Compositions and methods for detecting hyperfibrinolysis and monitoring and guiding treatment

Abstract

This invention relates to compositions and methods for detecting hyperfibrinolysis in a blood sample using a reagent including surfactant, an exogenous plasmin substrate, and optionally poly-lysine. Methods for detecting a fibrinolytic abnormality in a subject, monitoring the effectiveness of a treatment for hyperfibrinolysis, and guiding treatment of a patient in need of treatment for bleeding are also provided.

Description

Attorney Docket No.5470.979.WO COMPOSITIONS AND METHODS FOR DETECTING HYPERFIBRINOLYSIS AND MONITORING AND GUIDING TREATMENT STATEMENT OF PRIORITY

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 679,292, filed August 5, 2024, the entire contents of which are incorporated by reference herein. FIELD OF THE INVENTION

[0002] This invention relates to methods and compositions for detecting endogenous hyperfibrinolysis using a poly-lysine matrix as a co-factor, a surfactant, and a plasmin substrate. Methods for detecting a fibrinolytic abnormality, monitoring the effectiveness of a treatment for hyperfibrinolysis, and guiding treatment for bleeding are also provided. BACKGROUND OF THE INVENTION

[0003] Trauma represents the leading cause of mortality among Americans aged 1 to 46 years. In the general population, trauma accounted for 224,935 deaths in the United States in 2021, as reported by the Centers for Disease Control and Prevention (CDC), ranking it as the fourth leading cause of death. Trauma management incurs substantial economic costs, amounting to $670 billion annually in healthcare expenses. Notably, one-third of trauma- related fatalities are attributable to hemorrhage.

[0004] Fibrinolysis is a system responsible for the breakdown of fibrin in blood clots, essential for controlling coagulation and restoring blood flow after thrombosis. The primary biochemical event in fibrinolysis is the formation of the tissue plasminogen activator (tPA)- plasminogen complex on the surface of fibrin. This system is tightly regulated by several serine protease inhibitors (SERPINs), mainly Plasminogen Activator Inhibitor-1 (PAI-1) and α2- antiplasmin. In certain situations, such as massive trauma, burn, childbirth, or major surgery, this system may switch into a hyperactive state known as hyperfibrinolysis. Hyperfibrinolysis has been associated with dramatically increased mortality due to bleeding in trauma. Antifibrinolytic agents such as tranexamic acid (TXA) have proven effective in reducing mortality in trauma and childbirth and in reducing blood loss during surgery. However, for antifibrinolytic treatment to be effective, it must be administered as soon as possible; TXA given ≤1 hour after injury is more protective than when given 1–3 hours after injury, and TXA given after 3 hours is associated with increased risk of death (Napolitano (2017) Trauma Surg.Attorney Docket No.5470.979.WO Acute Care Open 2(1):e000056). Clinical trials and retrospective analyses of trauma cohorts have revealed that the fibrinolytic response varies among patients, with rates of hyperfibrinolysis reportedly ranging from 18% to almost 50%, depending on the measurement approach. One retrospective analysis has shown that antifibrinolytic treatment of trauma patients with no fibrinolysis abnormalities on admission may be harmful and cause increased mortality in that subpopulation. However, since fibrinolysis is naturally a very slow process, conventional techniques for measuring fibrinolysis require too much time (40-60 minutes for less sensitive assays such as thromboelastography (TEG) and up to 30 hours for sensitive tests such as euglobulin clot lysis time (ECLT). Considering that every 15-minute delay in antifibrinolytic treatment leads to a 10% reduction in survival benefits, the utility of these techniques is impractical.

[0005] A number of assays are known to estimate hypofibrinolysis, i.e., an abnormally low rate of fibrinolysis. In such assays, an exogenous plasminogen activator is added thereby overcoming the inhibitory capacity of the sample to measure how the sample is resistant to fibrinolysis activation. See, e.g., WO 2023 / 074864.

[0006] However, a rapid test for measuring hyperfibrinolysis is needed to identify patients who will benefit from treatment with an antifibrinolytic agent, thereby leading to increased survival rates after bleeding events such as trauma. The present invention addresses this need in the art. SUMMARY OF THE INVENTION

[0007] The present invention is based on the development of a rapid and sensitive assay for measuring hyperfibrinolysis. The assay is useful for identifying subjects with hyperfibrinolysis and other fibrinolysis-related conditions. Thus, one aspect of the invention relates to a method for detecting endogenous hyperfibrinolysis in a blood sample, comprising combining a cell- free blood-derived sample with a reagent comprising about 0.05% to about 1.6% surfactant, an exogenous plasmin substrate, and optionally about 0.005% to about 0.6% poly-lysine, wherein the reagent has a pH in the range of about 7.2 to about 7.8; and measuring, optionally at a temperature in the range of about 37°C to about 45°C, lysis of the exogenous plasmin substrate by plasmin in the cell-free blood-derived sample, thereby detecting hyperfibrinolysis in the blood sample.

[0008] Another aspect of the invention relates to a method for detecting a fibrinolytic abnormality in a subject, comprising combining a cell-free blood-derived sample from the subject with a reagent comprising about 0.05% to about 1.6% surfactant, an exogenous plasminAttorney Docket No.5470.979.WO substrate, and optionally about 0.005% to about 0.6% poly-lysine, wherein the reagent has a pH in the range of about 7.2 to about 7.8; measuring, optionally at a temperature in the range of about 37°C to about 45°C, lysis of the exogenous plasmin substrate by plasmin in the cell- free blood-derived sample from the subject, and comparing the lysis of the exogenous plasmin substrate by plasmin in the cell-free blood-derived sample from the subject to a reference standard, wherein a difference between the lysis of the exogenous plasmin substrate by the plasmin in the cell-free blood-derived sample and the reference standard is indicative of a fibrinolytic abnormality.

[0009] A further aspect of the invention relates to a method of monitoring the effectiveness of a treatment for hyperfibrinolysis in a subject in need thereof, comprising obtaining a cell- free blood-derived sample from the subject at two or more timepoints before, during, and / or after the treatment; combining each of the cell-free blood-derived samples with a reagent comprising 0.05% to 1.6% surfactant, an exogenous plasmin substrate, and optionally about 0.005% to about 0.6% poly-lysine, wherein the reagent has a pH in the range of 7.2 to 7.8; and measuring, optionally at a temperature in the range of about 37°C to about 45°C, lysis of the exogenous plasmin substrate by plasmin in each of the cell-free blood-derived samples; wherein an increase or no change in lysis of the exogenous plasmin substrate by plasmin over time indicates that the treatment is ineffective and a decrease in lysis of the exogenous plasmin substrate by plasmin over time indicates that the treatment is effective.

[0010] Another aspect of the invention relates to a method of guiding treatment of a patient in need of treatment for bleeding, said method comprising combining a cell-free blood-derived sample from the patient with a reagent comprising 0.05% to 1.6% surfactant, an exogenous plasmin substrate, and optionally about 0.005% to about 0.6% poly-lysine, wherein the reagent has a pH in the range of 7.2 to 7.8; measuring, optionally at a temperature in the range of about 37°C to about 45°C, lysis of the exogenous plasmin substrate by plasmin in the cell-free blood- derived sample; and administering to the patient an antifibrinolytic agent if hyperfibrinolysis is detected.

[0011] A further aspect of invention provides a method for detecting endogenous tissue plasminogen activator (tPA) activity in a blood sample, comprising combining a cell-free blood-derived sample with a reagent comprising about 0.05% to about 1.6% surfactant, an exogenous plasmin substrate, and optionally about 0.005% to about 0.6% poly-lysine, wherein the reagent has a pH in the range of about 7.2 to about 7.8; and measuring, optionally at a temperature in the range of about 37°C to about 45°C, lysis of the exogenous plasmin substrateAttorney Docket No.5470.979.WO by plasmin in the cell-free blood-derived sample thereby detecting endogenous tPA activity in the blood sample.

[0012] A hyperfibrinolysis assay composition comprising a cell-free blood-derived sample being tested and a reagent comprising 0.05% to 1.6% surfactant, an exogenous plasmin substrate, and optionally about 0.005% to about 0.6% poly-lysine, wherein the reagent has a pH in the range of 7.2 to 7.8 and is optionally devoid of thrombin, tissue-type plasminogen activator (tPA), and / or urokinase-type plasminogen activator (uPA), is also provided.

[0013] A hyperfibrinolysis assay kit is further provided, which comprises a reagent comprising 0.05% to 1.6% surfactant, an exogenous plasmin substrate, and optionally about 0.005% to about 0.6% poly-lysine, wherein the reagent has a pH in the range of 7.2 to 7.8 and is optionally devoid of thrombin, tissue-type plasminogen activator (tPA), and / or urokinase- type plasminogen activator (uPA).

