fibrinogen glycoprotein IIb / IIIa antagonists.

[0332] Coumarins are vitamin K antagonists. A prominent member of this class is warfarin (Coumadin). These anticoagulants are used to treat patients with deep vein thrombosis (DVT), pulmonary embolism (PE), and to prevent embolism in patients with atrial fibrillation (AF) and mechanical prosthetic heart valves. Other examples are acenocoumarol, phenprocoumon, atromentin, and phenindione.

[0333] Heparin is a biological substance usually made from pig intestine. It works by activating antithrombin III, which prevents blood from clotting with thrombin. A more highly processed preparation, low molecular weight heparin, is useful because it does not require monitoring of the APTT clotting parameter, as with enoxaparin (Clexane), and it has fewer side effects.

[0334] Fondaparinux is a synthetic sugar composed of five sugars (pentasaccharides) in heparin that bind to antithrombin and is an inhibitor of factor Xa. It is a smaller molecule than low molecular weight heparin. Another example is idraparinux sodium, which has a similar chemical structure and method of action to fondaparinux.

[0335] Drugs such as rivaroxaban, apixaban and edoxaban work by directly inhibiting factor Xa (unlike heparin and fondaparinux, which work through antithrombin activation).

[0336] Further examples include, but are not limited to, betrixaban from Portola Pharmaceuticals, darexaban (YM150) from Astellas, and more recently, retaxaban (TAK-442) and retaxaban (TAK-442) from Takeda. and Pfizer's elivaxaban (PD0348292).

[0337] Another type of anticoagulant is the direct thrombin inhibitors. Current members of this class include, but are not limited to, the bivalent drugs hirudin, lepirudin, and bivalirudin; and the monovalent drugs argatroban and dabigatran.

[0338] The antithrombin protein itself is used as an anticoagulant for protein therapy, which can be purified from human plasma or recombinantly produced (e.g., Atryn, which is a genetically modified (It is produced in the milk of goats that have been fermented.)

[0339] As pointed out above, anticoagulant administration, e.g., heparin, is the standard antithrombotic therapy indicated for acute venous thrombosis, thrombosis prophylaxis in postoperative (especially orthopedic) and bedridden patients, and flushing of venous lines to maintain patency. However, due to their efficacy, heparin and LMWH suffer from drawbacks. Uncontrolled bleeding as a result of simple exercise stress and associated contact with physical objects or at the surgical site is a major complication. In addition, approximately 5% (range up to 30%) of patients treated with heparin and approximately 2% of patients receiving unfractionated heparin (UFH) develop immune-mediated thrombocytopenia (HIT). This can be complicated by either bleeding (as a result of reduced platelet counts) or arterial and venous thrombosis due to intravascular platelet aggregation. The subject formulations and methods disclosed herein can prevent such undesirable effects of these anticoagulants.

[0340] More specifically, disseminated intravascular coagulation (DIG) is a pathological process characterized by widespread activation of the coagulation cascade resulting in clot formation in microvessels throughout the body. This can lead to impaired tissue blood flow and ultimately to multi-organ damage. In addition, the consumption of clotting factors and platelets during the clotting process can prevent normal blood clotting and cause severe bleeding from various sites.

[0341] In still some further embodiments, the subject matter disclosed herein provides methods applicable to the treatment, prevention, prophylaxis, amelioration, and inhibition of any bleeding associated with delivery or pregnancy, such as postpartum hemorrhage (PPH). Postpartum bleeding or postpartum hemorrhage (PPH) is often defined as the loss of more than 500 ml or 1,000 ml of blood within the first 24 hours after delivery. Signs and symptoms may initially include an increased heart rate, lightheadedness when standing, and increased respiratory rate. The condition may occur up to six weeks after delivery. The most common causes are decreased uterine contractions after delivery, not delivering all of the placenta, uterine lacerations, or poor blood clotting.

[0342] The causes of postpartum hemorrhage are commonly referred to as the "four T's": uterine atony, trauma, retained placenta, and coagulopathy: Tone: Uterine atony is the inability of the uterus to contract and can lead to continued bleeding. Retained placental tissue and infection can contribute to uterine atony. Uterine atony is the most common cause of postpartum hemorrhage. Trauma: Injury to the birth canal, including the uterus, cervix, vagina and perineum, which can occur even if the birth is properly monitored. All of these organs become highly vascularized during pregnancy, so bleeding can be substantial. Tissue: Retained tissue from the placenta or fetus can lead to bleeding. Thrombin: Bleeding disorders occur when there is a clotting defect, such as in those with diseases known as coagulopathy.

[0343] It should be understood that in some embodiments, the tPA and / or plasminogen free formulations of the subject matter disclosed herein and any methods using same may be applicable to the treatment and prevention of PPH as discussed above.

[0344] In still some further embodiments, the methods of the subject matter disclosed herein may also be applicable to the treatment of GPS. Goodpasture's syndrome (GPS) is a rare autoimmune disease in which antibodies attack the basement membrane of the lungs and kidneys, leading to pulmonary bleeding and renal failure. Depletion of fibrinolytic proteins such as tPA and / or plasminogen from bodily fluids, such as blood products, typically used to treat such patients, using the conjugates and methods of the subject matter disclosed herein may improve treatment.

[0345] In still some further embodiments, the methods of the presently disclosed subject matter may be applicable to the treatment of bleeding caused by blood vessel rupture.

[0346] In some specific embodiments, intra-articular injection of plasma or platelet-rich plasma is used to treat patients with knee osteoarthritis (OA). PRP injection into the knee has been shown to result in significant clinical improvement (Meheux CJ et al.). On the other hand, expression of urokinase-type plasminogen activator (PA), which degrades various extracellular matrix components such as collagen and aggrecan core protein, appears to be particularly important in the development of OA (Pap G et al. l.) Expression of stromelysin and urokinase-type plasminogen activator proteins in resection specimens and biopsies at different stages of knee osteoarthritis (Pap G. et al.). Thus, Injection of fibrinolytic protein-depleted fluids prepared by the conjugates and methods of the presently disclosed subject matter may prevent adverse effects and improve outcomes.

[0347] Still further, surgical procedures may be expected to cause severe bleeding in patients with inherited hemostatic disorders. Such patients may bleed excessively during or after surgery. It should be understood that for patients with inherited hemostatic disorders, the degree of surgical trauma as well as the degree of fibrinolytic activity at the surgical site must be considered. Thus, surgical procedures at sites rich in fibrinolytic activity, such as the oral cavity, nasopharyngeal cavity, and genitourinary system, especially the prostatic bed, may ultimately result in excessive bleeding in patients with inherited hemostatic disorders. Thus, in some embodiments, the method of the subject matter disclosed herein may be specifically applicable to such bleeding as well.

[0348] As mentioned above, the methods of the presently disclosed subject matter involve the administration or reintroduction of a body fluid or a preparation thereof having reduced or no fibrinolytic activity obtained by the methods discussed herein above, using a conjugate of the presently disclosed subject matter or any composition, device, device assembly, kit or system disclosed by the presently disclosed subject matter.

[0349] In some detailed embodiments, administration may be performed using an extracorporeal device. In yet some further embodiments, the fibrinolytic activity-free formulation may be administered to a subject using any device, apparatus, kit or system as discussed herein.

[0350] It should be understood that in some embodiments, formulations with reduced fibrinolytic activity as discussed above may be prepared from bodily fluids obtained from an allogeneic subject. In yet some further embodiments, particularly in the case of elective procedures (e.g., planned surgery), the subject may be treated with an autologous source of bodily fluid formulation.

[0351] "Autologous" blood donation, as used herein, is the concept that a recipient may donate blood for their own use if they are able to anticipate the need for blood and create a donation plan. Most commonly, this situation occurs during elective surgery. Autologous blood for transfusion can be obtained through preoperative donation. The term "allogeneic blood," as used herein, refers to blood collected from an unrelated donor of the same species. More specifically, in some embodiments, when the blood or blood products of the subject matter disclosed herein are obtained from at least one or more human subjects, allogeneic source means that the resulting product can be used in one or more other human individuals.

[0352] As noted above, the subject matter disclosed herein provides methods for treating bleeding, hemostatic disorders and any conditions related thereto. As used herein, "diseases," "disorders," "conditions," and the like, as they relate to the health of a subject, are used interchangeably and have the meanings ascribed to each and every such term.

[0353] The terms "associated" and "related to," used interchangeably herein when referring to a pathological condition, are understood to mean diseases, disorders, conditions, or any pathological condition that share a causal relationship, coexist with greater than chance frequency, or where at least one disease, disorder, condition, or pathological condition causes a second disease, disorder, condition, or pathological condition.

[0354] As mentioned above, the subject matter disclosed herein provides a method for treating a disorder as specified above. The term "treatment" as used herein means to ameliorate undesirable symptoms associated with a disease, to prevent the onset of such symptoms before they occur, to slow the progression of a disease, or to prevent the onset of a disease. "treatment" refers to administering a therapeutic amount of a composition of the subject matter disclosed herein that is effective to slow the progression of the hemostatic condition, slow the worsening of symptoms, promote the onset of remission, slow irreversible damage caused by the chronic phase of disease progression, delay the onset of said progressive phase, reduce the severity or cure the disease, improve survival or faster recovery, or prevent the disease from occurring, or a combination of two or more of the above. Treatment may be administered when the hemostatic condition first develops, or may be administered continuously, for example, by administering more than once a day, every day to every seven days, every seven days to every 15 days, every 15 days to every 30 days, every month to every two months, every two months to every six months, or even more frequently to achieve the therapeutic effects listed above.

[0355] The term "prevention" refers to preventing or reducing the risk of occurrence of a biological or medical event, specifically the risk of occurrence or recurrence of a disorder associated with bleeding that a researcher, veterinarian, physician or other clinician seeks to prevent in a tissue, system, animal or human, and the term "prophylactically effective amount" is intended to mean an amount of a pharmaceutical composition that will achieve this goal. Thus, in particular embodiments, the subject methods disclosed herein are particularly effective in preventing, i.e., preventing, pathologies associated with bleeding disorders. Thus, subjects to whom the composition is administered are less likely to develop symptoms associated with said bleeding disorders, and are also less likely to develop recurrences in subjects who have already experienced such symptoms in the past.

[0356] The term "improvement," as referred to herein, refers to the reduction in symptoms and improvement in the condition of a subject brought about by the compositions and methods of the presently disclosed subject matter, where said improvement may be manifested in the inhibition of, or a significant reduction in the magnitude of, a pathological process associated with a bleeding disorder as described herein, or an improvement in the physiological condition of an affected subject.

[0357] The term "inhibit" and all variations of this term are intended to encompass the limiting or prevention of the progression and exacerbation of a pathological condition or the progression of a pathological process, including the associated pathological condition or process.

[0358] The term "eliminate" optionally relates to the substantial eradication or elimination of pathological symptoms and possibly pathological causes according to the methods of the presently disclosed subject matter described below.

[0359] The terms "delay," "delaying the onset," "delay" and all variations thereof are intended to encompass slowing the progression and / or worsening of disorders associated with protein misfolding or protein aggregation, particularly bleeding disorders and symptoms thereof, slowing the progression, further worsening or development thereof, such that they appear later than in the absence of treatment according to the subject matter disclosed herein.

[0360] As stated above, treatment or prevention includes preventing or postponing the onset of the disease, preventing or postponing the onset of symptoms and / or reducing the severity of such symptoms that will or are expected to develop. It further includes ameliorating existing symptoms, preventing further symptoms, and ameliorating or preventing the underlying metabolic causes of the symptoms. The terms "inhibit," "ameliorate," "reduce," or "attenuate," as referred to herein, should be understood to refer to any one of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85%, about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, of a process, specifically, a bleeding disorder.

[0361] Single or multiple administrations on a daily, weekly or monthly schedule may be administered, with dose levels and pattern being selected by the treating physician. More specific embodiments typically involve use of 2-3 doses per week.

[0362] The subject matter disclosed herein relates to the treatment of subjects or patients in need thereof. By "patient" or "subject in need" is meant any organism that may be infected with the above-mentioned pathogens, including humans, domestic and non-domestic mammals, such as canine and feline subjects, bovine, simian, equine and murine subjects, rodents, poultry, aquaculture fish and exotic ornamental fish, for which the protective and preventative formulations, kit(s) and methods described herein are desired. It should be understood that the subject of treatment may also be any reptile or zoo animal.

[0363] By "mammalian subject" is meant any mammal for which the proposed therapy is desired, including human, equine, canine, and feline subjects, and more particularly humans. Specifically for non-human subjects, it should be noted that the subject methods disclosed herein may be carried out using injection (intravenous (IV), intra-arterial (IA), intramuscular (IM) or subcutaneous (SC)), drinking water, feed, spray, administration by oral irrigation, and direct administration to the digestive tract of a subject in need thereof.

[0364] It should be understood that the subject matter disclosed herein, in its further aspects, provides any body fluid preparation that exhibits reduced fibrinolytic activity and is prepared by any of the methods of the subject matter disclosed herein. Thus, the subject matter disclosed herein, in some embodiments, encompasses any mammalian body fluid preparation that lacks or has a reduced amount of at least one fibrinolytic protein, specifically at least one of tPA and / or plasminogen. In some embodiments, the preparation produced by the method of the subject matter disclosed herein lacks tPA. In yet some further embodiments, the preparation produced by the method of the subject matter disclosed herein lacks tPA and plasminogen. As noted above, such preparation produced by the method of the subject matter disclosed herein may be any body fluid, specifically mammalian blood, plasma or any blood product, that has been subjected to any of the procedures, conjugates, compositions, devices and systems described herein.

[0365] In yet another aspect, the subject matter disclosed herein provides a therapeutically effective amount of at least one blood and / or blood derived product having reduced fibrinolytic activity for use in a method for treating, preventing, prophylaxis, ameliorating, inhibiting bleeding, hemostatic disorders and any bleeding or pathological condition associated therewith in a subject in need thereof. It should be understood that such a bodily fluid preparation is prepared by a method of the subject matter disclosed herein. In still some further embodiments, such a bodily fluid preparation may be prepared by any of the conjugates of the subject matter disclosed herein, any composition thereof, or any device, set, kit, or system comprising a plurality of conjugates of the subject matter disclosed herein as defined herein.

[0366] In still some further embodiments, the blood and / or blood derived products used by the presently disclosed subject matter may be applicable to the treatment of any hemostatic disorder, particularly an inherited or acquired bleeding disorder, as defined by the presently disclosed subject matter as disclosed herein above.

[0367] Any of the conjugates described by the presently disclosed subject matter, any compositions thereof, and any devices, apparatus, kits, or systems described by the presently disclosed subject matter in the context of other aspects of the presently disclosed subject matter may be used in combination with any of the conjugates described by the presently disclosed subject matter in accordance with this aspect. It should be understood that the subject matter disclosed herein further encompasses any of the blood and / or blood derived products disclosed herein for use in accordance with any of the methods of treatment or any other method described herein for any of the conditions or disorders disclosed herein above.

[0368] All technical and scientific terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are intended to facilitate understanding of certain terms used frequently herein and are not intended to limit the scope of the present disclosure.

[0369] All definitions, and as used herein, must be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0370] The term "about" as used herein refers to values ​​that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% above and below the stated value, including integer values ​​and, where applicable, including non-integer values ​​as well, forming a continuous range. As used herein, the term "about" refers to ±10%.

[0371] The indefinite articles "a" and "an" are used herein in the specification and claims. When used herein, the term "a," "an," and "the" should be understood to mean "at least one" unless expressly dictated otherwise. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0372] The term "and / or," as used herein in the specification and claims, should be understood to mean "either or both" of the elements so conjoined, i.e., present in some cases as a coordinate conjunction and in other cases as a disjunction. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. Optionally, other elements may be present other than the elements specifically identified by the "and / or" clause, whether or not related to those elements specifically identified. Thus, as a non-limiting example, the phrase "A and / or B," when used in conjunction with open-ended language such as "comprising," in one embodiment refers to only A (optionally including elements other than B); in another embodiment refers to only B (optionally including elements other than A); in yet another embodiment refers to both A and B (optionally including other elements), and so forth.

[0373] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one of the number or listed elements, but also including more than one, and optionally including additional unlisted items. Only terms that clearly indicate otherwise, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," shall refer to the inclusion of exactly one of the number or listed elements. In general, the term "or" as used herein refers to exclusive alternatives (i.e., "either or, but not both") when preceded by a term of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, is a term used in patent law. "convert" has its ordinary meaning as used herein.

[0374] As used herein in the specification and claims, the phrase "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that elements other than those specifically identified in the list of elements to which the phrase "at least one" refers may optionally be present, whether or not related to the elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one, optionally including more than one, A, and no B (and optionally including elements other than B); in another embodiment to at least one, optionally including more than one, B, and no A (and optionally including elements other than A); in yet another embodiment to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements), and so forth.

[0375] It should also be understood that, unless expressly indicated otherwise, in any method claimed herein that includes two or more steps or actions, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are described.

[0376] Throughout this specification, as well as in the examples and claims which follow, unless the context otherwise requires, the terms "comprising," "including," "carrying," "having," "containing," and "involving" are used. "involving", "holding", "composed of" All transitional phrases, such as "," should be understood to be open-ended, i.e., to mean including, but not limited to, the following: As set forth in the US Patent Office Manual of Patent Examining Procedures, Thus, only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively. More specifically, the terms "comprises," "comprising," "includes," "including," "having" and variations thereof mean "including, but not limited to." The term "consisting of" means "including and not limited to." The term "consisting essentially of" means It means that a composition, method, or structure may include additional ingredients, steps, and / or moieties, provided that the additional ingredients, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0377] It should be noted that various embodiments of the subject matter disclosed herein may be provided in a range format. It should be understood that the description in range format is merely for convenience and brevity, and should not be construed as an inflexible limitation on the scope of the subject matter disclosed herein. Thus, a description of a range should be considered to have all the possible subranges specifically disclosed, as well as individual numbers within that range. For example, a description of a range such as 1-6 includes all the specifically disclosed subranges, such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 8 , 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is intended to include any recited numbers (fractional or integer) that are within the indicated range. The phrases "ranging between" a first and a second indicated number and "ranging from" a first indicated number to a second indicated number are used interchangeably herein and are intended to include the first and second indicated numbers and all fractional and integer numbers therebetween.

[0378] As used herein, the term "method" refers to manners, means, techniques and procedures for accomplishing a given task, including but not limited to manners, means, techniques and procedures that are either known to those of skill in the chemical, pharmacological, biological, biochemical and medical arts or that can be readily developed from known manners, means, techniques and procedures by such skilled artisans.

[0379] It is understood that certain features of the subject matter disclosed herein that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the subject matter disclosed herein that are described in the context of a single embodiment for brevity may also be provided individually or in any suitable subcombination or as suitable in any other described embodiment of the subject matter disclosed herein. Certain features described in the context of various embodiments should not be construed as essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0380] Various embodiments and aspects of the presently disclosed subject matter as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0381] It is to be understood that the subject matter disclosed herein, as disclosed and described, is not limited to the specific examples, method steps, and compositions disclosed herein, and thus the method steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing specific embodiments only, and is not intended to be limiting, since the scope of the subject matter disclosed herein will be limited only by the appended claims and equivalents thereof.

[0382] The following examples are representative of techniques employed by the inventors in carrying out aspects of the subject matter disclosed herein. While these techniques illustrate preferred embodiments for practicing the subject matter disclosed herein, it should be understood that one of ordinary skill in the art, in light of this disclosure, will recognize that numerous variations may be made without departing from the spirit and intended scope of the subject matter disclosed herein. EXAMPLES

[0383] Working Example Experimental procedure reagent: Sepharose 4B200 (Sigma Aldrich) Human plasma - healthy donor plasma was obtained from MDA Blood and / or human cryoprecipitate - cryoprecipitate was obtained from Hadassah Blood Bank.

[0384] method Bead Recovery (Method #1) 1. The beads are washed with 100 mM lysine pH=9.0 and 1M lysine for 2 min at 300 x g at RT. Washed three times with NaCl. 2. The beads were washed three times with 3M NaCl at 300×g for 2 minutes at room temperature (RT). 3. The beads were washed three times (x3) with H2O at 300 x g for 2 min at RT. 4. The beads were washed three times with 0.5 M NaOH at 300 x g for 2 min at RT. In the third wash, the beads are incubated with NaOH for 30 min. 5. The beads were washed 3 times with H2O for 2 min at 300 x g at RT. 6. The beads were washed 3 times with 0.5 M sodium citrate at 300 x g for 2 min at RT. 7. The beads were washed 3 times with a- 10 mM sodium citrate PH=6.8, b- 120 mM NaCl, c- 120 mM glycine at 300xg for 2 min at RT. 8. The beads were washed 3 times with H2O at 300xg for 2 min at RT. 9. The beads were washed 3 times with 20% ethanol + 1M NaCl. * After the third wash, 20% ethanol + 1M NaCl was added to give a 70% slurry, resuspended by gentle inversion, and stored at 4°C.

[0385] Bead Recovery with Peristaltic Pump (Method #2) 1- The filter was connected to a peristaltic pump. 2- 150 ml of each solution was run through the filter for 15 minutes: a- 100mM Lysine pH=9.0 and 1M NaCl b- 3M NaCl c- H2O d- 0.5M NaOH (incubate the filters with NaOH for 30 minutes) e-H2O f- 0.5M sodium citrate g- 10mM sodium citrate PH=6.8 120mM NaCl 120mM glycine h- H2O i- 20% ethanol + 1M NaCl All buffers were sterilized by autoclaving, except for NaOH.

[0386] PLG detection PLG detection in Example 7 was carried out using the kit: ab196262 PLG human simple step ELISA KIT.

[0387] [Table 1]

[0388] Reagent preparation: All reagents are brought to room temperature before use. Preparation of reagents used (for 36 well plates): 1x Wash Buffer PT (36 mL): 3.6 mL of 10x Wash Buffer PT + 32.4 mL of deionized water Antibody cocktail (1.8 mL): 180 µL of 10x capture antibody + 180 µL of 10x detection antibody + 1440 µL of antibody diluent CPI

[0389] Sample preparation: Plasma was serially diluted to 10 in sample diluent NS. 5 Diluted by: 1:100 plasma = 10 μL plasma + 990 μl sample diluent NS 1:10 5 Plasma = 1 μL plasma + 999 μl sample diluent NS

[0390] Standard preparation: 1- Stock standard solution (120 ng / mL): Reconstitute the PEG protein standard by adding 200 μL of water with a pipette. Keep at room temperature for 10 minutes and mix gently. 2- Label eight 1.5 mL tubes with standards 1 to 8. To tube 1, 225 μL of Sample Diluent NS was added. To tubes 2-8, 150 μL of sample diluent NS was added. 75 μL of stock standard solution was added to tube 1. 150 μL of standard was added to the next tubes (tubes 2-7). Standard #8 contains no protein and is the blank control.

[0391] Assay procedure: Remove the excess microplate strips from the plate frame and place them back into the foil bag with the desiccant pack, reseal and return to 4°C storage. 1-50 μL of all samples or standards were added to the appropriate wells. 2- 50 μL of antibody cocktail was added to each well. 3- The plate was sealed and incubated for 1 hour at room temperature on a plate shaker set at 400 rpm. 4- Each well was washed 3x with 350 μL 1x Wash Buffer PT (wash by aspirating or decanting from the wells, then dispense 350 μL 1x Wash Buffer PT into each well) 5-100 μL of TMB substrate was added to each well and incubated in the dark for 10 minutes on a plate shaker set at 400 rpm. 6- 100 μL of stop solution was added to each well. The plate was mixed by shaking on a plate shaker for 1 minute. 7- OD absorbance was read at 450 nm.

[0392] Thromboelastography (TEG) TEG Device TEG 5000 (60) (Haemonetics, Braintree, MA) (valid until October 31, 2019) Disposable cups and pins [HAE-07-052]

[0393] TEG Reagents: 1. WT-tPA 1mg / ml (Acytilyse 50mg) [Acytilyse 50mg, Boehringer Ingelheim; 1 vial of 2,333mg powder contains 50mg of active alteplase (WT-tPA)] 2. Calcium Chloride 0.2M [Haemonetics, Catalog No. 7003 Lot No.: 150597BA ] 3.Fresh frozen human citrate plasma 4. FVII Valin (1mg / ml stock) DPD-V304-037 Bulk B 5. Level I Control [Haemonetics, Catalog No. 8001, Lot No.: HMO3199 Expiration Date: October 2018] 6. Level II Control [Haemonetics, Catalog No. 8002, Lot No.: HMO3178 Expiration Date: October 2018] 7. PBS x 10, Biological Industries Lot Number: 1626505 8. TEG Hemostasis system dilution water Lot number: 0110-1404

[0394] The assay was performed according to the protocol "Clot lysis monitored by thromboelastography (TEG)". Prior to use of TEG, calibration was performed using level I and level II controls. The WT-tPA reagent was PBS x 1, 5 μl Wt-tPA (16.66 μM) + 595 μl Diluted 1:90 in PBS x 1 = working concentration 0.185 μM (final concentration in sample is 1.85 nM).

[0395] External bleeding model (tail amputation in mice) Animals and conditions: Species / strain: Mouse, c57 black Sex / number / age: Male, 24, 8 weeks old Supplier: Harlan Laboratories, Israel Body weight: Body weight was 20-25 g at the start of the study. The minimum and maximum body weights of the group were within ±10% of the group mean body weight. Acclimatization period: 7 days. Identification information: permanent marker (study duration 24 hours or less) and cage tag. Animal handling is performed in accordance with the National Institutes of Health (NIH) and the Association for Assessment and Accreditation of Laboratory Animal Care. All animals were kept in accordance with the AAALAC (Academic Animal Care and Administration) Regulations. Animals were housed in polysulfone (PSU) cages (4–6 mice / cage) with stainless steel grid tops and facilities for pelleted food and drinking water in clear polycarbonate bottles; bedding: steam-sterilized clean rice husks were used. Bedding material was replaced along with the cages at least twice a week. Diet: Animals were fed a commercial rodent diet ad libitum. Animals had free access to autoclaved drinking water obtained from the public water supply. Contaminants: None of the contaminants expected in the feed and water supplied are likely to have influenced the results of this study. Environmental conditions: Animals were housed under standard laboratory conditions with an adequate supply of fresh air. Animals were kept in a temperature and humidity controlled environment. The temperature range was 20-24°C, with a RH of 30-70% and a 12-hour light and 12-hour dark cycle. Veterinary Care: Animals were examined upon arrival for suitability for the study. As this was a 24-hour experiment, no veterinary follow-up was required after study initiation. Ethics Committee: The study was conducted in accordance with the Israel Animal Welfare Act after approval by the Israel Board for Animal Experiments. Responsible Party: 1. Facility Manager, overseeing all aspects of animal health and husbandry, and assisted by all animal program personnel. 2. The principal investigator has ensured that all research and technical staff are adequately trained and experienced in carrying out the required procedures.