[0014] These and other aspects of the invention are set forth in more detail in the description of the invention below. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 shows the timing of blood draw and fibrinolysis phenotypes. The y-axis represents fibrinolytic activity, with the center “physiologic fibrinolytic activity” portion representing a balanced level of fibrinolysis in which a patient has a fibrinolysis generation related to tissue injury and shock, a physiological response to thrombin generation and tissue ischemia. The x-axis represents time from injury. The “early blood draw” box represents the first time a trauma patient is encountered by a pre-hospital provider and intravenous access is obtained, which in most urban settings is around 10-20 min following injury. At this time, laboratory analysis of a patient with hyperfibrinolysis would have evidence of elevated D-dimer and plasmin antiplasmin (PAP) levels with elevated fibrinolysis. The hyperfibrinolysis phenotype (HYPER) is the least common following injury and has the highest mortality. Those patients with fibrinolysis shutdown (SHUTDOWN) would also have elevated D-dimer and PAP levels due to prior fibrinolysis activation, but have subsequently shutdown the system and viscoelastic assessment of fibrinolysis activity would be low. The patients with fibrinolysis shutdown have a higher prevalence than hyperfibrinolysis but a lower mortality. Those patients with physiological fibrinolysis (PHYS) due to their injury would have an elevation of D-dimer and PAP level and a moderate amount of fibrinolysis. These patients have a mortality rate that is roughly half of the patients in fibrinolysis shutdown. Patients lacking elevated D-dimer and PAP are either not severely injured or have hypofibrinolysis (HYPO), which is defined as aAttorney Docket No.5470.979.WO lack of activation of the fibrinolytic response to injury, which is a separate etiology than fibrinolysis shutdown. See Moore et al. (2019) Anesth. Analg. 129(3):762-773; Moore et al. (2020) ANZ J. Surg.90(4):413-414. ICU indicates intensive care unit.

[0016] FIG. 2 provides an illustration of a Fibrinolytic Activity Screening Test (FAST) assay according to some embodiments. SDS, sodium dodecyl sulfate; EKK-AMC, Boc-Glu- Lys-Lys-AMC; HBS, HEPES-buffered saline; tPA, tissue-type plasminogen activator; PLG, plasminogen; PLN, plasmin.

[0017] FIG. 3 shows the sensitivity of the FAST assay to the inclusion of exogenous recombinant tPA.

[0018] FIG. 4 shows the sensitivity of the FAST assay to determining the need for blood product transfusion in trauma as compared to ECLT.

[0019] FIG.5 shows that the FAST assay has a high negative predictive value: low 5-min rate – low chance of transfusion.

[0020] FIG.6 shows that results of the FAST assay with fresh plasma samples. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention will now be described with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0023] Amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three letter code, both in accordance with 37 CFR §1.822 and established usage. See, e.g., PatentIn User Manual, 99-102 (Nov.1990) (U.S. Patent and Trademark Office).

[0024] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.Attorney Docket No.5470.979.WO

[0025] To illustrate further, if, for example, the specification indicates that a particular amino acid can be selected from A, G, I, L and / or V, this language also indicates that the amino acid can be selected from any subset of these amino acid(s) for example A, G, I or L; A, G, I or V; A or G; only L; etc. as if each such subcombination is expressly set forth herein. Moreover, such language also indicates that one or more of the specified amino acids can be disclaimed. For example, in particular embodiments the amino acid is not A, G or I; is not A; is not G or V; etc. as if each such possible disclaimer is expressly set forth herein. Definitions

[0026] The following terms are used in the description herein and the appended claims.

[0027] As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0028] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0029] The term “about,” as used herein when referring to a measurable value such as an amount of polypeptide, dose, time, temperature, enzymatic activity or other biological activity and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

[0030] As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”

[0031] The term “therapeutically effective amount” or “effective amount,” as used herein, refers to that amount of a composition, compound, or agent of this invention that imparts a modulating effect, which, for example, can be a beneficial effect, to a subject afflicted with a disorder, disease or illness, including improvement in the condition of the subject (e.g., in one or more symptoms), delay or reduction in the progression of the condition, prevention or delay of the onset of the disorder, and / or change in clinical parameters, disease or illness, etc., as would be well known in the art. For example, a therapeutically effective amount or effective amount can refer to the amount of a composition, compound, or agent that improves a conditionAttorney Docket No.5470.979.WO in a subject by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0032] “Pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) refers to a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms “carrier” or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate-buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents. As used herein, the term “carrier” encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.

[0033] “Pharmaceutically acceptable,” as used herein, means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual along with the compositions of this invention, without causing substantial deleterious biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. The material would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art (see, e.g., Remington's Pharmaceutical Science; 21sted.2005).

[0034] The term “modulate,” “modulates,” or “modulation” refers to enhancement (e.g., an increase) or inhibition (e.g., a decrease) in the specified level or activity.

[0035] The term “enhance” or “increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve- fold, or even fifteen-fold and / or can be expressed in the enhancement and / or increase of a specified level and / or activity of at least about 1%, 5%, 10%, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more.

[0036] “Inhibit” or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 1%, 5%, 10%, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity or amount (at most, an insignificant amount, e.g., less than about 10% or even 5%).Attorney Docket No.5470.979.WO

[0037] “Treat,” “treating” and similar terms as used herein in the context of treating a subject refer to providing medical and / or surgical management of a subject. Treatment may include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically undertaken in an effort to alter the course of a disease (which term is used to indicate any disease, disorder, syndrome, or undesirable condition warranting or potentially warranting therapy) in a manner beneficial to the subject. The effect of treatment may include reversing, alleviating, reducing severity of, delaying the onset of, curing, inhibiting the progression of, and / or reducing the likelihood of occurrence or recurrence of the disease or one or more symptoms or manifestations of the disease. A therapeutic agent may be administered to a subject who has a disease or is at increased risk of developing a disease relative to a member of the general population. In some embodiments a therapeutic agent may be administered to a subject who has had a disease but no longer shows evidence of the disease. The agent may be administered e.g., to reduce the likelihood of recurrence of evident disease. A therapeutic agent may be administered prophylactically, i.e., before development of any symptom or manifestation of a disease. “Prophylactic treatment” refers to providing medical and / or surgical management to a subject who has not developed a disease or does not show evidence of a disease in order, e.g., to reduce the likelihood that the disease will occur, delay the onset of the disease, or to reduce the severity of the disease should it occur. The subject may have been identified as being at risk of developing the disease (e.g., at increased risk relative to the general population or as having a risk factor that increases the likelihood of developing the disease.

[0038] Grammatical variations of “administer,” “administration,” and “administering” to a subject include any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like. “Concurrent administration,” “administration in combination,” “simultaneous administration,” or “administered simultaneously” as used herein, means that the compounds are administered at the same point in time, overlapping in time, or one following the other. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same pointAttorney Docket No.5470.979.WO in time. “Systemic administration” refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject’s body (e.g., greater than 50% of the body), for example through entrance into the circulatory or lymph systems. By contrast, “local administration” refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject's body. Administration includes self- administration and the administration by another.

[0039] “Subjects” according to the present invention include mammals, avians, reptiles, amphibians, and fish. Mammalian subjects include but are not limited to humans, non-human primates (e.g., cynomolgus monkeys, rhesus monkeys chimpanzees, baboons, etc.), and non- human mammals such as dogs, cats, mice, hamsters, rats, horses, cows, pigs, rabbits, sheep and goats. In some embodiments, the subject is a laboratory animal. Human subjects include neonates, infants, juveniles, adults, and geriatric subjects. In some embodiments, the subject in need of treatment may include those at risk of hyperfibrinolysis or developing hyperfibrinolysis. A subject having an increased risk of hyperfibrinolysis may include a patient in need of treatment for bleeding, e.g., a patient with a burn injury, hemophilia, vascular tumor, hemoptysis, thrombocytopenia, fibrinogen deficiency, menorrhagia, coagulation disorder, epistaxis, massive trauma, undergoing or having undergone childbirth, or surgery.

[0040] As used herein, “endogenous” refers to any material derived from or produced within an organism, cell, tissue, or system. By comparison, the term “exogenous” refers to any material introduced into or produced outside an organism, cell, tissue, or system.

[0041] As used herein, the term “polypeptide” or “protein” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced byAttorney Docket No.5470.979.WO recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.

[0042] By an “isolated” polypeptide is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purpose of the invention, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0043] As used herein, the term “fibrinolysis” means the breakdown of a blood clot due to the conversion of inactive plasminogen in the clot to active plasmin. During fibrinolysis, active plasmin breaks down the fibrin mesh holding the clot together. An abnormally low rate of fibrinolysis is referred to as “hypofibrinolysis”. By comparison, an abnormal enhancement of fibrinolytic activity is referred to as “hyperfibrinolysis,” which may result in increased and sometimes fatal bleeding. Hyperfibrinolysis may be caused by increased plasminogen activator (PA) such as tPA or urokinase (u)-PA, or decreased fibrinolytic system inhibitor such as PAI- 1, thrombin-activated plasminogen inhibitor (TAFI), and / or α2-antiplasmin levels. In conventional assays, such as ECLT test, hyperfibrinolysis is defined as ECLT <4.6 hours, the 2.5th percentile of the reference range. Hyperfibrinolysis may be acquired or congenital. Congenital reasons for hyperfibrinolysis are rare and include deficiency of alpha-2-antiplasmin (alpha-2-plasmin inhibitor) and deficiency in plasminogen activator inhibitor type 1 (PAI-1). The affected individuals show a hemophilia-like bleeding phenotype. Acquired hyperfibrinolysis can occur in patient with liver disease, patients with severe trauma, patients undergoing major surgical procedures, and patients with other conditions.

[0044] The theoretical time course of fibrinolysis changes the various phenotypes of fibrinolysis after severe injury (FIG.1). With severe injury and shock, the expected response is activation of the fibrinolytic system to counterbalance early hypercoagulability. This occurs early after injury and often before prehospital providers arrive on the scene. After initial prehospital resuscitation, the phenotypes of post-injury fibrinolysis emerge. Patients who develop acute fibrinolysis shutdown will have a rapid transition to a low fibrinolytic state, while patients with physiologic fibrinolysis will have a more gradual decline in fibrinolytic activity. The hypofibrinolytic phenotype will have a blunted response to trauma and retain low fibrinolysis activity early after injury. Early blood draws (within an hour of injury) can stratify patients into respective phenotypes except for hypofibrinolysis and fibrinolysis shutdown. After resuscitation, all phenotypes converge into a low fibrinolytic state due to a post-Attorney Docket No.5470.979.WO resuscitation acquired fibrinolysis resistance from plasminogen activator inhibitor 1 (PAI-1) elevation. Patients who have sustained hyperfibrinolytic after initial in-hospital resuscitation efforts are unlikely to be alive several hours after injury. Obtaining blood samples several hours after provides a feedback on successful resuscitation efforts, but differentiating a patient’s initial fibrinolytic phenotype based on viscoelastic hemostatic assays is not possible as all prior phenotypes have converged to a fibrinolytic resistant state.