[0396] Establishment of a test model The animals were anesthetized with a mixture of ketamine and xylazine (100 and 10 mg / kg, respectively) according to their body weight. The animals were treated with the different treatments by intravenous injection: 200 μL saline, 200 μL plasma, and 200 μL plasminogen-depleted plasma. Following this, the animals were placed in a prone position. The distal 7 mm part of the tail was cut with a scalpel. The tail was immediately immersed in a 50 mL Falcon tube containing isotonic saline that had been prewarmed to 37°C in a water bath. The tail was oriented vertically with the tip positioned approximately 2 cm below the horizontal plane of the body. Each animal was monitored for 60 minutes to detect rebleeding, even if bleeding had stopped. A stopwatch was used to determine the bleeding time. If bleeding on / off cycles occurred, the total bleeding time during the 60 minutes was recorded. The experiment was terminated after 60 minutes in accordance with the regulations of the Institutional Animal Ethics Committee to avoid death during the experiment. Body weight, including tail snip, was recorded again and blood loss during the experiment was estimated from weight loss. Blood pellets were estimated after centrifugation of the tubes. At the end of the experiment, animals were sacrificed by anesthesia overdose.

[0397] The rodent species chosen is the commonly used laboratory strain, the c57 black healthy young adult animal. This mouse model is a first step in providing initial information regarding the efficacy of plasminogen-depleted plasma for the treatment of excessive bleeding.

[0398] A total of 24 mice across three groups were utilized. Each group contained eight animals. A control group received saline only, a second group received untreated plasma, and a third group received plasminogen-depleted plasma (PDP). Animals were randomized into each group. One animal from each group received either plasma or a control treatment, which was applied intravenously as a single 200 μL dose. The total number of animals was based on previous studies demonstrating that this was the minimum number of animals per group that would yield significant information regarding the amount of blood lost by the animals. Treatments were administered by intravenous injection.

[0399] Plasminogen-depleted cryoprecipitate with clear plasma: 1- ClearPlasma Assembly: Fill the filter with resin. Lock the filling inlet Set the flow regulator valve to OFF. Attach a clamp to the extension tube (with flow regulator). Close the clamp Attach the extension tube to the filter Attach the clamp to the collection bag Close the clamp Attach the collection bag to the filter. 1 ClearPlasma to 18ml resin: TXA conjugate TXA and / or cyclohexa Fill the carboxylic acid (90-100 μm superflow resin - conjugate 1. 2 Wash the resin with 60 ml of water. 3 Wash the resin with 60 ml of saline. 4 Connect the cryoprecipitate bag to the ClearPlasma 5 16 ml of cryoprecipitate is allowed to flow through the filter for 22 minutes. 6. Transfer the supernatant to a new tube - plasminogen-depleted cryoprecipitate.

[0400] 2- PLG detection E-80PMG-Human Plasminogen ELISA Kit, Lot No. 10-(ICL, Inc.) Instructions for use are from the kit insert. (1)

[0401] [Table 2]

[0402] Sample dilution: Prepare 1 / 5,000 dilutions of the samples in a serial dilution: 1:100 cryoprecipitate (1:100 = 5 μL cryoprecipitate (1:100 + 495 μl 1x diluent). 1:5000 cryoprecipitate (1:50 = 10 μL of diluted cryoprecipitate (1:100) + 490 μl 1× diluent). Mix thoroughly at each stage.

[0403] Assay procedure: 1 Bring all reagents to room temperature before use. 2 Pipette 100 μL of sample (in duplicate) into pre-designated wells. 3 Incubate the microtiter plate at room temperature for 60 minutes. Keep the plate covered and horizontal during incubation. 4 After incubation, aspirate the contents of the wells. 5 Fill wells completely with wash buffer, invert plate and then pour / swirl contents into waste container. Follow this by tapping wells firmly on absorbent paper to remove residual buffer. Repeat 3 times for a total of 4 washes. 6 Pipette 100 μL of appropriately diluted enzyme-antibody conjugate into each well. Incubate for 60 minutes at room temperature. Keep the plate flat, covered, in the dark during the incubation. 7 Wash and blot wells as described in steps 5 / 6. 8 Pipette 100 μL of TMB substrate solution into each well. 9 Incubate in the dark at room temperature for exactly 10 minutes. After 10 minutes, add 100 μL of stop solution to each well. 11 Determine the absorbance (450 nm) of the contents of each well.

[0404] Example 1 Resin synthesis Example 1.1 - Synthesis of Conjugate 1 [ka] Resin preparation and filter packing - scale-up This process was carried out according to the NHS-activated Sepharose Fast Flow (GE Healthcare Cat. No. 17-0906-02) product instructions.

[0405] The process began with a new, clean batch of naive beads received with proper documentation from the manufacturer.

[0406] The resin volume used in this protocol was 112 mL of drained resin (8 x 14 mL) in 8 x 50 mL tubes (70% resin slurry initially; 14 mL of drained resin per tube). Preparation, coupling and post-coupling washes were performed as cleanly as possible. Endotoxin washes and loading steps were performed in a clean environment.

[0407] Experimental equipment used 0.2 μm filter (e.g. Steritop), 3 x 1 L bottles (1 mM HCl; 0.1 M Tris-HCl, pH 8.5; 0.1 M acetate buffer, 0.5 M NaCl, pH 4.5), 2 x 0.5 L bottles (coupling buffer; 4 M urea), 4 x 1 L bottles (70% EtOH, 50 mM Tris-HCl pH 7.5; 50 mM Tris-HCl, 0.1 NaCl, pH 7.5, in pyrogen-free water, 20% EtOH), organic waste container, autoclaved spatulas, beakers, graduated cylinders, clean plastic pipettes, tubes. -Blow roller / rotator, 50mL catheter tip syringe, sterile ClearPlasma filter Casing

[0408] Reagents used Sodium bicarbonate (NaHCO3), sodium carbonate (Na2CO3), sodium chloride, tranexamic acid, 37% HCl, NaOH, Tris-HCl, acetic acid, sodium acetate, ethanol, urea, Ddw (double distilled water), pyrogen-free water

[0409] solution All final buffers and solutions were filtered through a 0.2 μm filter before use.

[0410] Coupling Buffer - 100 mL 0.2 M NaHCO3, 0.5 M NaCl, pH 8.3. A quantity of 1.68 g of sodium bicarbonate was weighed out and dissolved in 70 mL ddw. The pH was adjusted to 8.3 with (1 M NaOH or 1 M HCl). A quantity of 2.92 g of sodium chloride was weighed out and dissolved in the sodium bicarbonate solution. The volume was adjusted to 100 mL with DDW.

[0411] Ligand solution - 85 mL 25 mM (1 eq.) or 50 mM (2 eq.) or 125 mM (5 eq.) or 250 mM (10 eq.) tranexamic acid, in coupling buffer, pH adjusted to 6-9. The desired amount of tranexamic acid was dissolved in 85 mL 0.2 M NaHCO3, 0.5 M NaCl, pH 8.3. The pH was adjusted to the desired level. This solution was filtered through 0.2 μl.

[0412] Resin Primary Wash - 1.5L 1 mM HCl. In a 50 mL tube, add DDW to a volume of 44 mL. Add 1 mL HCL (approximately 37%, 12M) to make 0.25 M HCl. In a 2 L bottle, add 1400 mL DDW. Add 6 mL 0.25 M HCl. Adjust the volume to 1500 mL. Filter the solution through 0.2μ.

[0413] Resin Blocking Solution - 250 mL 0.3 M Tris-HCl, pH 8.5. A quantity of 11.82 g Tris-HCl was dissolved in 200 mL DDW. The pH was adjusted to 8.5 with 1 M NaOH or 1 M HCl. The volume was adjusted to 250 mL with ddw. The solution was 0.2μ filtered.

[0414] Basic Wash - 1.5L 0.1M Tris-HCl, pH 8.5. A quantity of 23.64g Tris-HCl was dissolved in 1300mL DDW. The pH was adjusted to 8.5 with 1M NaOH or 1M HCl. The volume was adjusted to 1500mL with ddw. The solution was 0.2μ filtered.

[0415] Resin Acidic Wash - 1.5L 0.1M Acetate Buffer, 0.5M NaCl, pH 4.5. A quantity of 12.3g of sodium acetate was dissolved in 1300mL DDW. The pH was adjusted to 4.5 with 1M NaOH or 0.1M acetic acid (1.15mL glacial acetic acid brought to 200mL total with ddw). A quantity of 43.83g of sodium chloride was dissolved. The volume was adjusted to 1500mL. The solution was filtered through 0.2μ.

[0416] Endotoxin Wash Solution (EW) 1 - 600 mL 70% EtOH. Mix 420 mL high quality ethanol with 180 mL DDW.

[0417] EW2 - 600 mL 50 mM Tris-HCl pH 7.5. A quantity of 3.63 g Tris-HCl was dissolved in 500 mL DDW. The pH was adjusted to 7.5 with 1 M HCl. The volume was adjusted to 600 mL with DDW.

[0418] EW3 - 300mL 4M Urea in Pyrogen Free Water. A quantity of 72g of Urea was dissolved in 200mL DDW for irrigation. The volume was adjusted to 300mL with irrigation water.

[0419] EW4 - 600 mL 50 mM Tris-HCl, 0.1 NaCl, pH 7.5 in pyrogen-free water. A quantity of 3.63 g Tris-HCl was dissolved in 500 mL DDW for irrigation. The pH was adjusted to 7.5 with 1 M HCl. A quantity of 3.51 g sodium chloride was dissolved in the buffer. The volume was adjusted to 600 mL with water for irrigation.

[0420] Endotoxin Free Storage Solution (EFS) - 550 mL 20% EtOH in pyrogen free water. A volume of 110 mL of high quality ethanol was mixed with 440 mL water for irrigation.

[0421] 1M HCl - pH adjusted. 4.17mL 37% HCl was diluted to 50mL with DDW.

[0422] 1M NaOH-pH adjustment. 2g NaOH was diluted in 50mL DDW.

[0423] A solution made with irrigation water was used to adjust the pH of the endotoxin-free solution.

[0424] preparation - The resin was resuspended (NHS-activated Sepharose beads, 70% slurry in 100% isopropanol) and 8 x 14 mL was transferred to 8 x 50 mL PP tubes. - The tubes were spun down at 400g for 3 minutes. - The storage solvent was aspirated. - The tube was filled to 45mL with 1mM HCl and the resin was resuspended. - The tubes were spun down at 400g for 3 minutes and aspirated. - The wash was repeated 4 more times (total of 5 washes).

[0425] Coupling - 7mL of resin was added to each tube. - pH adjusted to 7.5-8 (if necessary). - The tubes were gently rotated continuously for 2-4 hours (room temperature) / overnight (4°C). - The resin was spun down, aspirated, and blocked with 25 mL (each tube) 0.1 M Tris-HCl, pH 8.5 for 2-4 h with rotation.

[0426] Washing The resin was spun down and aspirated. Add 30 mL 0.1 M Tris-HCl pH 8-9, resuspend, spin down, and aspirate. Add 30 mL 0.1 M acetate buffer, 0.5 M NaCl, pH 4-5, resuspend, spin down, and aspirate. These washes (Tris to acetate) were repeated five times. This process: If continued for an additional hour - washing steps (add, spin down, aspirate) were performed twice with 30mL 20% EtOH and stored as a 50% slurry in 20% EtOH. When proceeding directly to the endotoxin wash - add 30mL 70% EtOH (EW1).

[0427] Endotoxin Wash If the resin was stored in 20% EtOH, it was spun down, aspirated, and 30 mL 70% EtOH (EW1) was added. The resin was incubated in 70% EtOH for 30 minutes (with rotation). The resin was washed and 30 mL 50 mM Tris-HCl pH 7.5 (EW2) was added. The resin was washed and 30 mL 50 mM Tris-HCl pH 7.5 was added and the resin was incubated (rotating) for 30 minutes. The resin was washed and 15 mL 4M urea (EW3) was added. The resin was washed and 15 mL 4M urea was added and the resin was incubated (rotating) for 30 minutes. The resin was washed and 30 mL 50 mM Tris-HCl, 0.1 M NaCl, pH 7.5 (EW4) was added. The resin was washed and 30 mL 50 mM Tris-HCl, 0.1 M NaCl, pH 7.5 was added and the resin was incubated for 30 minutes (rotating). The resin was washed and 30 mL 20% EtOH (EFS) was added. The wash was repeated. The resin was stored as a 70% slurry in 20% EtOH (EFS) until loading.