[0045] An uncomplicated assay for use in patients with significant bleeding is needed to rapidly exclude hyperfibrinolysis mediated by tPA release and therefore predict severe bleeding that may require a blood transfusion. However, the detection of hyperfibrinolysis that is mediated by tPA release is often difficult. Moreover, to be beneficial to a patient who may need to be treated with an antifibrinolytic agent (e.g., during surgery or trauma), the detection of hyperfibrinolysis is preferably very rapid. Accordingly, the invention stems, in part, from an attempt to reduce the amount of time required to detect endogenous hyperfibrinolysis, and provides methods and reagents for rapidly detecting endogenous hyperfibrinolysis in a cell- free blood-derived sample. In one aspect, the invention provides an assay method for detecting endogenous hyperfibrinolysis in a blood sample by combining a cell-free blood-derived sample with a reagent comprising about 0.05% to about 1.6% surfactant, an exogenous plasmin substrate, and optionally about 0.005% to about 0.6% poly-lysine, wherein the reagent has a pH in the range of about 7.2 to about 7.8; and measuring, optionally at a temperature in the range of about 37°C to about 45°C, lysis of the exogenous plasmin substrate by plasmin in the cell-free blood-derived sample, thereby detecting endogenous hyperfibrinolysis in the blood sample. Advantageously, the assay method does not require the use of exogenous enzymes such as thrombin, urokinase-type plasminogen activator (uPA), and / or tPA, can be carried out in about 2 to 10 minutes, and is useful in identifying patients who are unlikely to require early transfusion or may require a massive transfusion, e.g., those at risk of early death from hemorrhage and should receive treatment with an anti-fibrinolytic agent. In addition, the assay method may be used as a rapid screen for hyperfibrinolysis in subjects with an uncharacterized bleeding disorder. The assay method can be performed using a small volume of cell-free blood- derived sample (e.g., about 40 to 80 microliters of plasma) and includes reagents that are non- proteinaceous, stable, and inexpensive, thereby rendering the assay adaptable to a point-of-care device.

[0046] In some embodiments, a cell-free blood-derived sample is taken from a source. The source can be any source including a donor bag or directly from a subject patient. In some embodiments, the cell-free blood-derived sample is from a human or other animal (e.g.,Attorney Docket No.5470.979.WO veterinary animal, laboratory animal, companion animal, or exotic animal). In some embodiments, blood is collected into blood collection tubes according to standard / common clinical practice. In some embodiments, the blood may be collected and used without anti- coagulant or, in other embodiments, may contain an anti-coagulant (such as citrate, heparin, low molecular weight heparins, synthetic pentasaccharides, EDTA, other calcium chelating agent, thrombin inhibitors, or the like). In some embodiments, the blood may be citrate blood (e.g., whole blood collected into a container containing 3.2% citrate). The blood sample may be processed to remove all or a portion of the cells in the blood sample (e.g., centrifugation to separate the blood cells from the liquid portion) thereby providing a “cell-free blood-derived sample,” “blood-derived sample,” or “blood product,” e.g., plasma, serum, treated serum, or a combination thereof. In some embodiments, the sample is a blood-derived sample from whole blood, e.g., plasma, anti-coagulated plasma, platelet-poor plasma, anti-coagulated platelet-poor plasma, cryoprecipitate, etc.

[0047] In some of the methods described herein, hyperfibrinolysis may be measured in a blood-derived sample where platelet function has been reduced (e.g., by treating the sample with a platelet inhibitor). For the methods herein, reduced platelet function does not mean that the blood-derived sample does not have any platelet function at all. Rather, the blood-derived sample with reduced platelet function simply has reduced platelet function as opposed to normal whole blood. For example, a blood-derived sample with reduced platelet function includes a blood-derived sample that has a platelet function that is at least 25% less, or at least 50% less, or at least 75% less, or at least 90% less platelet function than whole blood. Reduced platelet function can be assessed by a reduction in the aggregation of platelets to one another during blood clotting (e.g., in the presence of Kaolin and calcium).

[0048] In some embodiments, platelet function is reduced by contacting a blood-derived sample with a platelet function inhibitor. Non-limiting platelet function inhibitors include, but are not limited to, adenosine diphosphate (ADP) receptor inhibitors (e.g., clopidogrel, prasugrel, ticagrelor, ticlopidine), phosphodiesterase inhibitors (e.g., cilostazol), glycoprotein IIb / IIIa receptor inhibitors (e.g., abciximab, eptifibatide, and tirofiban), adenosine reuptake inhibitors (e.g., dipyridamole) and thromboxane inhibitors, including thromboxane synthase inhibitors and thromboxane receptor antagonists (e.g., tertroban). Any of these platelet function inhibitors (or combinations thereof) can be used in the methods described herein.

[0049] In some embodiments, the blood-derived sample is physically manipulated to reduce the number of platelets. For example, whole blood may be centrifuged (e.g., at 1000 rpm for 10 minutes) to remove some or most of the platelets. The platelet-rich plasma will float on theAttorney Docket No.5470.979.WO top of the blood in the supernatant. This supernatant can be removed (e.g., by aspiration) leaving the platelet-reduced or -poor plasma at the bottom of the tube. In another method, platelet-reduced blood may be obtained by contacting whole blood with platelet-specific antibodies attached to a solid surface. The platelets will selectively bind to the solid surface, and the platelet-reduced blood can be obtained. For example, antibodies that specifically bind to the glycoprotein IIb / IIIa receptor (which is expressed on platelets but not on red blood cells) may be used. As a further alterative, platelets may be removed from whole blood using a membrane filter (e.g., a 0.8 μm polycarbonate filter). In some embodiments, the cell-free blood- derived sample is free or substantially free of platelets. In some embodiments, a platelet-free or platelet-poor blood-derived sample is a sample with, e.g., less than 10 X 103platelets / μL, preferably less than 10 X 102platelets / μL, less than 100 platelets / μL, or most preferably an undetectable number of platelets (e.g., approximately zero platelets).

[0050] In another embodiment, the blood-derived sample being analyzed has a volume between 2 µL and 400 µL (e.g., from 2 µL to 10 µL, 10 µL to 20 µL, 15 µL to 50 µL, 50 µL to 100 µL, 100 µL to 250 µL, or from 200 µL to 500 µL). In still other embodiments, the sample being analyzed has a volume between 10 µL and 100 µL.

[0051] In some aspects, the blood-derived sample of choice is then mixed or combined with a reagent comprising about 0.05% to about 1.6% surfactant and an exogenous plasmin substrate, wherein the reagent has a pH in the range of about 7.2 to about 7.8. In other aspects, the blood-derived sample of choice is mixed or combined with a reagent comprising about 0.005% to about 0.6% poly-lysine, about 0.05% to about 1.6% surfactant and an exogenous plasmin substrate, wherein the reagent has a pH in the range of about 7.2 to about 7.8. The reagent may be provided in the form of a liquid or solid (e.g., lyophilized). The reagent may be prepared by combining solid and / or liquid forms of each of poly-lysine, surfactant, and exogenous plasmin substrate, and optionally drying the reagent (e.g., by lyophilization) if a solid reagent is desired. The reagent may be provided in a blood collection tube, vessel or container, e.g., as a dry coating on the bottom of a blood collection tube, vessel or container. When provided in the form of a solid (e.g., powder) or coating on the bottom of a tube, vessel or container, the reagent may be reconstituted, e.g., with water or buffer, prior to the addition of the blood-derived sample. Alternatively, reagent in the form of a solid (e.g., powder) or coating may be dissolved in the blood-derived sample. In some embodiments, blood is collected into a blood collection tube, vessel or container (optionally containing an anti- coagulant) according to standard / common clinical practice and a reagent solution is added to the blood. In some embodiments, blood is collected into a blood collection tube, vessel orAttorney Docket No.5470.979.WO container (optionally containing an anti-coagulant), processed to obtain a blood-derived sample (e.g., plasma, anti-coagulated plasma, platelet-poor plasma, or anti-coagulated platelet-poor plasma) according to standard / common clinical practice, and a solution of the reagent is added to the cell-free blood-derived sample.

[0052] In some embodiments, the step of combining a cell-free blood-derived sample with a reagent described herein involves adding the cell-free blood-derived sample of choice to lyophilized reagent, adding lyophilized reagent to the cell-free blood-derived sample of choice, or collecting the cell-free blood-derived sample of choice in a tube, vessel or container (e.g., a cup or a cuvette) containing lyophilized reagent. In some embodiments, the step of combining a cell-free blood-derived sample with a reagent described herein involves adding the cell-free blood-derived sample of choice to a solution of the reagent, adding a solution of the reagent to the cell-free blood-derived sample of choice, or collecting the cell-free blood-derived sample of choice in a tube, vessel or container containing a solution of the reagent. In some embodiments, the assay may employ a microfluidic device or multi-well plate (e.g., a black 96- well plate).