[0428] filling All the resin from the tubes was pooled into one container. If the beads had settled, the container was gently swirled to ensure a uniform suspension. - The suspension was filled into a sterile 50 mL catheter-tipped syringe. - The casing was gently filled with the suspension. If topping up was necessary, the same method as above was used. - When the filter was nearing full, the outlet luer lock cover was opened to allow excess storage solution volume to drain from the filter. After the resin compartment was filled, the outlet was closed. The filled products were stored according to product specifications.

[0429] Example 1.2 - Synthesis of Conjugate 2 [ka] The reaction for the preparation of conjugate 2 is shown diagrammatically in FIG.

[0430] More specifically, the following procedures were used: 1. A quantity of 4.2 ml of Sepharose 4B200 (Sigma Aldrich) was passed through a glass funnel filter using a shaker and washed with acetone. Sepharose contained 1 ml of reactive functional groups, therefore 4.2 ml was considered to contain 4.2 mmol. 2. Succinic anhydride (0.42, 4 mmol) was added to a slurry of beads in CH2Cl2 (3 ml) followed by pyridine (0.339 ml). The mixture was shacked overnight. 3. The beads were filets and washed with acetone. 4. The beads were suspended in CH2Cl2 and N-Hydroxylsuccinamide (NHS) (0.483 g) was added, followed by EDC (0.8 g). The mixture was shaken overnight and then filtered. 5. This product was suspended in DMF (3 ml) and N,N-diisopropylethylamine (0 .54 g) and 4-(aminomethyl)cyclohexanecarboxylic acid (0.66 g) were added. The mixture was shaken overnight. The product was washed with acetone and sent for further testing.

[0431] Example 1.3 - Synthesis of Conjugate 3 ("TXA-Glyoxal Agarose Resin") [ka] procedure 1. Wash the glyoxal agarose beads with distilled water using a glass filter. 2. Prepare ligand solutions and test for activity and / or absorbance at 280 nm. Add 3.1 ml glyoxal agarose beads to 9 ml TXA solution in pH 10.05 buffer. 4. Stir gently and check pH frequently. Remove aliquots of suspension and assay for activity or absorbance at 280 nm. 5. Continue gentle stirring for several hours or until the activity reading remains constant (indicating complete immobilization). Avoid magnetic stirrers. Note: Longer immobilization times favor stronger biomolecule / bead reactions and stability, but may result in undesirable distortions. 6. Once the activity / absorbance has stabilized, add 10 mg solid sodium borohydride to the suspension and stir in an open container at room temperature for 30 minutes to allow the hydrogen to escape. Do not perform this step near an open flame; if possible, do so near an extractor fan. 7. Remove excess borohydride by washing the suspension with 25 mM phosphate buffer pH 7.0 using a vacuum filter. The suspension is then washed thoroughly with distilled water and filtered to dryness. 8. Ligand-coupled glyoxal agarose beads should be stored at 4-10°C in 20% ethanol in water containing a preservative.

[0432] Example 1.4 - Synthesis of Conjugate 4 ("TXA-ECH Agarose Resin") [ka] Conjugation of tranexamic acid (TXA) with ECH-agarose beads (G-Biosciences, #786-1223) 1. Prepare ligand solution - 50 mM TXA in ddw, pH adjusted to 5.2 with HCl. Wash the 2.2 mL resin with 10 mL ddw. Spin down by centrifugation for 3 min at 500 g. 3. Wash the resin with 160 mL 0.5M NaCl. 4. Add 4 mL of ligand solution to the resin after draining. 5. Add EDC (coupling agent) to give 100 mM in the final reaction. 6. Rotate for 1 hour and adjust pH to 5.0 with HCl. Continue rotating overnight. 7. Spin down the resin and decant. 8. Wash the resin with three cycles of alternating solutions: a. 0.1M acetate, 0.5M NaCl, pH 4.0 b. 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.0 9. Wash twice with ddw. 10. Wash once, then store in 20% EtOH at 2-8°C.

[0433] Example 1.5 - Synthesis of Conjugate 5 [ka] Reactant - Sodium cyanoborohydride [ka] In the first step, a Schiff base is prepared, which is then reduced with sodium cyanoborhydride (NaCNBH3) or preferably sodium borohydride (NaBH4) to give the final material. The double bond in the Schiff base is characterized by an infrared absorbance at 1590-1690 cm-1. The carbonyl in the aldehyde is the starting material, characterized by an infrared absorbance at 2700-2900 cm-1. The final material also requires tranexamic acid.

[0434] More specifically, the following procedures were used: 1. Glyoxal agarose beads were washed with distilled water using a glass filter. 2. Ligand solutions were prepared and tested for activity and / or absorbance at 280 nm. 3. A volume of 1 ml of glyoxal agarose beads was added to 9 ml ligand solution in pH 10.05 buffer. If the ligand was unstable at room temperature, the following steps were carried out in a cold room. 4. Gentle mixing was performed and the pH was checked frequently. Aliquots of the suspension were withdrawn and assayed for activity or absorbance at 280 nm. 5. Gentle stirring was maintained for several hours or until the activity reading remained constant (indicating complete immobilization). Magnetic stirrer was avoided. NOTE: Longer immobilization times favor stronger biomolecule / bead reactions and stability, but may also result in undesirable distortions. 6. When the activity / absorbance becomes constant, add 10 mg of solid sodium borohydride to the suspension. The hydrogen was allowed to escape by adding 100% ethanol and stirring at room temperature in an open vessel for 30 minutes. This step was never carried out near a flame, but rather near an exhaust fan whenever possible. 7. Excess borohydride was removed by washing the suspension with 25 mM phosphate buffer pH 7.0 using a vacuum filter. The suspension was then washed thoroughly with distilled water and filtered to dryness. 8. Ligand-coupled glyoxal agarose beads were stored at 4-10°C in 20% ethanol in water containing a preservative.

[0435] Example 1.6 - Synthesis of Conjugate 6 TXA-Sepharose Resin [ka] 1.4.2 ml of Sepharose beads were passed through a glass funnel filter using a shaker and washed with acetone. 2. Succinic anhydride (0.42, 4 mmol) was added to a slurry of beads in CH2Cl2 (3 ml) followed by pyridine (0.339 ml). The mixture was shacked overnight. 3. The beads were filets and washed with acetone. 4. The beads were suspended in CH2Cl2 and N-Hydroxylsuccinamide (NHS) (0.483 g) was added, followed by EDC (0.8 g). The mixture was shaken overnight and then filtered. 5. This product was suspended in DMF (3 ml) and N,N-diisopropylethylamine (0.54 g) and 4-(aminomethyl)cyclohexanecarboxylic acid (0.66 g) were added. The mixture was shaken overnight. The final beads were washed twice with acetone and then with 70% ethanol, centrifuged, and suspended in 20% ethanol.

[0436] Example 2 Determination of the activity of newly synthesized TXA-conjugated ECH-agarose beads, Sterogene Superflow and glyoxal agarose beads-conjugate 1 material: Plasma: Plasma from healthy donors was obtained from the MDA Blood Bank. The plasma used was Rh negative and negative for various viral antigens (e.g., HBV, HCV, HTLV, HIV). ·beads: 1. TXA-conjugated glyoxal agarose beads synthesized by the procedure described in Example 1 (Preparation of Conjugate 1) 2. TXA-conjugated ECH-agarose beads (new beads), Daren laboratories 3. Sterogene Superflow (sterogene beads) Equipment: Shaker: KRS-3016 (MRC) ELISA reader: 800TS (BioTek) The experiments were performed in a non-sterile environment. ·Solution preparation: Binding buffer (30ml): Sodium citrate (10 mM) - 0.3 ml NaCl (120mM; date of preparation: September 25, 2017) - 1.2ml DDW=28.5ml

[0437] Plasminogen depletion 1. The beads were already stored in 20% ethanol and 70% resin. A 1 ml amount of the beads was transferred to a 15 ml tube. 2. Fill the tube with water, and 3. The tube was centrifuged at 300 xg for 2 minutes at room temperature. 4. The supernatant was removed. 5. These steps were repeated three times. 6. Suspend the beads in binding buffer * : a-10mM sodium citrate PH=7.16 b-120mM sodium chloride 7. The centrifugation step was carried out as in section 3. 8. The supernatant was removed. 9. These steps were repeated twice. 10. A volume of 1 ml of 74E plasma was added to the beads. 11. The mixture was mixed thoroughly by gentle inversion and incubated at room temperature for 2 hours (plate shaker- shaking at 80 rpm). 12. A centrifugation step was performed as in section 3 and the supernatant was transferred to a new tube containing plasma-depleted plasminogen. 13. Plasma depletion was tested using an ELISA assay. 14. The remaining plasma (including undepleted and depleted) was incubated at -20°C.

[0438] PLG detection The kit Ab108893 Human Plasminogen ELISA Kit (Abacam®) was used according to the manufacturer's instructions.

[0439] Plates were prepared as follows in Table 3 (1:20,000 plasma dilution):

[0440] [Table 3]

[0441] Reagent preparation All reagents were equilibrated to room temperature (18-25 °C) before use. Fresh reagents were prepared immediately before use. 1- 1x Diluent M: 1:10 dilution of 1.5ml of 10x Diluent M concentrate with 15ml of reagent grade water. Mix gently and thoroughly. 2- 1x Wash Buffer: 2ml of 20x Wash Buffer, 1:20 dilution with 38ml of reagent grade water for concentrate. Mix gently and thoroughly. 3- 1x Biotinylated Plasminogen Detection Antibody: First spin the 50x Biotinylated Plasminogen Antibody vial to collect the bottom contents. Add 12μl of 50x stock Biotinylated Plasminogen Antibody to 588μl of 1x Diluent M. Gently but thoroughly. Mix. 4- 1x SP conjugate: 100x streptavidin-peroxidase conjugate (SP conjugate) is briefly spun down and 6 μl of conjugate is diluted 1:100 in 594 μl of 1x Diluent M.

[0442] Standard preparation: 1- The standards were equilibrated to room temperature. 2- Seven tubes were labeled #2 to #8. 3- Add 120 μL of 1x Diluent M to tubes #2 through #8. 4- Prepare standard #2, add 120 μL of standard #1 to tube #2 and mix gently. 5- Prepare standard #3, add 120 μL of standard #2 to tube #3 and mix gently. 6- Prepare subsequent serial dilutions using the table below as a guide. 7- 1x Diluent M serves as the zero standard, 0 ng / mL (tube #8).

[0443] Sample preparation The samples are serially diluted 1:20,000 in 1x Diluent M: a- 1:100 plasma = 5 μL plasma + 495 μl 1×Diluent M b- 1:20,000 plasma = 5 μL of diluted plasma + 995 μL 1×Diluent M

[0444] The following procedures were used: 1- All reagents, working standards and samples were prepared as instructed. The assay was performed at room temperature (18-25°C). 2- The excess microplate strips were removed from the plate frame and immediately placed back into the foil pouch containing the desiccant. The pouch was tightly resealed to minimize exposure to water vapor and stored in a vacuum desiccator. 3- A volume of 50 μL of plasminogen standard or sample was added to each well. The wells were covered with sealing tape and incubated for 1 hour. The timer was started after the last sample was added. 4- Five manual washing steps with 200 μL 1× Wash Buffer were performed, each time inverting the plate and decanting the contents (liquid was completely removed by tapping 14 times on absorbent paper towels). 5- A volume of 50 μL of 1× biotinylated plasminogen antibody was added to each well and incubated for 1 hour. 6- The microplate was washed as described above. 7- A volume of 50 μL of 1×SP conjugate was added to each well and incubated for 30 minutes. The microplate reader was turned on and the program was set up in advance. 8- The microplate was washed as described above. 9-Add a volume of 50 μL of colorimetric substrate per well and incubate for approximately 12 minutes or until optimal blue color intensity occurs (gently tap the plate to ensure adequate mixing and break up any air bubbles in the wells with the pipette tip) 10- A volume of 50 μL of stop solution was added to each well. The color changed from blue to yellow. 11- The absorbance was immediately read in a microplate reader at a wavelength of 450 nm. The following results were observed:

[0445] · PLG concentration: Calibration curve results * are summarized in Table 4 and shown in Figure 3.

[0446] [Table 4]

[0447] The formulas given in Table 5 were obtained. The concentrations and depletion percentages of the samples were calculated accordingly and are given in Table 6.

[0448] [Table 5]

[0449] [Table 6]

[0450] The PLG concentration in undepleted plasma appears to be slightly higher than the normal range of plasminogen concentration in human plasma (153.1-174.9 μg / ml, according to the kit instruction manual). Plasminogen levels were 9.0-10.0 μg / ml after incubation with TXA Superflow recovery beads. More than 2% were depleted, more than 90% after incubation with YA2-2 beads, and TXA conjugates. After incubation with TXA-conjugated ECH agarose beads, the depletion was 38%. Recovery was achieved with TXA Superflow beads and Y2-2 beads (TXA-conjugated glyoxal agarose). The TXA-conjugated ECH agarose beads showed high efficiency in removing plasminogen from plasma, whereas the TXA-conjugated ECH agarose beads showed low efficiency.