[0053] In some embodiments, the reagent has a pH in the range of about 7.2 to about 7.8, e.g., in the range of about 7.2, 7.3, 7.4 to about 7.5, 7.6, 7.7, or 7.8, or any range or value therebetween. In some embodiments, the pH is about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7 or about 7.8. In some embodiments, a reagent as described herein may be added to an anticoagulant in a blood collection tube and be reconstituted (e.g., prior to being mixed with a cell-free blood-derived sample) with a solution or buffer suitable for providing the reagent having a pH in the range of about 7.2 to about 7.8. Examples of suitable buffers include, but are not limited to, HEPES-buffered saline (HBS), phosphate-buffered saline (PBS), Tris- buffered saline (TBS), and the like. In some embodiments, the pH of the reagent is about 7.5.

[0054] One or more surfactants are included in the reagent at a low concentration to preferentially disrupt the interactions / conformations of fibrinolytic system inhibitors (e.g., PAI-1, TAFI, and / or α2-antiplasmin) thereby reducing the need to further fractionate the blood (e.g., plasma) sample or otherwise reduce or eliminate inhibitor activity. In some embodiments, the reagent includes, one, two, three, four, five, six, seven, eight or more different surfactants. In some embodiments, the one or more surfactants are independently non-ionic surfactants or ionic surfactants. In some embodiments, the reagent comprises, consists of, or consists essentially of one or more non-ionic surfactants. In some embodiments, the reagent comprises, consists of, or consists essentially of one or more ionic surfactants. In some embodiments, theAttorney Docket No.5470.979.WO reagent comprises, consists of, or consists essentially of one or more non-ionic surfactants and one or more ionic surfactants.

[0055] Non-ionic surfactants include, but are not limited to, t- octylpheoxypolyethoxyethanol (TRITON™ X-100), TERGITOL™ (poly(ethylene glycol-ran- propylene glycol) monobutyl ether) and other alcohol ethoxylates, polyoxyethylenesorbitan monolaurate (TWEEN® 20), nonylphenol polyoxyethylene ether (NONIDET® P10 or IGEPAL® CA-630), decyldimethylphosphine oxide (APO-10), cyclohexyl-n-ethyl-β-D- maltoside, cyclohexyl-n-hexyl-β-D-maltoside, cyclohexyl-n-methyl-β-D-maltoside, n- decanoylsucrose, n-decyl-β-D-glucopyranoside, n-decyl-β-D-maltopyranoside, n-decyl-β-D- thiomaltoside, digitonin, n-dodecanoyl sucrose, n-dodecyl-β-D-glucopyranoside, n-dodecyl-β- D-maltoside, polyoxyethylene (10) dodecyl ether (GENAPOL® C-100), isotridecanol polyglycol ether (GENAPOL® X-80), isotridecanol polyglycol ether (GENAPOL® X-100), heptane-1,2,3-triol, n-heptyl-β-D-glucopyranoside, n-heptyl-β-D-thioglucopyranoside, and combinations thereof. In some embodiments, the reagent comprises t- octylpheoxypolyethoxyethanol (TRITON™ X-100).

[0056] Ionic surfactants (e.g., anionic, cationic, or zwitterionic surfactants) include, but are not limited to, sodium cholate, sodium deoxycholate, sodium caprylate (octanoate), chenodeoxycholic acid, cholic acid, dehydrocholic acid, docusate sodium, docusate sodium salt, glycocholic acid hydrate, glycodeoxycholic acid monohydrate, glycolithocholic acid ethyl ester, N-lauroylsarcosine sodium salt, N-lauroylsarcosine, lithium dodecyl sulfate, calcium propionate, 1-octanesulfonic acid sodium salt, sodium 1-butanesulfonate, sodium chenodeoxycholate, sodium cholate hydrate, sodium 1-decanesulfonate, sodium 1- decanesulfonate, sodium deoxycholate, sodium deoxycholate monohydrate, sodium dodecylbenzenesulfonate, sodium dodecyl sulfate (SDS), sodium glycochenodeoxycholate, sodium glycocholate hydrate, sodium 1-heptanesulfonate, sodium hexanesulfonate, sodium 1- nonanesulfonate, sodium octyl sulfate, sodium pentanesulfonate, sodium 1-propanesulfonate hydrate, sodium taurodeoxycholate hydrate, sodium taurohyodeoxycholate hydrate, sodium tauroursodeoxycholate, taurocholic acid sodium salt hydrate, taurolithocholic acid 3-sulfate disodium salt, TRITON™ X-200, TRITON™ QS-15, TRITON™ QS-44, TRITON™ XQS- 20, TRIZMA® dodecyl sulfate, ursodeoxycholic acid, alkyltrimethylammonium bromide, amprolium hydrocholoride, benzalkonium chloride, benzethonium hydroxide, benzyldimethylhexadecylammonium chloride, benzyldodecyldimethylammonium bromide, choline p-toluenesulfonate salt, dimethyldioctadecylammonium bromide, dodecylethyldimethylammonium bromide, dodecyltrimethylammonium bromide,Attorney Docket No.5470.979.WO ethylhexadecyldimethylammonium bromide, Ggirard's reagent, hexadecylpyridinium bromide, hexadecylpyridinium chloride monohydrate, hexadecylpyridinium chloride monohydrate, hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium p- toluenesulfonate, hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium p- toluenesulfonate, methylbenzethonium chloride, myristyltrimethylammonium bromide, oxyphenonium bromide, N,N′,N′-polyoxyethylene (10)-N-tallow-1,3-diaminopropane, tetraheptylammonium bromide, tetrakis(decyl)ammonium bromide, thonzonium bromide, LUVIQUAT™ FC370, LUVIQUAT™ HM 552, LUVIQUAT™ HOLD, LUVIQUAT™ MS 370, LUVIQUAT™ PQ 11PN, sulfobetain-10, sulfobetain-16, 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS), and combinations thereof. In some embodiments, the reagent comprises sodium dodecyl sulfate (SDS).

[0057] In some embodiments, the surfactant is included in the reagent at a final concentration in the range of about 0.05% to about 1.6% (v / v), e.g., in the range of about 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, to about 0.45%, 0.5%, 0.55%, 0.60%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.05%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, or 1.6% (v / v), or any value or range therebetween. In some embodiments, the surfactant is included in the reagent at a final concentration of about 0.05%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.60%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%,1.0%, 1.05%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, or 1.6% (v / v). In some embodiments, more than one surfactant, e.g., two or more surfactants, is included in the reagent. In some embodiments, each of the two or more surfactants is independently included in the reagent at a final concentration in the range of about 0.01% to about 0.79% (v / v), e.g., in the range of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, to about 0.45%, 0.5%, 0.55%, 0.60%, 0.65%, 0.7%, 0.75%, 0.76%, 0.77%, 0.78%, 0.79% (v / v), or any value or range therebetween, wherein the total concentration of the two or more surfactants in the reagent is in the range of about 0.05% to about 1.6% (v / v), or any value or range therebetween. In some embodiments, the reagent includes a first surfactant and a second surfactant, wherein the ratio of the first surfactant to the second surfactant is in the range of about 1:10 to about 10:1, e.g., 10:1, 9;1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. In some embodiments, the reagent includes a first surfactant and a second surfactant, where the first surfactant and second surfactant are included in equal amounts. By way of illustration, a first surfactant is provided at a final concentrationAttorney Docket No.5470.979.WO of about 0.2% (v / v) and a second surfactant is provided at a final concentration of about 0.2% (v / v).

[0058] To detect plasmin activity and the rate of fibrinolysis, the reagent also includes an exogenous plasmin substrate. Plasmin is a trypsin-like enzyme that breaks down the fibrin matrix by cleaving Arg-Xaa and Lys-Xaa bonds. In some embodiments, the plasmin substrate is a peptide, e.g., a peptide comprising between 3 and 20 amino acid residues, between 3 and 10 amino acid residues, or between 3 and 6 amino acid residues (e.g., 3, 4, 5, 6, 7, 8, 9, 10 to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acid residues, or any value or range therebetween). In some embodiments, the plasmin substrate is a peptide comprising a carboxy-terminal arginine residue. In some embodiments, the plasmin substrate is a peptide comprising a carboxy-terminal lysine residue, e.g., a peptide having the sequence (Xaa)n-Lys, wherein n is an integer between 2 and 19. Exemplary peptides that may be used to measure plasmin activity include, but are not limited to, peptides having the amino acid sequence of Ala-Phe-Lys, Val- Leu-Lys, Glu-Lys-Lys, Glu-Phe-Lys, or Gly-Pro-Lys.

[0059] In some embodiments, the exogenous plasmin substrate comprises a detectable label such that lysis (cleavage) of the plasmin substrate causes release of the detectable label. In some embodiments, the detectable label is a chromophore such as 4-nitroanilide (pNA). In some embodiments, the exogenous plasmin substrate is a chromogenic peptide. Examples of chromogenic peptide substrates of plasmin include, but are not limited to, H-D-Val-Leu-Lys- pNA (S-2251; Friberger et al. (1978) Haemostasis 7(2-3):138-45), pyroGlu-Phe-Lys-pNA (CoaChrom Diagnostica GmbH, Austria), and Tosyl-Gly-Pro-Lys-pNA (Zedira GmbH, Germany). In some embodiments, the detectable label is a fluorescent label, e.g., a fluorophore or fluorochrome such as 7-amino-4-methylcoumarin (AMC; excitation 360 nm / emission 460 nm), 7-amino-4-trifluoromethylcoumarin (AFC; excitation 376 nm / emission 482 nm) and 6- amino-1-naphthalene-sulfonamide (ANSN; excitation 352 nm / emission 470 nm). In some embodiments, the exogenous plasmin substrate is a fluorogenic peptide. Examples of fluorogenic peptide substrates of plasmin include, but are not limited to, Suc-Ala-Phe-Lys- AMC (Bachem), H-D-Val-Leu-Lys-AFC (AnaSpec), D-Ala-Phe-Lys-ANSNH (Cryopep), Boc-Val-Leu-Lys-MCA (Bachem), Boc-Glu-Lys-Lys-AMC (Bachem), and Boc-Glu-Lys- Lys-MCA (Bachem). Electrochemical detection of plasmin activity via a ferrocene-modified peptide may also be used. See, e.g., Castrillo et al. (2015) Electroanalysis 27(3):789-798.