[0451] Example 3 Determination of activity of new conjugated GE beads, Sterogene Superflow beads material: Plasma: Plasma from healthy donors was obtained from the MDA Blood Bank (see Appendix 1). Beads: 1- Conjugate 1 ("TXA conjugated GE beads") - pH 6.5, 7.5, 8.5. See Example 1 for synthesis protocol. 2- "Sterogene Post-Recovery TXA Superflow Beads" [Conjugate-6] Method #1: Beads were used in a pig experiment and allowed to recover. Equipment: 1- Shaker: KRS-3016 (MRC) 2- ELISA reader: 800TS (BioTek). The experiment was performed in a non-sterile environment.

[0452] Plasminogen depletion 1.a. Sterogene TXA Superflow beads are already in 20% ethanol and 70% resin. A 1 ml amount of the beads was transferred to a 15 ml tube. b. 1 ml of each reaction of TXA-conjugated GE beads already in 20% ethanol and 70% resin 2. The tube was filled with water. 3. Centrifuge at 300 x g for 2 minutes at RT. 4. The supernatant was removed. 5. Repeat 3 times 6. The beads were suspended in binding buffer. * : a- 10mM sodium citrate PH=7.16 b- 120mM sodium chloride 7. Centrifuge as in Section 3. 8. The supernatant was removed. 9. Repeat twice 10. 1 ml of 27K plasma was added to the beads. 11. Mix thoroughly by gently inverting the tube and incubate at room temperature for 2 hours (on a plate shaker - shaking at 80 rpm). 12. Centrifuge as in section 3 and transfer the supernatant to a new tube - Plasma depleted plasminogen 13. Plasma depletion was tested using an ELISA assay. 14. The remaining plasma (undepended and depleted) was incubated at -20°C.

[0453] PLG detection The kit Ab108893 Human Plasminogen ELISA Kit (Abacam®) was used according to the manufacturer's instructions.

[0454] Plates were prepared as follows (1:20,000 plasma dilution).

[0455] [Table 7]

[0456] Reagent preparation: Equilibrate all reagents to room temperature (18-25°C) before use. Fresh reagents immediately before use. 1x Diluent M: Dilute 2ml of 10x Diluent M concentrate 1:10 with 18ml of reagent grade water. Mix gently but thoroughly. 1× Wash Buffer: Dilute 4 ml of 20× Wash Buffer Concentrate 1:20 with 76 ml of reagent grade water. Mix gently but thoroughly. 1x Biotinylated Plasminogen Detection Antibody: 50x Biotinylated Plasminogen Antibody Biotinylated Plasminogen Antibody Spin the flask to collect contents at the bottom. Add 18 μl of 50× stock biotinylated plasminogen antibody to 882 μl of 1× Diluent M. Mix gently but thoroughly. 1x SP Conjugate: 100x streptavidin-peroxidase conjugate (SP conjugate) was spun down briefly and 9 μl of the conjugate was diluted 1:100 with 891 μl of 1x Diluent M. Standards were prepared as detailed in Example 2. The calibration curve results are summarized in Table 4 and shown in Figure 2. The formulas shown in Table 5 were obtained.

[0457] Sample preparation: Samples were diluted 1:20,000 with 1x Diluent M in serial dilutions: a- 1:100 plasma = 5 μL plasma + 495 μl 1×Diluent M b- 1:20,000 plasma = 5 μL of diluted plasma + 995 μL 1×Diluent M

[0458] Assay procedure: 12- All reagents, working standards and samples were prepared as instructed. Reagents were equilibrated to room temperature before use. Assays were performed at room temperature (18-25°C). 13- The excess microplate strips were removed from the plate frame and immediately placed back into the foil pouch containing the desiccant. The pouch was tightly resealed to minimize exposure to water vapor and stored in a vacuum desiccator. 14- 50 μL of plasminogen standard or sample was added per well. The wells were covered with sealing tape and incubated for 1 hour. The timer was started after the last sample was added. 15- 5 manual washes with 200 μL 1× Wash Buffer, inverting the plate each time and decanting the contents (remove all liquid by tapping the plate on absorbent paper towels 4 times). 16-Add 50 μL of 1× biotinylated plasminogen antibody to each well and incubate for 1 hour. 17- Wash the microplate as described above. 18-Add 50 μL of 1×SP conjugate to each well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. 19- Wash the microplate as described above. 20-Add 50 μL of colorimetric substrate per well and incubate for approximately 12 minutes or until optimal blue color intensity occurs (gently tap plate to ensure adequate mixing and break up any air bubbles in wells with pipette tip). 21- 50 μL of stop solution was added to each well. The color changed from blue to yellow. 22- The absorbance was immediately read in a microplate reader at a wavelength of 450 nm.

[0459] · PLG concentration: The concentration and percentage depletion of the samples were calculated according to the formulas detailed in Table 5 and are shown in Table 8.

[0460] [Table 8]

[0461] The PLG concentration in undepleted plasma appears to be slightly higher than the normal range for plasminogen concentrations in human plasma. Ink of plasma with TXA Superflow beads recovered using method #1 Approximately 24% of the plasminogen was depleted after incubation with beads recovered using method #2, and over 93% after incubation with beads recovered using method #3. Therefore, the 24% depleted beads must be recovered again, and the 93% beads can be used again in pig experiments. TXA-conjugated GE beads synthesized at PH=6.5 or PH=7.5 showed over 60% PLG depletion, while those synthesized at PH=8.5 showed 54% depletion. This experiment demonstrates that the reaction is more efficient at PH=6.5. Future experiments will be performed overnight or with reactions of different ligand concentrations.

[0462] Example 4 Plasma filtration with ClearPlasma TXA superflow, conjugate 1 Filter preparation: 1-The filter was filled with 25 ml of resin. 2-100 ml of 70% ethanol was poured onto the filter and incubated for 30 minutes. Wash twice with 3-70% ethanol. Washed three times with 4-20% ethanol Stored at 5-4°C.

[0463] Resin activation: The resin was washed three times with DDW and twice with binding buffer) 10 mM sodium citrate + 120 mM sodium chloride).

[0464] Plasma filtration: The human plasma bag was connected to the filter and the entire volume (200 ml) was passed through the filter into the receiving bag over a period of 1 hour.

[0465] The results are shown in Table 9 below.

[0466] [Table 9]

[0467] As shown in Table 9, filtration of plasma effectively depleted plasminogen.

[0468] ClearPlasma™ Instruments TXA conjugated beads and / or cyclohexane Carboxylic acid agarose beads = conjugate 1. material: Plasma: Plasma from healthy donors was obtained from the Magen David Adorn (MDA) Blood Bank. -ClearPlasma™ device: Use a 3 ml Pasteur pipette to apply the cone to the cartridge. Conjugate cyclohexane carboxylic acid = conjugate 2 was packed into agarose beads; the resin was washed with 150 ml of saline and stored at 2-8 °C. -Equipment: ELISA reader: 800TS (BioTek)

[0469] A volume of 250 ml of plasma was connected to the device using a luer lock and allowed to flow through the device at a rate of 2 drops / sec for approximately 30 minutes. The depleted plasma was collected in a sterile cap. Samples from the raw and depleted plasma were taken for analysis and the remainder was frozen at -20°C. E-80 PMG-Human Plasma Plasminogen concentrations were determined using the plasminogen ELISA kit, lot number 9 (ICL, Inc.).

[0470] [Table 10]

[0471] Greater than 96% of PEG was removed from plasma after filtration with the ClearPlasma™ device described above. It looks like it has been.

[0472] Example 5 Plasminogen levels from cryoprecipitate using ClearPlasma Determining the effectiveness of Clear Plasma in depleting Cryoprecipitate is an important blood product used in the treatment of hemorrhages in general and hemorrhages in particular. Therefore, the ClearPlasma device converts plasminogen activator (PAC) from cryoprecipitate. The ability to deplete gen was determined.

[0473] material: a. ClearPlasma: plastic filter (Pentracor Inc.), TXA-conjugated Superflow resin (Sterogene; Lot: 1608:88), extension line with flow regulator (Qosina; Lot: 159299), blood collection bag (Fresenius; Lot: FA17H30126). b.Equipment: i. Shaker: Catalog number KRS-3016; Serial number: SH30000003; Manufacturer: MRC. ii. ELISA Reader: Catalog number 800TS; serial number: 1709201B; Manufacturer: BioTek iii.Centrifuge: Catalog number Z383K; serial number: 31030005; Manufacturer: HERMLE.a. c. Cryoprecipitate: Cryoprecipitate was obtained from Hadassah Blood Bank (details of cryoprecipitate in Appendix 1). (Y 2002 18 170007 O Rh positive)

[0474] The experiment was performed as described in the Experimental Procedures. Data were collected using cryopreservation through ClearPlasma. A single pass of the cryoprecipitate unit (29 ml) reduced the plasminogen concentration of the cryoprecipitate from 155.87 to 2.7 μg / ml, a reduction of approximately 98%, without any effect on the clotting factors (see Figure 3). Thus, the ClearPlasma device is well suited for use in depleting plasminogen from cryoprecipitate. Depletion of plasminogen from cryoprecipitate provides a novel formulation with improved ability to treat bleeding conditions.

[0475] Example 6 Determination of active plasminogen concentrations in vivo Briefly, fresh blood was collected from healthy volunteers. 50 μl of tPA (6 μM) or normal saline (NS) was added to 1.8 ml of fresh blood and the blood was analyzed using a Hemochron 401 instrument. In parallel experiments, 50 μl of tPA (6 μM) or NS was added to 1.8 ml plasminogen-free plasma.

[0476] To determine the efficacy of the conjugates of the presently disclosed subject matter, two parameters were evaluated: clotting time and total clot lysis time.

[0477] Clotting time Clotting time was inversely proportional to the concentration of active plasminogen.

[0478] In control blood, the clotting time was 113±27 seconds (n=7), whereas in blood treated with tPA it was 255±41 seconds (n=8). In plasminogen-depleted blood, the clotting time in the absence of tPA was 110±16 (n=6), whereas in the presence of tPA it was 118+18 (n=7).

[0479] Total clot lysis time The presence of a clot was determined by simple observation and by reinserting the tube containing the clot into the machine.

[0480] After 14±5.2 min (n=6), the clot had disappeared from control blood treated with tPA, whereas in control blood treated with NS, the clot was intact 2 h after complete clot formation.

[0481] In plasminogen-depleted blood with or without tPA, the clots were intact at 2 hours after complete clot formation. These results clearly demonstrate the efficacy of the conjugates of the presently disclosed subject matter.

[0482] Example 7 Evaluating the efficacy of plasma apheresis in a porcine model of hepatic laceration The study demonstrated the effectiveness of ClearPlasma™ and plasminogen activator in comparison to normal plasma. The aim of this study was to investigate the benefits of gen-depleted plasma. The study was conducted at Biotechfarm Ltd. (Israel).

[0483] the purpose The objective was to evaluate the efficacy of PDP administration against blood loss in a porcine liver laceration model.

[0484] Study Endpoints Blood loss was measured 30 minutes after the 4 cm liver laceration and judged according to the grading scheme presented below.

[0485] [Table 11]

[0486] Research period 8-9 hours for each pig

[0487] Study design Fourteen female domestic pigs were assigned to four groups (control, normal plasma, ClearPlasma™ and TXA)

[0488] The groups were treated as follows:

[0489] Control Group 1: 1. Induction of general anesthesia 2. Plasma collection over 90 minutes 3. Induction of liver laceration 30 min after the end of plasma collection 4. Suturing the abdominal wall and skin 30 minutes after induction of liver laceration

[0490] Control group 2: 1. Induction of general anesthesia 2. Induction of liver laceration 30 min after the end of plasma collection 3. Suturing the abdominal wall and skin 30 minutes after induction of liver laceration

[0491] Control group 3: 1. Induction of general anesthesia 2.Treatment with TXA 3. Induction of liver laceration 30 min after the end of plasma collection 4. Suturing the abdominal wall and skin 30 minutes after induction of liver laceration

[0492] Study Group: 1. Induction of general anesthesia 2. Plasma collection over 90 minutes - filtration with ClearPlasma™ (test set) 3. Induction of liver laceration 30 min after the end of plasma collection 4. Suturing the abdominal wall and skin 30 minutes after induction of liver laceration

[0493] [Table 12]

[0494] Study Procedure 1. Buprenorphine was administered before induction of anesthesia. 2. Anesthesia - Animals were sedated with ketamine, xylazine and atropine and then intubated with an endotracheal tube; anesthesia was maintained with isoflurane in oxygen. 3. A catheter was placed in an accessible ear vein for vascular access. 4. A central venous cannula (CVC) was introduced into each jugular vein for plasma sampling. 5. Plasma was collected for 90 minutes. Then, plasma transfusion with or without ClearPlasma™ was performed. Blood was carried out. 6. 30 min after plasma collection, the liver laceration model was induced as follows: - The pig was placed in supine position. - The abdominal area was clipped and scrubbed. - A midline incision was made. - The right lobe of the liver was exposed and exteriorized, and after ensuring an adequate working area, it was rinsed with saline. - A grid pattern of lacerations was made, 4 cm (length), 1 cm apart and 0.5 cm deep (liver pieces were weighed). 7. Blood loss was measured by determining the weight difference between the dry sponge and the bloody sponge after model introduction. The weight difference was expressed as blood loss in grams. 8. When applicable, time to hemostasis (TTH) was measured. 9. 30 minutes after the introduction of the laceration, the abdominal wall and skin were sutured.