[0060] In some embodiments, the exogenous plasmin substrate is included in the reagent at a final concentration in the range of about 0.1 mM to about 1.0 mM, e.g., in the range of about 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, 0.35 mM, 0.4 mM, 0.45 mM, 0.5 mM, 0.55Attorney Docket No.5470.979.WO mM, to about 0.6 mM, 0.65 mM, 0.7 mM, 0.75 mM, 0.8 mM, 0.85 mM, 0.9 mM, 0.95 mM, 1.0 mM, or any value or range therebetween. In some embodiments, the exogenous plasmin substrate is included in the reagent at a final concentration of about 0.4 mM, about 0.45 mM, about 0.5 mM, about 0.55 mM, about 0.6 mM, about 0.65 mM, about 0.7 mM, about 0.75 mM, or about 0.8 mM.

[0061] In some aspects, poly-lysine is included in the reagent to provide a matrix for potentiating the activation of plasminogen and / or enhance the interactions between plasminogen, plasmin and tPA in the cell-free blood-derived sample. Use of a poly-lysine matrix as a co-factor saves time as there is no need to wait for fibrin formation. In addition, poly-lysine is easy to store, is stable, and facilitates standardization of the assay. In some embodiments, the poly-lysine is exogenous to the blood-derived sample. In some embodiments, the poly-lysine may be relatively pure. In some embodiments, the poly-lysine is poly-L-lysine, poly-D-lysine or a variant thereof. In some embodiments, the poly-lysine is poly-L-lysine or a variant thereof. A poly-lysine may include, e.g., poly(2-hydroxyethyl methacrylate)-lysine. In some embodiments, the reagent is devoid of poly-D-lysine. In some embodiments, the purity of the exogenous poly-lysine may be greater than or equal to about 90%, greater than or equal to about 91%, greater than or equal to about 92%, greater than or equal to about 93%, greater than or equal to about 94%, greater than or equal to about 95%, greater than or equal to about 96%, greater than or equal to about 97%, greater than or equal to about 98%, or greater than or equal to about 99%.

[0062] In some embodiments, the poly-lysine is a polymer having an average molecule weight in the range of about 30K to greater than 300K, e.g., about 30K to about 70K, about 70K to about 150K, or about 150K to about 300K. In some embodiments, the poly-lysine is a polymer having an average molecule weight of about 30K, about 40K, about 50K, about 60K, about 70K, about 80K, about 90K, about 100K, about 110K, about 120K, about 130K, about 140K, about 150K, about 160K, about 170K, about 180K, about 190K, about 200K, about 210K, about 220K, about 230K, about 240K, about 250K, about 260K, about 270K, about 280K, about 290K, about 300K, or more, or any value or range therebetween.

[0063] In some embodiments, the poly-lysine is included in the reagent at a final concentration in the range of about 0.005% to about 0.6% (w / v) poly-lysine, e.g., in the range of about 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.30% to about 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60% (w / v), or any value or range therebetween. In some embodiments, the poly-lysine is included in the reagent at a final concentration of about 0.005%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, aboutAttorney Docket No.5470.979.WO 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5% or about 0.6% (w / v).

[0064] According to some embodiments, the reagent comprises one or more surfactants, and a plasmin substrate in a buffer at pH 7.5. In other embodiments, the reagent comprises poly- lysine, one or more surfactants, and a plasmin substrate in a buffer at pH 7.5. In other embodiments, the reagent comprises poly-lysine, SDS, TRITON™ X-100, and a plasmin substrate in a buffer at pH 7.5. In a further embodiment, the reagent comprises 0.067% poly- lysine, 0.4% surfactant, and 0.6 mM plasmin substrate in a buffer at pH 7.5. In some embodiments, the reagent comprises 0.067% poly-lysine, 0.2% SDS, 0.2% TRITON™ X-100, and 0.6 mM plasmin substrate in a buffer at pH 7.5. In some embodiments, the reagent comprises 0.067% poly-lysine, 0.4% surfactant, and 0.6 mM Boc-Glu-Lys-Lys-AMC in HBS buffer at pH 7.5. In some embodiments, the reagent comprises 0.067% poly-lysine, 0.2% SDS, 0.2% TRITON™ X-100, and 0.6 mM Boc-Glu-Lys-Lys-AMC in HBS buffer at pH 7.5. In some embodiments, the reagent is devoid of exogenous thrombin, exogenous tPA, and / or exogenous uPA.

[0065] Once the cell-free blood-derived sample and reagent have been combined, lysis of the exogenous plasmin substrate by plasmin in the cell-free blood-derived sample is measured, e.g., with a fluorimeter or spectrometer including, e.g., a plate reader. In some embodiments, lysis of the exogenous plasmin substrate is continuously or intermittently measured (e.g., every 10, 20, 30, 40, 50, or 60 seconds). In some embodiments, lysis of the exogenous plasmin substrate by plasmin in the cell-free blood-derived sample is measured immediately after combining the same. In some embodiments, lysis of the exogenous plasmin substrate by plasmin is measured for a relatively short period of time. It should be understood that, in some embodiments, the duration of assays described herein may be less than the conventional time indicated for measurement of certain assay readout features (e.g., fibrinolysis features such as LY30). For instance, in some embodiments, measurements are taken for a period of about 2 to about 15 minutes, e.g., about 2 to 10 minutes, or any value or range therebetween. In some embodiments, lysis of the exogenous plasmin substrate is measured for no more than about 10 minutes, e.g., less than or equal to about 10 minutes, less than or equal to about 9 minutes, less than or equal to about 8 minutes, less than or equal to about 7 minutes, less than or equal to about 7 minutes, less than or equal to about 5 minutes, or less than or equal to about 3 minutes.

[0066] The assay may be performed at any suitable temperature. In some aspects, the assay may be performed at a temperature preferably at or above physiologically relevant temperatures. In some embodiments, the assay may be performed at the subject's bodyAttorney Docket No.5470.979.WO temperature (e.g., greater than or equal to about 37°C). In some embodiments, the assay may be performed at a temperature in the range of about 37°C to about 45°C, e.g., about 37°C, 38°C, 39°C, 40°C, 41°C to 42°C, 43°C, 44°C, 45°C, or any value or range therebetween. In some embodiments, the assay is performed at a temperature of about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, or about 45°C. In some embodiments, the assay is performed at about 42°C. In some aspects, the assay may be carried out in a temperature-uncontrolled environment, e.g., at room temperature. In some aspects, the assay may be carried out, e.g,, in an ultrasound-based device, e.g., at about 37°C or at room temperature.

[0067] Changes in fluorescence resulting from plasmin-mediated cleavage of a fluorogenic plasmin substrate or changes in color due to cleavage of a chromogenic plasmin substrate are measured thereby detecting hyperfibrinolysis in the blood-derived sample. In some embodiments, changes in fluorescence or color are detected over time and the rate of fibrinolysis is determined. In general, the assay results of a test sample may be compared to a healthy control(s) as well as a blank control. The blank control is not a sample from a subject. Comparison of kinetic measurements (e.g., numerical data, plots, or traces) to a reference standard (e.g. fibrinolysis of a healthy cell-free blood-derived sample, or standard curve) allows for the phenotypic assessment of a subject or predisposition toward a phenotype to be determined. Phenotypes may include normal or healthy fibrinolysis or hyperfibrinolysis. Parameters that may be used to determine a fibrinolytic abnormality using an assay described herein include the rate, amount of signal at a fixed time point, maximum signal, minimum signal and the like. In some embodiments, the assay uses a fluorescent plasmin substrate and uses relative fluorescence units (RFU) for assessing fibrinolytic activity, e.g., calculated as a percent increase from baseline (RFU@5min) / ((baseline RFU)*0.01). In some embodiments, a higher rate of fibrinolysis compared to a reference standard may be indicative of hyperfibrinolysis. In some embodiments, a cutoff based on C-statistics analysis of a cohort of blood-derived samples is used. By way of illustration, frozen plasma samples were shown to have a cutoff of 9.1 RFU / minute. However, the cutoff may be adjusted depending on the samples being tested, e.g., fresh versus previously frozen. In some embodiments, a rate of fibrinolysis in a cell-free blood-derived sample that is about 5% or more (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more, or any value or range therebetween) than the rate of fibrinolysis of a reference standard, may be indicative of hyperfibrinolysis in a cell-free blood-derived sample. In some aspects, a rate ofAttorney Docket No.5470.979.WO fibrinolysis comprising a cutoff of 121% plasmin activity from baseline may be indicative of hyperfibrinolysis in a cell-free blood-derived sample.