[0495] Physiological Parameter Monitoring Before plasma sampling application (baseline), immediately after sampling application, immediately before induction of liver laceration, during bleeding and immediately after skin suturing: - Esophageal temperature, - mean arterial pressure, - Heart rate, - oxygen saturation, - activated clotting time, - Blood sample for CBC, - Hematology, biochemistry and coagulation parameters, including prothrombin time (PT) and partial thromboplastin time (PTT). Plasma collection before application (baseline), 30 minutes after induction of liver laceration (just before suturing) and at the end of the study (5 hours after suturing): - Detailed clinical signs: After recovery from anesthesia Before plasmapheresis: - Weight

[0496] supportive care During recovery, the animals were placed on a skin-heating blanket.

[0497] Euthanasia Animals were sacrificed 5 hours after suturing. Note: Two animals with persistent bleeding 60 minutes after induction of the liver laceration (i.e., immediately prior to suturing) were humanely euthanized.

[0498] Clinical Observation Method Behavioral attributes include, but are not limited to, the following: 1. No blood, urine or fecal excretion, discolored urine (if applicable), diarrhea or absence of feces (constipation). 2. Signs of illness or injury, lethargy, vomiting, excessive salivation, abnormal posture, pain, lameness, discomfort, unwillingness or inability to move. 3. Neurological Severity Score (NSS) 4. Further evaluation was performed whenever necessary based on clinical observations.

[0499] Equipment Haemonetics MCS®+, syringes, needles and surgical glassware, sphygmomanometer, oximeter. All diluents and solutions for cleaning and rinsing the device or parenteral injection assembly were handled in a manner that ensured their sterility, pyrogen-free and protection from contamination.

[0500] result In all experiments, pigs underwent plasma collection using the Haemonetics MCS®+ system. As depicted in Figure 4, whole blood was collected, centrifuged, red blood cells were immediately reinfused into the pig, and up to 700 ml of plasma was collected and either filtered using ClearPlasma™ or filtered. The pigs were placed under anesthesia and blood was taken from a vein and introduced into the plasmapheresis system. The red blood cells were then reinfused into the animals and, in the test animals, the plasma was filtered by ClearPlasma™ and reinfused into the animals. Detailed results from each pig are detailed in Table 13.

[0501] [Table 13]

[0502] [Table 14]

[0503] Experiment 1: Plasma from pig 144 was filtered using ClearPlasma™ and pig 144 Plasma from pig 143 was transfused into pig 143 after mock filtration. A 4 cm liver resection was then performed and blood volume and bleeding time were recorded. This experiment shows that depleting plasminogen levels reduces bleeding. Although the liver resection size was smaller in the control pigs, bleeding was greater in the pigs receiving PDP. In addition, clinical assessment was improved and pulses in the control pigs were higher and more erratic when compared to the pigs receiving PDP.

[0504] Experiment 2: Effect of PDP compared to normal plasma and TXA. Three pigs were studied as follows: pig 145 was treated with normal plasma, pig 146 was treated with plasminogen-depleted plasma, and pig 147 was treated with tranexamic acid (TXA). The results demonstrate that PDP reduces bleeding volume when compared to normal plasma. In addition, there was a small difference in bleeding volume with TXA compared to normal plasma.

[0505] Experiment 3: Effect of PDP compared to normal plasma and TXA. The results demonstrate that PDP reduces bleeding volume when compared to normal plasma.

[0506] Experiment 4: Effect of PDP compared to normal plasma and TXA. The results demonstrate that PDP reduces bleeding volume when compared to normal plasma.

[0507] Experiment 5: Reduction of bleeding after liver section in pigs treated with PDP. Pigs were anesthetized and plasma was collected as described in the Materials and Methods section. Pigs were treated with normal plasma, PDP, TXA 16 mg / kg, or left untreated. A 4 cm liver section was then performed using a scalpel as described in the Materials and Methods section. The results demonstrate that PDP reduces bleeding when compared to normal plasma.

[0508] Figure 5 shows the amount of blood loss as seen over a 30 minute period after liver laceration. As can be seen in Figure 5, the amount of blood loss was approximately 10% with ClearPlasma compared to pigs receiving normal plasma. Decreased by more than 50%.

[0509] To verify the reduction in fibrinolytic protein activity such as plasminogen and tPA, thromboelastography (TEG) was performed (see Figures 6A-6L). (TEG) is a method to test blood clotting efficiency. Pigs were numbered: 169 - control, 172 - plasmapheresis and plasminogen depletion; 173 - plasmapheresis only. As can be seen in Figure 6J, only pig 172 had no fibrinolytic response after tPA administration. The results demonstrate a significant reduction in fibrinolytic proteins such as plasminogen and tPA.

[0510] In conclusion, all studies have demonstrated that the use of ClearPlasma™ has significantly improved the survival of patients receiving standard plasma transfusions. Clinical outcomes were significantly improved and the pigs lost less blood compared to pigs that had not been treated with sucrose.

[0511] Example 8 Safety evaluation of administration of plasminogen-depleted plasma against blood loss in Clexane-treated animals in a porcine liver laceration model This study was carried out at Biotechfarm Ltd. (Israel).

[0512] the purpose To evaluate the safety of administration of plasminogen-depleted plasma against blood loss in a porcine liver laceration model.To investigate the effect of plasminogen depletion in animals under anticoagulant treatment.To provide a lack of risk of developing thromboembolic complications in patients undergoing blood thinner therapy (Clexane).

[0513] Study Endpoints Blood loss was measured 30 min after 4CM liver laceration.

[0514] Study design In this study, by comparing fibrinolytic protein-depleted plasma with normal plasma in Clexane-treated animals, we confirmed that treatment with PDP does not lead to the development of deep vein thrombosis (DVT). Species and sex: Domestic female pigs, 40-50 kg at the start of the study Number of groups: 4 groups (3 control groups and 1 test device group) Group size: n=5 (females) for control group; n=5 (females) for test device group Total number of animals: 20

[0515] Group and treatment composition: Control group 1: 1. Untreated controls: induction of general anesthesia 2. Plasma collection for 90 minutes 3. Induction of liver laceration 30 minutes after the end of plasma collection 4. Suturing of the abdominal wall and skin 30 minutes after induction of liver laceration.

[0516] Control group 2: 1. 4000 units Clexane: Pigs were injected with 4000 units Clexane 10 hours before surgery. 2. Induction of general anesthesia. 3. Induction of liver laceration 30 minutes after the end of plasma collection. 4. Suturing of the abdominal wall and skin 30 minutes after induction of liver laceration.

[0517] Control group 3: 1. Fresh plasma and 4000 units of Clexane: Pigs were injected with 4000 units of Clexane 10 hours before surgery. 2. Induction of general anesthesia. 3. Treatment with TXA. 4. Induction of liver laceration 30 minutes after the end of plasma collection. 5. Suturing of the abdominal wall and skin 30 minutes after induction of liver laceration.

[0518] Test groups: 1. Plasminogen-depleted plasma and 4000 units of Clexane: Pigs were injected with 4000 units of Clexane 10 hours before surgery. 2. Induction of general anesthesia. 3. Plasma collection over 90 minutes - filtration with ClearPlasma™ (test device group). 4. End of plasma collection. 5. Induction of liver laceration 30 minutes after the end of the experiment. 6. Suturing of the abdominal wall and skin 30 minutes after the induction of liver laceration.

[0519] Study Procedure Ten hours before treatment with PDP or normal plasma, ) syringe, 4,000 IU (40 mg) / 0.4 ml solution for injection in a prefilled syringe was injected intravenously (IV) into pigs. Analgesia: Buprenorphine; Anesthesia: Animals were sedated with ketamine, xylazine and atropine, then intubated with an endotracheal tube and anesthesia was maintained with isoflurane in oxygen.

[0520] An accessible ear vein was catheterized for vascular access, and a central venous cannula (CVC) was introduced into each jugular vein for plasma sampling applications.

[0521] Plasma collected during a 90 minute period. This plasma was then treated with ClearPlasma™ or The blood was transfused without treatment.

[0522] Thirty minutes after application of the plasma sampling, liver laceration was induced as follows: - The pig was placed in supine position, Shaving and scrubbing the abdominal area, - A midline incision was made, -The right lobe of the liver was exposed and exteriorized to provide sufficient working space and was rinsed with saline. - A grid pattern of lacerations was made, 4 cm (length), 1 cm apart and 0.5 cm deep, and the liver pieces were weighed. Blood loss was measured by determining the weight difference between the dry and bloody sponges after model introduction. Weight difference was expressed as blood loss in grams. Time to hemostasis (TTH) was measured, if applicable. Thirty minutes after the introduction of the laceration, the abdomen was sutured, followed by the skin.

[0523] Physiological parameter monitoring: Before applying plasma collection: Baseline, immediately after sampling application, immediately before induction of liver laceration, during bleeding and immediately after suturing the skin. Blood samples for esophageal temperature, mean arterial pressure, heart rate, oxygen saturation, activated clotting time CBC Hematology, biochemistry and coagulation parameters including prothrombin time (PT) and partial thromboplastin time (PTT): Plasma collection pre-application - baseline, 30 minutes after induction of liver laceration (just before suturing) and at the end of the study, 5 hours after suturing Detailed clinical signs: After recovery from anesthesia Body weight: once, before plasma apheresis

[0524] Supportive care: Buprenorphine was administered prior to induction of anesthesia. Animals were placed on a skin-heating blanket during recovery.

[0525] Terminal examination: Animals were sacrificed 5 hours after suturing. Note: Any animals with persistent bleeding for more than 60 minutes after induction of the liver laceration (i.e., immediately prior to suturing) were humanely euthanized.

[0526] Study duration: 8-9 hours for each pig

[0527] Clinical Observation Methods: Behavioral attributes included, but were not limited to: 1. Blood, urine and fecal excretion, discolored urine (if applicable), diarrhea, absence of feces (constipation). 2. Signs of illness or injury, lethargy, vomiting, excessive salivation, abnormal posture, pain, lameness, discomfort, unwillingness or inability to move. 3. Neurological Severity Score (NSS). 4. Further evaluation was performed whenever indicated based on clinical observations.

[0528] Equipment: Haemonetics MCS®+, syringes, needles and surgical glassware, sphygmomanometer, oximeter. All diluents and solutions for washing and rinsing the device or parenteral injection assembly were handled in a manner that ensured their sterility and pyrogen-free. Ensure that all test solutions are protected from contamination.

[0529] In all experiments, pigs underwent plasma collection using the Haemonetics MCS®+ system. As depicted in Figure 5, whole blood was collected and centrifuged, red blood cells were immediately reinfused into the pig, and up to 700 ml of plasma was collected and either filtered or purified using ClearPlasma™. The pigs were placed under anesthesia and venous blood was taken and introduced into the plasmapheresis system. Following this, red blood cells were returned to the animals and purified by ClearPlasma™. The plasma was filtered and finally the animals were rebled.

[0530] result:

[0531] [Table 15]

[0532] [Table 16]

[0533] [Table 17]

[0534] As expected, when animals were treated with Clexane, the amount of extraverted blood increased by 18 The data also showed that administration of one unit of fresh plasma (FP) to animals increased blood loss from 274 cc to 596 cc. Furthermore, depletion of plasminogen from the FP reduced the magnitude of blood loss from 596 cc to 128.7 cc.

[0535] Postmortem data indicate that PDP-treated animals did not develop thrombotic or thromboembolic events. No clots were noted in the lungs, heart, or spleen of PDP-treated animals. The conclusion regarding the absence of thromboembolic events is further supported by the absence of D-dimer in PDP-treated animals.

[0536] The data show that plasminogen-depleted plasma is an effective antihemorrhagic treatment, even in animals treated with Clexane. The data also show that plasminogen PDP is effective, even in animals treated with Clexane. Thus, PDP may be a good solution for patients on anticoagulant therapy who need to undergo emergency surgery. The data also suggest that patients on PDP can be treated with Clexane without the risk of increased bleeding; such a conclusion is appropriate for patients who need to be treated with anticoagulants to prevent or treat DVT in the postoperative period. The data also show that patients can be treated simultaneously with Clexane and PDP, thereby preventing both bleeding and thrombotic events.

[0537] Example 9 In vitro characteristics of plasminogen-depleted plasma (PDP) compared with FFP The coagulation parameters of plasminogen depleted plasma (PDP) and fresh frozen plasma (FFP) were calculated according to experimental procedures and compared as detailed in Table 17. A volume of 215 ml of fresh frozen plasma (FFP) was run through the ClearPlasma (denoted as PDP in Table 17; unprocessed plasma). (Compare with FFP for plasma.) Up to 80% of the PLG appears to be depleted in the PPD.

[0538] [Table 18]

[0539] All experiments were performed in triplicate, and statistics were calculated using Student's t-test (two-tailed, equal variance).

[0540] In addition, several tests on specific blood components were performed to determine the coagulation and biochemical status of treated and untreated plasma (tests on nutrient and protein content). The tests were performed at the Institute of Hematology and Biochemistry, Benay-zion. The results are shown in Table 18. There were no significant differences in biochemical parameters between FFP and PDP, therefore ClearPlasma It did not appear to affect the nutrient and / or protein content levels.