[0068] In clinical samples, a difference between lysis of the exogenous plasmin substrate by plasmin in a cell-free blood-derived sample and the reference standard may be indicative of a fibrinolytic abnormality. Accordingly, in some embodiments is provided a method for detecting a fibrinolytic abnormality in a subject, comprising combining a cell-free blood- derived sample from the subject with a reagent comprising about 0.05% to about 1.6% surfactant, an exogenous plasmin substrate, and optionally about 0.005% to about 0.6% poly- lysine, wherein the reagent has a pH in the range of about 7.2 to about 7.8; measuring, optionally at a temperature in the range of about 37°C to about 45°C, preferably at about 42°C, lysis of the exogenous plasmin substrate by plasmin in the cell-free blood-derived sample from the subject, and comparing the lysis of the exogenous plasmin substrate by plasmin in the cell- free blood-derived sample from the subject to a reference standard, wherein a difference between the lysis of the exogenous plasmin substrate by the plasmin in the cell-free blood- derived sample and the reference standard is indicative of a fibrinolytic abnormality. In some embodiments, more rapid cleavage of the plasmin substrate as compared to the reference standard is indicative of hyperfibrinolysis, which may lead to bleeding complications that can be treated by the administration of a therapeutic agent that strengthens a blood clot or slows the dissolution of a blood clot. Thus, in some embodiments, if hyperfibrinolysis is detected, the method further comprises administering to the subject at least one antifibrinolytic agent. In some embodiments, the subject is a trauma patient, optionally the subject has undergone a massive trauma, burn injury, childbirth or surgery or the subject has hemophilia, vascular tumor, hemoptysis, thrombocytopenia, fibrinogen deficiency, menorrhagia, coagulation disorder, orepistaxis. In some aspects, the subject is a female with acute obstetric coagulopathy (AOC). In some embodiments, lysis of the exogenous plasmin substrate by plasmin in the cell- free blood-derived sample from the subject is measured within 3 hours (e.g., 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1 hour, or 0.5 hour) of the trauma. In some embodiments, the antifibrinolytic agent is administered to the subject within 3 hours of the trauma, optionally about 2.5 hours, about 2 hours, about 1.5 hours, about 1 hour, or about 0.5 hour of the trauma.

[0069] A method of monitoring the effectiveness of a treatment for hyperfibrinolysis in a subject in need thereof is also provided, the method comprising the steps of obtaining a cell- free blood-derived sample from the subject at two or more timepoints before, during, and / or after the treatment; combining each of the cell-free blood-derived samples with a reagent comprising 0.05% to 1.6% surfactant, an exogenous plasmin substrate, and optionally 0.005%Attorney Docket No.5470.979.WO to 0.6% poly-lysine, wherein the reagent has a pH in the range of 7.2 to 7.8; and measuring, optionally at a temperature in the range of about 37°C to about 45°C, preferably at about 42°C, lysis of the exogenous plasmin substrate by plasmin in each of the cell-free blood-derived samples; wherein an increase or no change in lysis of the exogenous plasmin substrate by plasmin over time indicates that the treatment is ineffective and a decrease in lysis of the exogenous plasmin substrate by plasmin over time indicates that the treatment is effective. In some embodiments, a cell-free blood-derived sample from the subject is obtained before and during treatment. In some embodiments, a cell-free blood-derived sample from the subject is obtained before and after treatment. In some embodiments, a cell-free blood-derived sample from the subject is obtained before, during and after treatment. In some embodiments, the treatment comprises an antifibrinolytic agent.

[0070] A method for detecting endogenous tPA activity in a blood-derived sample is also provided, the method comprising combining a cell-free blood-derived sample with a reagent comprising about 0.05% to about 1.6% surfactant, an exogenous plasmin substrate, and optionally about 0.005% to about 0.6% poly-lysine, wherein the reagent has a pH in the range of about 7.2 to about 7.8; and measuring, optionally at a temperature in the range of about 37°C to about 45°C, preferably at about 42°C, lysis of the exogenous plasmin substrate by plasmin in the cell-free blood-derived sample thereby detecting endogenous tPA activity in the blood- derived sample. The assay method includes an artificial co-factor for tPA-mediated activation of plasminogen (i.e., poly-lysine) and measures plasmin as a read out. This is in contrast to conventional assays where exogenous tPA or uPA is added.

[0071] In some embodiments, a method of guiding treatment of a patient in need of treatment for bleeding is also provided, said method comprising the steps of combining a cell- free blood-derived sample from the patient with a reagent comprising 0.05% to 1.6% surfactant, an exogenous plasmin substrate, and optionally about 0.005% to about 0.6% poly- lysine, wherein the reagent has a pH in the range of 7.2 to 7.8; measuring, optionally at a temperature in the range of about 37°C to about 45°C, preferably at about 42°C, lysis of the exogenous plasmin substrate by plasmin in the cell-free blood-derived sample; and administering to the patient an antifibrinolytic agent if hyperfibrinolysis is detected. In some embodiments, the method provides for selecting an appropriate therapeutic, dose of therapeutic, and / or therapeutic treatment regime, e.g., duration of treatment. In some embodiments, the patient has a burn injury, hemophilia, vascular tumor, hemoptysis, thrombocytopenia, fibrinogen deficiency, menorrhagia, coagulation disorder, epistaxis, massive trauma, undergone childbirth, or surgery. In some embodiments, lysis of theAttorney Docket No.5470.979.WO exogenous plasmin substrate by plasmin in the cell-free blood-derived sample is measured within 3 hours (e.g., 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1 hour, or 0.5 hour) of the onset of bleeding.

[0072] In accordance with one or more methods provided herein, an antifibrinolytic agent is administered. “Antifibrinolytics” or “antifibrinolytic agents” include agents that are a class of drug that are inhibitors of fibrinolysis. Examples of antifibrinolytic agents include, but are not limited to, aminocaproic acid (ε-aminocaproic acid) and tranexamic acid. These lysine-like drugs interfere with the formation of the fibrinolytic enzyme plasmin from its precursor plasminogen by plasminogen activators (primarily t-PA and u-PA), which takes place mainly in lysine rich areas on the surface of fibrin. Additional anti-plasmin therapies include, e.g., nafamostat and camostat, which in addition to inhibiting plasmin activity, can act as anti- coagulants for disseminated intravascular coagulation. In addition, patients with mild to moderate forms of hemophilia, von Willebrand disease, uremic platelet dysfunction or chronic liver disease undergoing surgery may benefit from treatment with desmopressin acetate (DDAVP), which is a synthetic analog of vasopressin that shortens the bleeding time. Furthermore, aprotinin is a broad-spectrum serine protease inhibitor that inhibits factor XII, kallikrein, plasmin, and PAR1 receptors and has been shown to reduces bleeding. In some embodiments, an antifibrinolytic agent may be used alone or in combination with another antifibrinolytic agent or other therapeutic agent.

[0073] In some embodiments, the antifibrinolytic agent is a plasminogen inhibitor. In some embodiments, the plasminogen inhibitor is tranexamic acid (TXA). In some embodiments, the antifibrinolytic agent is aminocaproic acid (also known as Amicar, ε-aminocaproic acid, or 6- aminohexanoic acid). In some embodiments, the antifibrinolytic agent is aprotinin.

[0074] Inhibitors of fibrinolysis (including those listed above) are well known and can be used at known concentrations. In various embodiments, the antifibrinolytic agent is administered at a concentration in range of between about 2.5 µg / mL to about 250 µg / mL. For those inhibitors of fibrinolysis that are used therapeutically in human patients, dosages are well known and may be based on individual characteristics of the patient (e.g., state of overall health, weight, gender, and age).

[0075] Antifibrinolytic agents may be formulated according to known methods for preparing pharmaceutically useful compositions, such as by admixture with a pharmaceutically acceptable carrier. Suitable carriers and their formulations are described, for example, in Remington's Pharmaceutical Science (current edition). In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will containAttorney Docket No.5470.979.WO an effective amount of a first antifibrinolytic agent either alone, or in combination with a second antifibrinolytic agent, other therapeutic, and / or a suitable amount of carrier. Examples of suitable carriers, diluents, or excipients include, e.g., sterile water, physiological saline, glucose, or the like. The compositions may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.

[0076] In some embodiments, hyperfibrinolysis assay compositions and assay kits are disclosed. In some embodiments, a hyperfibrinolysis assay composition is provided, which comprises a cell-free blood-derived sample being tested and a reagent comprising 0.05% to 1.6% surfactant (e.g., SDS, TRITON™ X-100, TERGITOL™, or a combination thereof) and an exogenous plasmin substrate (optionally comprising a detectable label, e.g., a fluorescent label), wherein the reagent has a pH in the range of 7.2 to 7.8. In some embodiments, a hyperfibrinolysis assay composition is provided, which comprises a cell-free blood-derived sample being tested and a reagent comprising 0.005% to 0.6% poly-lysine, 0.05% to 1.6% surfactant (e.g., SDS, TRITON X-100, TERGITOL™, or a combination thereof), and an exogenous plasmin substrate (optionally comprising a detectable label, e.g., a fluorescent label), wherein the reagent has a pH in the range of 7.2 to 7.8. In some embodiments, a hyperfibrinolysis assay kit is provided that comprises a reagent comprising 0.05% to 1.6% surfactant (e.g., SDS, TRITON™ X-100, TERGITOL™, or a combination thereof) and an exogenous plasmin substrate, wherein the reagent has a pH in the range of 7.2 to 7.8. In some embodiments, a hyperfibrinolysis assay kit is provided that comprises a reagent comprising 0.005% to 0.6% poly-lysine, 0.05% to 1.6% surfactant (e.g., SDS, TRITON™ X-100, TERGITOL™, or a combination thereof), and an exogenous plasmin substrate, wherein the reagent has a pH in the range of 7.2 to 7.8. The kit may also include any solvents, solutions, buffer agents, acids, bases, salts, additives, etc. needed for the assay. In some aspects, the hyperfibrinolysis assay composition and assay kit is devoid of thrombin, tPA, and / or uPA. In some cases, the kits include a sample collection container free of a subject sample for testing and configured to contain such sample, which container also contains the reagent comprising 0.05% to 1.6% surfactant, an exogenous plasmin substrate, and optionally 0.005% to 0.6% poly-lysine. In some embodiments, the sample collection container is coated with or otherwise includes the reagent in dry (lyophilized) form. In some embodiments, the sample collection container includes the reagent in liquid form. In some embodiments, the assay compositions or kits may be used to perform a hyperfibrinolytic activity screening test as provided in one orAttorney Docket No.5470.979.WO more of the methods described herein. The kit may also include instructions on how to use the materials in the kit. The subject sample is typically provided by the user of the kit. In some embodiments, the assay uses a microfluidic device.