[0541] [Table 19]

[0542] All experiments were performed in triplicate. Statistics were calculated using Student's t-test (two-tailed, equal variance).

[0543] In addition to clots, lysis in both PPD and FFD was monitored by thromboelastography (TEG) as detailed in the Experimental Procedures section. The results are shown in Figure 7. PDP appears to abolish fibrinolytic activity in human plasma.

[0544] Example 10 Mouse Safety Study - Evaluation of Antifibrinolytic Technology in a Tail Bleeding Assay The study was carried out at Biocell Ltd. (Israel).

[0545] the purpose The purpose of this study was to investigate the safety of intravenous injection of plasminogen-free plasma in mice.

[0546] Study Endpoints The safety of the plasminogen-depleted plasma was determined by blood markers (blood counts, biochemistry) and clinical observations (skin, hair, eye / mucous membrane examination, nervous system, physical activity and general behavior). These parameters were measured before injection, 48 hours after injection and 7 days after injection.

[0547] animal 12 Animals Species / strain: Mouse: C57 Black Sex / number / age: Male, 10-12 weeks old Supplier: Harlan Laboratories, Israel

[0548] ethics committee This study was supported by the Israel Board for Animal Experiments. In compliance with the Israel Animal Welfare Act after approval by the Israeli government carried out.

[0549] Study design In this experiment, each mouse was injected with 200 μl of one of the treatments. 1. Healthy 12-week-old C57 black mice were anesthetized with a mixture of ketamine and xylazine (100 and 10 mg / kg, respectively) and their body weights were measured. 2. Mice were randomly divided into 3 different groups (4 mice per group) and injected with 200 μl of: 1. Saline - Group 1 2. Normal plasma – Group 2 3. Plasminogen-free plasma - Group 3 (ClearPlasma (C57 black mouse derived) Plasma filtered through a 50-mL filter. 3. 48 hours after injection, 2 mice from each group were sacrificed to determine the safety of this injection; blood was collected in EDTA K3 tubes for blood collection and sent for blood testing (4°C). 4. After 7 days, 2 mice from each group were sacrificed to determine the safety of the injection; blood was collected in EDTA K3 tubes for blood sampling and sent for blood testing (4°C). 5. Mouse blood was examined for complete blood count (CBC), LDH, AGT, ALT and complete biochemistry analysis.

[0550] Physiological markers demonstrate that intravenous injection of plasminogen-depleted plasma did not affect mouse physiology. Complete blood count (CBC), blood biochemistry, body weight, morphological appearance, and animal behavior were examined and found to be normal in all groups. Thus, according to the results of this study, the use of plasminogen-depleted plasma appears to be safe.

[0551] Bleeding test in mice Plasminogen depletion was also performed in mice: plasma from C57 black mice was subjected to ClearPlasma (control plasma was not treated). Then, plasminogen levels Eliza [Kit: ab198511-Plasminogen Total (PLG) Mouse ELISA Kit This was determined by the plasminogen elimination assay, which shows 97% depletion of plasminogen (as shown in the table below).

[0552] [Table 20]

[0553] A tail-cut bleeding study in mice was performed according to the procedure described in the Experimental Procedures section. This non-GLP study aimed to investigate the use of the ClearPlama™ device and the benefits of plasminogen-depleted plasma compared to normal plasma. The study was conducted at Biocell Ltd. (Israel).

[0554] the purpose To examine the effect of PDP on bleeding rate in mice.

[0555] Study Endpoints The amount of blood lost was graded using the following standard grading scheme:

[0556] [Table 21]

[0557] Briefly, mice were injected with saline or unprocessed plasma (FFP) or plasma lacking plasminogen (processed plasma or PDP). Animals were placed in a prone position. The distal 10 mm portion of the tail was cut with a scalpel. The tail was immediately immersed in a 50 mL tube containing isotonic saline pre-warmed to 37°C in a water bath. The tail was oriented vertically with the tip positioned approximately 2 cm below the horizontal plane of the body. Each animal was monitored for 20 min (to detect possible rebleeding events, even if bleeding had stopped).

[0558] Bleeding time The tails of the participating mice were clipped at 7 mm and the time it took for the bleeding to stop was measured. The study was performed in three groups, with one mouse from each treatment group per repetition. The results show that the PDP mice had the shortest bleeding times in all repetitions of the study except for the second one.

[0559] Bleeding amount The tails of the mice were clipped at 7 mm and the time it took for bleeding to stop was measured. The study was performed in three groups, with one mouse from each treatment group per repetition. Blood was collected, centrifuged, and A ruler was used to measure the size. The results show that PDP mice had the least amount of bleeding in all replicates of this study except for #2 (see Figure 8).

[0560] Blood cell pellet size 24 hours after bleeding test Blood cell pellets from the bleeding study were centrifuged and the supernatant aspirated. Pellet size was then measured using a ruler. Statistics were calculated using one-way ANOVA followed by post-hoc LSD / SCHELF (p<0.05 considered significant). Figure 9 shows the pellet size results from each mouse. Figure 10 summarizes the statistical analysis of the pellet size measurements. PDP appears to reduce bleeding in mice by over 40%. These results show that PDP (ClearPlasma™) significantly improved bleeding time and bleed time in treated mice compared to the control group. has been demonstrated to reduce blood loss and

[0561] Example 11 ClearPlasma reduces both plasminogen and tPA protein levels in pigs lower Female pigs underwent plasma apheresis under anesthesia using the Haemonet lcs mcs+ system. Two groups of pigs were studied: a control group and a test group. In the test group, the pigs' plasma was filtered with ClearPlasma. Both groups underwent the same procedure, i.e., volume of blood filtered: 700 ml, treatment with anticoagulant [Anticoagulant Citrate Dextrose Solution Mixture a], time: approximately 100 minutes, and volume of blood advanced (ml): approximately 700 ml of plasma was collected. After these procedures, the plasma was reinfused into the animals.

[0562] Plasma samples from each group were analyzed for plasminogen depletion (ELISA kit: ab108893 human plasminogen, as detailed in the Experimental Procedures section) and tPA depletion (wild type tPA sandwich ELISA# Technozym T-PA AG EDTA Elisa Kit 96 TC12007). Figure 1 As shown in Figures 1A to 11B, plasma samples filtered through ClearPlasma showed high levels of plasminogen and Both tPA and tPA appear to be depleted.

[0563] Example 12 Plasma pH and Conductivity the purpose To compare the pH and conductivity of PDP and donor plasma.

[0564] material Plasma: Plasma from healthy donors Terminally sterilized ClearPlasma™ devices Equipment: pH meter, conductivity meter

[0565] The bag containing human plasma was connected to the ClearPlasma™ device and the entire volume was then quenched for 1 hour. A volume (200 ml) was passed through the device into a receiving bag. The pH and conductivity of the PDP and plasma were measured. After the plasma was processed through the ClearPlasma™ device, as detailed in Table 21, , slight changes in pH and conductivity values ​​were observed.

[0566] [Table 22]

[0567] Example 13 Safety-related analysis · Blood compatibility Terminally sterilized ClearPlasma™ devices (polysaccharide polymers in a polycarbonate housing) The particles of Rimer have been tested by in vitro hemolysis test: whole blood hemoglobin concentration measurement (cyanmethemoglobin method). The test was performed by Envigo (Israel). The study was performed in accordance with the OECD Good Laboratory Practice (GLP) principles (as revised in 1997), ENV / MC / CHEM(98)17.

[0568] The in vitro hemolysis test measures whole blood hemoglobin concentration using the cyanmethemoglobin method. Pooled blood from three rabbits was incubated with a negative control (glass vial, batch: 16491) and a positive control (nitrile examination glove, batch: 41010104) and the test article (ClearPlasma™). Incubation was performed at 37±1°C for 3 hours. Afterwards, the hemoglobin content in the plasma was measured to determine hemolysis.

[0569] The % hemolysis of the negative control (glass vial, batch: 16491) was -0.5% and therefore graded as non-hemolytic.

[0570] The % hemolysis of the positive control (nitrile examination glove, batch: 41010104) was 91.1% and therefore the grade was assessed as hemolytic.

[0571] The negative and positive controls met the eligibility criteria and therefore the test was validated.

[0572] The percent hemolysis index of the test product ClearPlasma™ (batch number: 18-0001) was −0.3% and −0.1% and is considered to be non-hemolytic.

[0573] Under the conditions of this study, the calculated hemoglobin content of the ClearPlasma( Parts A and B of 18-0001 (Batch Number: 18-0001) are considered non-hemolytic.

[0574] Material-borne pyrogenicity testing The aim of this study was to provide general information regarding the detection of material-mediated pyrogenicity of plasminogen-depleted plasma.

[0575] This study was conducted by American Preclinical Services, LLC (MN, USA).

[0576] This study United States Pharmacopeia (USP) <151> Pyrogen Test Regulatory Standards, ISO 10993-11:2017 Biological Evaluation of Medical Devices ion of Medical Devices) Part 11: Tests for Systemic Toxicity This was carried out in accordance with.

[0577] A total of three animals were used. Baseline control body temperatures were established for each animal within 30 minutes prior to injection. The test article (plasminogen-depleted plasma, Lot No. 001) was incubated at 37±2°C. The mixture was warmed and injected intravenously into the lateral ear vein of each animal at 10 ml / kg within 10 minutes. The temperature of each animal was recorded at 30 minute intervals between 1 and 3 hours after injection. Based on the results of this study, the test article showed no evidence of material-mediated pyrogenicity in rabbits.

[0578] Bacterial endotoxin testing The objective of this study was to determine the water extracts prepared with terminally sterilized ClearPlasma™ devices and beads. The aim of the study was to estimate the concentration of bacterial endotoxin (a pyrogen) in the blood.

[0579] The study was conducted by Milouda & Migal Laboratories (Israel).

[0580] The device and the beads were tested separately.

[0581] The devices without beads were incubated for 1 hour with 50 ml LAL reagent water per sample, which had been heated to 37° C. prior to testing. Extracts from the samples were then tested for bacterial endotoxins using the kinetic turbidimetric LAL test method.

[0582] The results for the two samples tested were 0.00860EU / ml and 0.01380EU / ml.

[0583] Three vials of beads in 30% ethanol were then tested as follows: Each sample was diluted 1:10 in LAL Reagent Water and incubated for 1 hour at 15-30°C. Samples were centrifuged at 5,000 RPM for 10 minutes. The supernatants (dilution 1:10) were tested for bacterial endotoxins using the kinetic turbidimetric LAL test method. Results were <0.05EU / ml for each test.

[0584] Maximum summary results for [instrument + beads] are USP <161> The results were below the limit value (20 EU / device) established by: [0.01380EU / ml x 50ml] + [<0.05EU / ml x 27ml (to fill the device)] = 0.69EU / device + [<1.35]EU / device = <2.04EU / device

[0585] LAL testing is performed on each product batch as part of release testing.

[0586] In the method claims which follow, the alphanumeric and roman numerals used to designate the claim steps are provided for convenience only and do not imply any particular order to performing the steps.

[0587] Finally, it should be noted that the phrase "comprising" as used throughout the appended claims should be interpreted to mean "including, but not limited to."

[0588] While examples in accordance with the subject matter disclosed herein have been shown and disclosed, it will be understood that many changes can be made therein without departing from the spirit of the subject matter disclosed herein.

[0589] Example 14 Efficacy and Safety of ClearPlasma™ in Patients with Acute Upper Gastrointestinal Bleeding Clinical trials to determine A randomized, double-blind, controlled trial will be conducted to determine the safety and efficacy of PDP filtered by the ClearPlasma™ device in patients with acute upper gastrointestinal bleeding. This will be a non-inferiority The primary outcome was safety: thromboembolic events (venous or arterial). The study aimed to determine the following parameters: 1) Total blood loss after plasma transfusion [time frame: first 48 hours after surgery]. Blood loss is determined by RBC blood units transfused [time frame: 2 weeks], plasma units transfused [time frame: 2 weeks], hemoglobin drop-compared to baseline, platelet units transfused [time frame: 2 weeks] and rebleeding episodes during the 2-week follow-up; 2) Length of hospital stay [time frame: during the 2-week follow-up]; 3) mortality associated with plasma transfusion.

[0590] The study will be conducted at several centers (multicenter) in phase 1 / 2 with a total duration of 12 months (each patient will be in the study for 2 weeks). The study will include 2 arms: a) Transfusion of 250-500ml of PDP, + normal plasma if necessary; b) Normal plasma transfusion.

[0591] The patient population concerns patients with acute upper gastrointestinal bleeding (UGIB) diagnosed by melena / presence of blood in gastric lavage / hematemesis. The sample size is 30 patients (15 in each group). The sample size calculation is based on a review of medical literature in similar indications and clinical designs, with descriptive analysis.

[0592] Patients will be screened for eligibility for the study by evaluation of inclusion and exclusion criteria. Screening procedures will include collection of demographic data, medical history, physical examination, and vital signs.

[0593] To be included in the study, patients had to be aged 18 years or older, had a physician-diagnosed acute upper gastrointestinal bleeding (less than 24 hours), and provided written informed consent. The informed consent process followed the recommendations of ISO 14155:2011.