[0077] The hyperfibrinolysis assays described herein may be used for a wide variety of clinical applications. Advantageously, a Fibrinolytic Activity Screening Test or FAST assay described herein can identify patients at high-risk for severe bleeding and death from too much fibrinolysis (hyperfibrinolysis), providing answers in, for example, 5-10 minutes that can be used by clinicians to make life-saving, time-dependent, personalized medical decisions regarding the administration of antifibrinolytic therapies and / or specific blood products targeted at a patient's specific pathologic abnormality. In some embodiments, the hyperfibrinolysis assays described herein may be used in the treatment and / or diagnosis of cardiovascular disease (e.g., myocardial infarction, cerebrovascular accident), cancer, traumatic injury, liver disease, pre- and post-organ transplant, obstetrics, gynecology, end- stage renal disease, hemodialysis, and diseases requiring or potentially requiring any surgical intervention where bleeding is a risk, as well as general medical screening and wellness evaluations (e.g., routine physical examinations). In some embodiments, the hyperfibrinolysis assays herein may be used to assess an exacerbation of postpartum hemorrhage (PPH), e.g., associated with acute obstetric coagulopathy (AOC). AOC is characterized by very high plasmin / antiplasmin complexes (R=0.8) and rapid depletion of functional fibrinogen and factor V. Detecting the presence of AOC provides for early intervention, e.g., tranexamic acid administration and the optimal approach for replacing coagulation factors, thereby preventing worsening of PPH.

[0078] Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention. EXAMPLE 1: Assessing Parameters of FAST Assay

[0079] Current techniques for assessing fibrinolysis are not rapid enough to be used as a decision-making tool in trauma management, as even a one-hour delay in decision-making causes a 40% reduction in antifibrinolytic treatment efficacy. Moreover, many assays (e.g., turbidity, plasmin generation, halo, and HYPHEN GFC) use exogenous tPA to expedite the readout, leading to a loss of sensitivity to endogenous fibrinolysis. Additionally, using active enzymes such as tPA complicates point-of-care applications, as active enzymes require specialAttorney Docket No.5470.979.WO storage conditions (e.g., -80°C freezers) and may lose activity over time, leading to standardization issues.

[0080] We have developed a novel approach, referred to herein as the Fibrinolytic Activity Screening Test (FAST), for measuring fibrinolysis that is rapid (5 minutes readout) and sensitive to bleeding outcomes in trauma. This method is based on observations that: (a) poly- lysine can replace fibrin as a matrix for tPA-plasminogen complex formation, eliminating the need to rely on clotting thereby saving time and avoiding the use of active enzymes to initiate clotting; (b) a combination of detergents such as SDS and TRITON™-X100, at low concentrations, has been demonstrated to preferentially affect SERPINs' conformation without loss of target enzyme activity and may even release some of the active enzymes from the enzyme-inhibitor complex; and (c) plasmin and tPA activity can be maximized at temperatures higher than normal body temperatures and at above physiological pH (FIG.2). Accordingly, to prepare a reagent useful in rapidly assessing fibrinolysis, multiple variables were tested including (a) different types of poly-lysine (poly-L-lysine, poly-D-lysine); (b) various poly- lysine concentrations, ranging from 0.005% to 0.6% final; (c) different SDS and TRITON™- X100 concentrations, ranging from 0.01 to 0.4%; (d) various combinations of SDS and TRITON™-X100 (SDS alone, TRITON™-X100 alone, or both); (e) different pH levels, ranging from 6.9 to 7.8; and (f) different temperatures, ranging from 25°C to 45°C.

[0081] Based upon this analysis, an assay was developed that allows for the fastest detection of fibrinolysis with maximum sensitivity to endogenous fibrinolytic activity. For the FAST assay, 40 µL of the plasma sample is mixed in a black 96-well plate (non-binding) with 80 µL of the pre-made reagent solution (buffered to pH 7.5) containing 1000 µL poly-L-lysine (0.1% w / v stock solution), 30 µL SDS (0.2% final), 160 µL TRITON™-X100 (0.2% final), 6 µL of a specific plasmin fluorogenic substrate (Boc-EKK-AMC, 0.6 mM final), 150 µL 1X HBS and 150 µL 10X HBS (pH 7.45). The plasma sample and reagent are mixed 4 times with a pipette to homogeneity. Immediately afterward, kinetic measurements of fluorescent signal generation were taken every 30 seconds at 360 excitation / 460 emission in a pre-heated plate-reader at 42°C for 5 minutes.

[0082] The FAST assay was tested to determine sensitivity to exogenous tPA. To normal pooled plasma was added 1000 pM, 100 pM, 10 pM or 0 pM recombinant tPA (rtPA). The plasma samples were mixed with the reagent containing poly-L-lysine, 0.2% SDS, 0.2% TRITON™-X100, Boc-EKK-AMC substrate and HBS (pH 7.5) in a 96-well black plate. Fluorescence (excitation / emission 360 / 460) was immediately read every 30 seconds in a plate reader at 42°C (FIG.3).Attorney Docket No.5470.979.WO

[0083] To evaluate the FAST assay in comparison with other fibrinolytic parameters measured by different approaches and to assess its association with bleeding outcomes in trauma, we used a cohort of 161 trauma patients collected at the Mayo Clinic (obtained on admission, prior to any treatment) and 42 normal control individuals collected at UNC Chapel Hill and the Mayo Clinic. As a bleeding outcome in the trauma cohort, we used the need for blood product transfusion during hospitalization. Plasma samples were mixed with the reagent containing poly-L-lysine, 0.2% SDS, 0.2% TRITON™-X100, Boc-EKK-AMC substrate and HBS (pH 7.5) in a 96-well black plate. Notably, no rtPA was added. Fluorescence (excitation / emission 360 / 460) was immediately read every 30 seconds in a plate reader at 42°C The FAST readout showed good correlations with fibrinolytic parameters such as ECLT, plasmin-antiplasmin complexes, and D-Dimer. Moreover, the test demonstrated high sensitivity to the need for blood product transfusion (sensitivity 0.902, 95%CI 0.720-0.957) and a high negative predictive value (NPV 0.924, 95%CI 0.835-0.967) (FIGS. 4-6). This indicates that the absence of low fibrinolytic activity by the FAST assay was associated with a very low chance of developing massive bleeding requiring blood product administration during hospitalization. Without being bound by theory, it is believed that patients who demonstrate low fibrinolytic activation by the FAST assay and thus have a low risk of bleeding are candidates for not receiving antifibrinolytic treatment such as tranexamic acid (TXA). We believe that this approach in management can reduce the additional mortality associated with TXA treatment in trauma patients with normal fibrinolysis. EXAMPLE 2: Exemplary FAST Assay Protocol

[0084] Reagents and Supplies: CLS3356 - Corning®96 Well Storage Microplate round bottom, black polypropylene. Poly-L lysine (0.1% w / v stock). SDS (10% stock). TRITON™- X100 (make a 2% stock in advance, must be fully dissolved before use). Boc-Glu-Lys-Lys- AMC plasmin substrate (Bachem) (150 mM stock – dilute in H2O+DMSO 50:50, water first (heat to 37°C and mix well, then same volume of pre-heated, freshly opened 100% DMSO); make 20 µL aliquots and store at -20°C.10x HBS pH 7.5: 0.1M HEPES, 1.5 M NaCl in diH2O; adjust pH to 7.5 with HCl; store at room temperature in the dark; and check pH before use.

[0085] Samples: Citrated, platelet-poor plasma (prepared by International Society on Thrombosis and Haemostasis (ISTH) recommendations). Avoid using samples that were previously thawed, as it will decrease the fibrinolytic activity.

[0086] Preparation of the main components of the assay:Attorney Docket No.5470.979.WO 1) To pre-make four FAST plates (rows A to E) (enough for 120 samples, 30 samples per plate in duplicates). a. Further dilute the substrate in freshly opened 100% DMSO – 1040 µL DMSO + 80 µL stock substrate. Mix well. b. Using an automatic dispenser pipette, load 4 μL of diluted substrate into each well from A1 to E12 in four plates. Seal plates with plate sealing film and mark the filled wells. Label wells “for FAST,” add the date, and your initials. Store at +4°C for up to 1 month. (if fresh DMSO was used, the solution with the substrate should freeze at +4°C). 2) To pre-make the master mix: • 200 mL poly-L lysine (0.1%) • 34 mL diH2O • 30 mL 10xHBS (pH 7.5) • 6 mL SDS (10%) • 30 mL TRITON™-X100 (2%) Mix well but avoid excessive shaking. The solution will appear cloudy. Store at room temperature in the dark for up to 3 months. Gently mix before each use.

[0087] Experimental protocol: 1) Enter the settings for the plate reader: a. Set the temperature to 42°C (preset in advance, to allow the plate reader to reach 42°C). b. Add a 40-second orbital shake (mid-fast) before the first reading. c. Set up a kinetic, fluorometric reading: • duration 5 minutes • interval 1 minute • excitation 360nm • emission 460nm. 2) Thaw the plasma at room temperature. For 400-500 μL aliquots, thaw for 30 minutes. Mix the plasma with a pipette to redissolve potential precipitate. 3) Bring the FAST plate from the +4°C refrigerator. 4) Load 40 μL of plasma into the FAST plate. 5) Gently shake the bottle with the master mix (without foaming). 6) Add 80 μL of the “master mix” with a multichannel pipette, avoiding bubbles. Since the activity starts very quickly, try to load the whole plate in less than 1 minute. When theAttorney Docket No.5470.979.WO reading is finished, transfer the kinetic data to MICROSOFT EXCEL® and process it using the following formula: =[cell at 5min] / ([cell at 1 minute] / 100). Samples without hyperfibrinolysis should have a FAST plasmin activity value between 90 and 120.

[0088] The foregoing examples are illustrative of the present invention, and are not to be construed as limiting thereof. Although the invention has been described in detail with reference to preferred embodiments, variations and modifications exist within the scope and spirit of the invention as described and defined in the following claims.

Claims

Attorney Docket No.5470.979.WO WHAT IS CLAIMED IS:

1. A method for detecting endogenous hyperfibrinolysis in a blood sample, comprising: combining a cell-free blood-derived sample with a reagent comprising about 0.05% to about 1.6% surfactant and an exogenous plasmin substrate, wherein the reagent has a pH in the range of about 7.2 to about 7.8; and measuring lysis of the exogenous plasmin substrate by plasmin in the cell-free blood- derived sample thereby detecting endogenous hyperfibrinolysis in the blood sample.

2. The method of claim 1, wherein the blood-derived sample is obtained from a subject.

3. A method for detecting a fibrinolytic abnormality in a subject, comprising: combining a cell-free blood-derived sample from the subject with a reagent comprising about 0.05% to about 1.6% surfactant and an exogenous plasmin substrate, wherein the reagent has a pH in the range of about 7.2 to about 7.8; measuring lysis of the exogenous plasmin substrate by plasmin in the cell-free blood- derived sample from the subject, and comparing the lysis of the exogenous plasmin substrate by plasmin in the cell-free blood- derived sample from the subject to a reference standard, wherein a difference between the lysis of the exogenous plasmin substrate by the plasmin in the cell-free blood-derived sample and the reference standard is indicative of a fibrinolytic abnormality.

4. The method of claim 3, wherein the fibrinolytic abnormality is hyperfibrinolysis.

5. The method of claim 4, wherein the subject is a trauma patient, optionally the subject has undergone a massive trauma, burn injury, childbirth, or surgery.

6. The method of claim 5, wherein lysis of the exogenous plasmin substrate by plasmin in the cell-free blood-derived sample from the subject is measured within 3 hours of the trauma.Attorney Docket No.5470.979.WO 7. The method of any one of claims 4-6, wherein if hyperfibrinolysis is detected, the method further comprises administering to the subject at least one antifibrinolytic agent.

8. The method of claim 7, wherein the antifibrinolytic agent is administered to the subject within 3 hours of the trauma.

9. A method of monitoring the effectiveness of a treatment for hyperfibrinolysis in a subject in need thereof, comprising: obtaining a cell-free blood-derived sample from the subject at two or more timepoints before, during, and / or after the treatment; combining each of the cell-free blood-derived samples with a reagent comprising 0.05% to 1.6% surfactant and an exogenous plasmin substrate, wherein the reagent has a pH in the range of 7.2 to 7.8; and measuring lysis of the exogenous plasmin substrate by plasmin in each of the cell-free blood-derived samples; wherein an increase or no change in lysis of the exogenous plasmin substrate by plasmin over time indicates that the treatment is ineffective and a decrease in lysis of the exogenous plasmin substrate by plasmin over time indicates that the treatment is effective.

10. The method of claim 9, wherein the treatment comprises an antifibrinolytic agent.

11. A method of guiding treatment of a patient in need of treatment for bleeding, said method comprising: combining a cell-free blood-derived sample from the patient with a reagent comprising 0.05% to 1.6% surfactant and an exogenous plasmin substrate, wherein the reagent has a pH in the range of 7.2 to 7.8; measuring lysis of the exogenous plasmin substrate by plasmin in the cell-free blood- derived sample; and administering to the patient an antifibrinolytic agent if hyperfibrinolysis is detected.Attorney Docket No.5470.979.WO 12. The method of claim 11, wherein the patient has a burn injury, hemophilia, vascular tumor, hemoptysis, thrombocytopenia, fibrinogen deficiency, menorrhagia, coagulation disorder, epistaxis, massive trauma, undergone childbirth, or surgery.

13. The method of claim 11 or claim 12, wherein lysis of the exogenous plasmin substrate by plasmin in the cell-free blood-derived sample is measured within 3 hours of the onset of bleeding.

14. The method of any one of claims 11-13, wherein the antifibrinolytic agent is administered to the subject within 3 hours of the onset of bleeding.

15. A method for detecting endogenous tissue plasminogen activator (tPA) activity in a blood sample, comprising: combining a cell-free blood-derived sample with a reagent comprising about 0.05% to about 1.6% surfactant and an exogenous plasmin substrate, wherein the reagent has a pH in the range of about 7.2 to about 7.8; and measuring lysis of the exogenous plasmin substrate by plasmin in the cell-free blood- derived sample thereby detecting endogenous tPA activity in the blood sample.

16. The method of any preceding claim, wherein lysis of the exogenous plasmin substrate is measured for about 2 to about 15 minutes, optionally lysis of the exogenous plasmin substrate is measured for no more than about 10 minutes.

17. The method of any preceding claim, wherein the cell-free blood-derived sample is free of platelets.

18. The method of any preceding claim, wherein the surfactant comprises sodium dodecyl sulfate, polyethylene glycol tert-octylphenyl ether, poly(ethylene glycol-ran- propylene glycol) monobutyl ether, or a combination thereof.

19. The method of any preceding claim, wherein lysis of the plasmin substrate releases a detectable label.

20. The method of claim 19, wherein the detectable label is a fluorescent label.Attorney Docket No.5470.979.WO 21. The method of claim 20, wherein the plasmin substrate comprises a fluorogenic peptide, optionally a peptidyl-4-methylcoumaryl-7-amide (MCA) or 7-amino-4- methylcoumarin (AMC), comprising a carboxy-terminal lysine residue.

22. The method of claim 21, wherein the plasmin substrate comprises Boc-Val-Leu- Lys-MCA, Boc-Glu-Lys-Lys-MCA, or Boc-Glu-Lys-Lys-AMC.

23. The method of any one of claims 7, 8, or 10-22, wherein the antifibrinolytic agent is aminocaproic acid, tranexamic acid, nafamostat, aprotinin, or a combination thereof.

24. The method of any preceding claim, wherein the reagent further comprises about 0.005% to about 0.6% poly-lysine.

25. The method of any preceding claim, wherein lysis of the exogenous plasmin substrate by plasmin is measured at a temperature in the range of about 37°C to about 45°C.

26. The method of any preceding claim, wherein the reagent is devoid of thrombin, tissue-type plasminogen activator (tPA), and / or urokinase-type plasminogen activator (uPA).

27. A hyperfibrinolysis assay composition comprising a cell-free blood-derived sample being tested and a reagent comprising 0.05% to 1.6% surfactant and an exogenous plasmin substrate, wherein the reagent has a pH in the range of 7.2 to 7.

8.

28. The hyperfibrinolysis assay composition of claim 27, wherein the cell-free blood-derived sample is free of platelets.

29. A hyperfibrinolysis assay kit comprising a reagent comprising 0.05% to 1.6% surfactant and an exogenous plasmin substrate, wherein the reagent has a pH in the range of 7.2 to 7.

8.

30. The hyperfibrinolysis assay composition of claim 27 or claim 28, or the hyperfibrinolysis assay kit of claim 29, wherein the reagent further comprises about 0.005% to about 0.6% poly-lysine.Attorney Docket No.5470.979.WO 31. The hyperfibrinolysis assay composition of claim 27, 28, or 30 or the hyperfibrinolysis assay kit of claim 29 or 30, wherein the reagent is devoid of thrombin, tissue- type plasminogen activator (tPA), and / or urokinase-type plasminogen activator (uPA).

32. The hyperfibrinolysis assay composition of claim 27, 28, 30 or 31, or the hyperfibrinolysis assay kit of claim 29, 30 or 31, wherein the surfactant comprises sodium dodecyl sulfate, polyethylene glycol tert-octylphenyl ether, poly(ethylene glycol-ran- propylene glycol) monobutyl ether, or a combination thereof.

33. The hyperfibrinolysis assay composition of any one of claims 27, 28, or 30-32, or the hyperfibrinolysis assay kit of claim 29 or 30-32, wherein the plasmin substrate comprises a detectable label.

34. The hyperfibrinolysis assay composition or hyperfibrinolysis assay kit of claim 33, wherein the detectable label is a fluorescent label.

35. The hyperfibrinolysis assay composition or hyperfibrinolysis assay kit of claim 34, wherein the plasmin substrate comprises a fluorogenic peptide, optionally a peptidyl-4- methylcoumaryl-7-amide (MCA) or 7-amino-4-methylcoumarin (AMC), containing a carboxy-terminal lysine residue.

36. The hyperfibrinolysis assay or hyperfibrinolysis assay kit of claim 35, wherein the plasmin substrate comprises Boc-Val-Leu-Lys-MCA, Boc-Glu-Lys-Lys-MCA, or Boc- Glu-Lys-Lys-AMC.

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