[0594] The following parameters will exclude patients from the study: pregnancy, administration of plasma infusions in the previous month, known renal failure creatinine clearance <30 ml / min, arterial or venous thrombosis in the last 3 months, history of allergic reaction to plasma, participation in another clinical trial, and anticoagulant treatment with warfarin, apixaban, rivaroxaban, dabigatran, low molecular weight heparin, etc.

[0595] The visit / assessment schedule was baseline, 8-12 hours after transfusion (including blood sample), the next morning (day 2), 72 hours, and 14 days. Baseline assessments included blood pressure, pulse, blood work including complete CBC, PT, INR, PTT, and biochemistry. The treatment schedule was a one-time transfusion, and the follow-up schedule was 12 hours, 24 hours, 72 hours, and 2 weeks after transfusion.

[0596] Administration of ClearPlasma™ should be based on ABO blood type compatibility. For this purpose, ClearPlasma™ Blood Type AB can be considered a universal plasma. ClearPlasma™ must be administered by intravenous infusion after thawing using an infusion set equipped with a filter. Aseptic technique must be used throughout the infusion.

[0597] Possible adverse effects are allergic reactions and fever. Patients may be forced to change their treatment. Participants may be withdrawn upon the occurrence of a serious adverse event that delays the study, withdrawal of consent, or at the discretion of the investigator.

[0598] Data from this study will be recorded on Case Report Forms (CRFs) in accordance with the Code of Federal Regulations to allow for the systematic capture, review, management, storage, analysis, and reporting of clinical research data.

[0599] The following study sites will be monitored: a) before the start of the study (i.e., the study start visit); b) Early in the study after the first patient enrollment and completion of the CRF; c) after 10–15 patients have been enrolled during the course of the study; d) After the last patient is enrolled (i.e., end-of-study visit).

[0600] Additional on-site monitoring visits may occur due to incomplete CRF reporting, poor data quality, or an excessive number of patient attrition or bias.

[0601] The study may also be subject to audit by the Sponsor or its designee, and investigation by appropriate regulatory authorities. Investigators must consent to audits or inspections of study-related records and must permit direct handling of source materials with due regard for data protection and medical confidentiality.

[0602] The study data will be imported into the CRF with all variables included according to the final approved protocol. The data will be compiled in compliance with ICH-GCP (Good Clinical Practices), ISO 14155:2011 Clinical Trials of Medical Devices in Human Subjects - Clinical investigation of medical devices for human subjects - Good Clinical Practice and requirements of 21 CFR Part 11 The information is collected, recorded, organized, and reported in accordance with FDA standards, such as:

Claims

1. A therapeutically effective amount of at least one blood and / or blood-derived product having reduced fibrinolytic activity, said blood and / or blood-derived product lacking plasminogen and tPA, and said product suitable for use in a method for treating, preventing, prophylactically preventing, ameliorating or inhibiting bleeding, hemostatic disorders and any bleeding or pathological condition related thereto in a subject in need thereof, said blood and / or blood-derived product being prepared by an ex vivo or in vitro method of depleting at least one fibrinolytic protein from one or more mammalian body fluids or any preparation thereof, said method comprising: (i) subjecting said one or more body fluids to an affinity depletion procedure specific for said at least one fibrinolytic protein; and (ii) recovering the body fluid depleted in said at least one fibrinolytic protein obtained in step (i); Including; the affinity depletion procedure comprises contacting the body fluid with an effective amount of a plurality of conjugates or a composition comprising the plurality of conjugates, or applying the body fluid to a device, set of devices, kit or system comprising the plurality of conjugates or composition, wherein each conjugate comprises a particle, at least one linker and at least one amino acid, derivative or analog thereof, the plurality of conjugates comprises at least two different conjugates, and the amino acid, derivative or analog thereof is 4-(aminomethyl)-cyclohexanecarboxylic acid (tranexamic acid), and the conjugate is (a) 【Chemical 1】 (In the formula, 【Chemistry 2】 represents a particle), (b) 【Chemistry 3】 (In the formula, 【Chemistry 4】 represents a particle) and (c) 【Chemistry 5】 (In the formula, 【Chemistry 6】 represents a particle) One of the A therapeutically effective amount of at least one blood and / or blood-derived product having reduced fibrinolytic activity. (a) the particles and / or linkers of the at least two different conjugates are different; (b) the particles have an average particle size of about 90 μm to about 150 μm; (c) the particles are at least one of polysaccharide beads, glass beads, cotton beads, plastic beads, nylon beads, latex beads, magnetic beads, paramagnetic beads, superparamagnetic beads, starch beads, silicon beads, PTFE beads, polystyrene beads, gallium arsenide beads, gold beads, or silver beads; and (d) the particles are agarose beads or sepharose beads; At least one of A therapeutically effective amount of at least one blood and / or blood-derived product with reduced fibrinolytic activity according to claim 1.

3. The conjugate, 【Chemistry 7】 (In the formula, 【Chemistry 8】 represents a particle), A therapeutically effective amount of at least one blood and / or blood-derived product with reduced fibrinolytic activity according to claim 1 or 2.

4. The device, a housing having at least one fluid inlet port and at least one fluid outlet port; Including; the housing comprises at least one chamber, the at least one chamber defining a control volume in fluid communication with the at least one fluid inlet port and the at least one fluid outlet port; the control volume contains a plurality of particle populations including at least a first population of first particles and a second population of second particles; - said first particles differ in size from said second particles; at least one of the first particle and the second particle is a conjugated particle conjugated to an amino acid, a derivative thereof, or an analog thereof, wherein the amino acid, a derivative thereof, or an analog thereof is tranexamic acid, the conjugated particle is defined by a plurality of conjugates or compositions, and each conjugate comprises a particle, at least one linker, and at least one amino acid, a derivative thereof, or an analog thereof, the plurality of conjugates comprises at least two different conjugates, and the amino acid, a derivative thereof, or an analog thereof is 4-(aminomethyl)-cyclohexanecarboxylic acid (tranexamic acid), and the conjugate is (a) 【Chemistry 9】 (In the formula, 【Chemistry 10】 represents a particle); (b) 【Chemistry 11】 (In the formula, 【Chemistry 12】 represents a particle); and (c) 【Chemistry 13】 (In the formula, 【Chemistry 14】 represents a particle) A therapeutically effective amount of at least one blood and / or blood-derived product with reduced fibrinolytic activity according to any one of claims 1 to 3, which is one of:

5. (a) The body fluid is at least one of whole blood, plasma, or a blood-derived product containing at least one coagulation factor; (b) the blood-derived product is at least one of whole blood, plasma, fresh frozen plasma (FFP), platelet-rich plasma (PRP), and cryoprecipitate; (c) the fibrinolytic protein is at least one of plasminogen and tissue plasminogen activator (tPA); (d) the method further comprises measuring the amount of plasminogen in the fibrinolytic protein-depleted body fluid collected in step (ii) by determining at least one of the clotting time and the total clot lysis time in the fibrinolytic protein-depleted body fluid; and / or (e) the method is for use in the preparation of at least one blood and / or blood-derived product having reduced fibrinolytic activity; A therapeutically effective amount of at least one blood and / or blood-derived product with reduced fibrinolytic activity according to any one of claims 1 to 4.

6. A therapeutically effective amount of at least one blood and / or blood-derived preparation having reduced fibrinolytic activity according to any one of claims 1 to 5, wherein the hemostatic disorder is a hereditary or acquired bleeding disorder.

7. (i) the inherited hemostatic disorder is a disorder caused by at least one of a deficiency of at least one coagulation factor and an undetermined bleeding tendency, or (ii) the acquired hemostatic disorder is at least one of bleeding caused by surgery, bleeding caused by trauma, acute gastrointestinal bleeding, bleeding associated with burns, hemorrhagic stroke, lung injury caused by emphysema and COPD, bleeding associated with childbirth, and bleeding caused by fibrinolytic or thrombolytic therapy; A therapeutically effective amount of at least one blood and / or blood-derived product with reduced fibrinolytic activity according to any one of claims 1 to 6.

8. A therapeutically effective amount of at least one blood and / or blood-derived preparation with reduced fibrinolytic activity as described in claim 7, wherein the deficiency of at least one coagulation factor is a deficiency of at least one of factor XI, factor X, factor V, factor VII, factor II (prothrombin), and factor I (fibrinogen).

9. A therapeutically effective amount of at least one blood and / or blood-derived product having reduced fibrinolytic activity for use in a method for treating, preventing, prophylactically preventing, ameliorating or inhibiting bleeding, hemostatic disorders and any bleeding or pathological condition related thereto in a subject in need thereof, wherein said blood and / or blood-derived product lacks plasminogen and tPA; The method comprises: (A) depleting at least one fibrinolytic protein from one or more mammalian body fluids or any preparation thereof, wherein said method comprises: (i) subjecting said one or more body fluids to an affinity depletion procedure specific for said at least one fibrinolytic protein; and (ii) recovering the body fluid depleted in said at least one fibrinolytic protein obtained in step (i); and (b) administering to the subject a fibrinolytic protein-depleted body fluid, wherein the affinity depletion procedure comprises contacting the body fluid with an effective amount of a plurality of conjugates or a composition comprising the plurality of conjugates, or applying the body fluid to a device, set of devices, kit, or system comprising the plurality of conjugates or composition, wherein each conjugate comprises a particle, at least one linker, and at least one amino acid, derivative, or analog thereof, the plurality of conjugates comprising at least two different conjugates, and the amino acid, derivative, or analog thereof is 4-(aminomethyl)cyclohexanecarboxylic acid (tranexamic acid), and the conjugate is (a) 【Chemistry 15】 (In the formula, 【Chemistry 16】 represents a particle); (b) 【Chemistry 17】 (In the formula, 【Chemistry 18】 represents a particle); and (c) 【Chemistry 19】 (In the formula, 【Chemistry 20】 represents a particle) One of the A therapeutically effective amount of at least one blood and / or blood-derived product with reduced fibrinolytic activity for use, comprising:

10. (a) the particles and / or the linkers of the at least two different conjugates are different; (b) the particles have an average particle size of about 90 μm to about 150 μm; (c) the particles are at least one of polysaccharide beads, glass beads, cotton beads, plastic beads, nylon beads, latex beads, magnetic beads, paramagnetic beads, superparamagnetic beads, starch beads, silicon beads, PTFE beads, polystyrene beads, gallium arsenide beads, gold beads, or silver beads; and (d) the particles are agarose beads or sepharose beads; 10. A therapeutically effective amount of at least one blood and / or blood-derived product with reduced fibrinolytic activity for use according to claim 9, which is at least one of:

11. The conjugate of claim 1 【Chemical 21】 (In the formula, ​ 11. A therapeutically effective amount of at least one blood and / or blood-derived product with reduced fibrinolytic activity for use according to claim 9 or 10, wherein the blood and / or blood-derived product is any one of the following:

12. The device, a housing having at least one fluid inlet port and at least one fluid outlet port; Including; the housing comprises at least one chamber, the at least one chamber defining a control volume in fluid communication with the at least one fluid inlet port and the at least one fluid outlet port; the control volume contains a plurality of particle populations including at least a first population of first particles and a second population of second particles; - said first particles differ in size from said second particles; at least one of the first particle and the second particle is a conjugate particle conjugated to an amino acid, a derivative thereof or an analog thereof, wherein the amino acid, a derivative thereof or an analog thereof is tranexamic acid, the conjugate particle is defined by a plurality of conjugates or compositions, and each conjugate comprises a particle, at least one linker and at least one amino acid, a derivative thereof or an analog thereof, the plurality of conjugates comprises at least two different conjugates, and the amino acid, a derivative thereof or an analog thereof is 4-(aminomethyl)-cyclohexanecarboxylic acid (tranexamic acid), and the conjugate is (a) 【Chemical 23】 (In the formula, 【Chemistry 24】 represents a particle); (b) 【Chemistry 25】 (In the formula, 【Chemical 26】 represents a particle); and (c) 【Chemical 27】 (In the formula, 【Chemical 28】 represents a particle) A therapeutically effective amount of at least one blood and / or blood-derived product with reduced fibrinolytic activity for use according to any one of claims 9 to 11, which is one of:

13. A therapeutically effective amount of at least one blood and / or blood-derived preparation having reduced fibrinolytic activity for use according to any one of claims 9 to 12, wherein the hemostatic disorder is an inherited or acquired bleeding disorder.

14. (i) The inherited hemostatic disorder is a disorder caused by at least one of a deficiency of at least one coagulation factor and an undetermined bleeding tendency, or (ii) A therapeutically effective amount of at least one blood and / or blood-derived product with reduced fibrinolytic activity for use according to any one of claims 9 to 13, wherein the acquired hemostatic disorder is at least one of bleeding caused by surgery, bleeding caused by trauma, acute gastrointestinal bleeding, bleeding associated with burns, hemorrhagic stroke, lung injury due to emphysema and COPD, bleeding associated with childbirth and bleeding resulting from fibrinolytic or thrombolytic therapy.

15. A therapeutically effective amount of at least one blood and / or blood-derived product with reduced fibrinolytic activity for use as described in claim 14, wherein the deficiency of at least one coagulation factor is a deficiency of at least one of factor XI, factor X, factor V, factor VII, factor II (prothrombin) and factor I (fibrinogen).

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