Freeze-dried platelet derivative compositions for treating antiplatelet-induced coagulation disorders - Patents.com

Abstract

Provided herein are methods and compositions for treating a coagulation disorder in a subject. Such methods may include administering to a subject in need thereof an effective amount of a composition comprising platelets or, in an exemplary embodiment, a platelet derivative, and in a further exemplary embodiment, a freeze-dried platelet derivative (FDPD), for example because the subject has been administered an anticoagulant. Exemplary embodiments of such methods and platelet derivatives used therein, as well as various characteristics of numerous additional aspects and embodiments, are provided herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 150,338, filed February 17, 2021, U.S. Provisional Application No. 63 / 275,937, filed November 4, 2021, U.S. Provisional Application No. 63 / 276,420, filed November 5, 2021, U.S. Provisional Application No. 63 / 264,227, filed November 17, 2021, and U.S. Application No. 17 / 673,773, filed February 16, 2022 (which is a continuation-in-part of U.S. Application No. 16 / 994,377, filed August 14, 2020, and PCT / US2020 / 046525, filed August 14, 2020). U.S. Application No. 16 / 994,377, filed August 14, 2020, claims priority to U.S. Provisional Application No. 62,887,923, filed August 16, 2019, and U.S. Provisional Application No. 63,065,337, filed August 13, 2020. PCT / US2020 / 046525, filed August 14, 2020, claims priority to U.S. Provisional Application No. 62,887,923, filed August 16, 2019, and U.S. Provisional Application No. 63,065,337, filed August 13, 2020. Each of the applications listed in this paragraph is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present disclosure relates to the use of platelet derivatives as a treatment for antithrombotic-induced coagulopathy. The use of antiplatelet agents can increase the likelihood of bleeding. [Background technology]

[0003] background Antiplatelet drugs (also referred to herein as antiplatelet agents) are common in the adult U.S. population and use multiple mechanisms to inhibit platelet action. Antiplatelet drugs are used to treat and / or prevent a number of cerebrovascular and cardiovascular diseases.

[0004] However, antiplatelet drugs are involved in a large number of drug-related adverse events (ADEs). Overdose and adverse events associated with these drugs pose a risk of serious bleeding and related complications to the patient population. In addition, subjects treated with antiplatelet drugs face additional complications during surgery because the drug may need to be tapered before surgery, while cessation of therapy may expose the subject to an increased risk of heart attack, stroke, or death.

[0005] Thus, there is a need in the art for treatment of coagulation disorders, such as antiplatelet-induced coagulation disorders, as well as solutions for preparing subjects taking antiplatelet drugs for surgery. Summary of the Invention

[0006] Thus, the use of antithrombotic agents (i.e., antiplatelet and / or anticoagulant agents) can increase a subject's likelihood of bleeding. Here, we demonstrate that platelet derivatives can circumvent or overcome this inhibition to restore hemostasis. Thus, in exemplary embodiments, freeze-dried platelet derivatives (FDPDs) and compositions comprising the same are provided, which can reduce this increased likelihood of bleeding in a subject, and in certain exemplary embodiments, can circumvent or overcome this inhibition of platelets by such antithrombotic agents to restore hemostasis.

[0007] Provided herein are methods and compositions for treating a coagulation disorder in a subject. Such methods can include administering to a subject in need thereof an effective amount of a composition comprising platelets, or in exemplary embodiments, a platelet derivative, and in further exemplary embodiments, FDPD, for example, because the subject has been administered an anticoagulant. Various characteristics of exemplary embodiments of such FDPDs and methods, as well as numerous additional aspects and embodiments, are provided herein.

[0008] Further details regarding aspects and embodiments of the present disclosure are provided throughout this patent application. The preceding paragraphs in this Summary section are not an exhaustive list of aspects and embodiments disclosed herein. The sections and section headings are for ease of reading and are not intended to limit the combinations of disclosed methods, compositions, and kits or functional elements thereof across sections. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows light transmission aggregometry of cangrelor ("Cang") in platelet-rich plasma, expressed as integrated aggregation curves, induced by 10 μM adenosine diphosphate (ADP) activation with or without increasing concentrations of cangrelor. [Figure 2] 1 shows the effect of cangrelor, ADP, or their combination on platelet occlusion using T-TAS® technology. [Figure 3] 3 is a bar graph of the area under the curve (AUC) for the data sets from Figure 2. Replicate data sets from Figure 2 are presented as means. [Figure 4] 3 is a bar graph of occlusion times for the dataset from Figure 2. Replicate datasets from Figure 2 are presented as averages. [Figure 5] 1 shows the effect of FDPD ("thromb"; 300,000 / μL) supplemented to platelet-rich plasma in the presence and absence of ADP and cangrelor after 60, 90, or 115 minutes of rehydration on platelet occlusion using T-TAS® technology. [Figure 6] 6 is a bar graph of AUC for the datasets from Figure 5. The replicate datasets from Figure 5 are shown as the mean. [Figure 7] 6 is a bar graph of occlusion times for the data sets from Figure 5. Replicate data sets from Figure 5 are shown as averages. [Figure 8]1 is a bar graph of AUC from aggregation experiments of platelets (at a concentration of 250,000 platelets per μL) treated with collagen (10 μg / mL) and various concentrations of eptifibatide ("Epti"). [Figure 9] 1 shows the effect of eptifibatide at various concentrations on whole blood using T-TAS® technology. [Figure 10] 1 shows the effect of FDPD ("Tsome") supplementation (approximately 200,000 / μL) on whole blood with and without various concentrations of eptifibatide using T-TAS® technology. [Figure 11] 11 is a bar graph of occlusion times for the data set from FIG. 10. [Figure 12] 11 is a bar graph of AUC for the dataset from FIG. 10. [Figure 13] Figure 1 shows that FDPD (various lots) occlude in the presence of eptifibatide in platelet-poor plasma (PPP). [Figure 14] 14 is a bar graph of AUC for the datasets from Figure 13. The replicate datasets from Figure 13 are shown as the mean. [Figure 15] 14 is a bar graph of occlusion times for the datasets from Figure 13. The replicate datasets from Figure 13 are shown as averages. [Figure 16] 1 is a bar graph of AUC from aggregation experiments of platelets treated with collagen (10 μg / mL) or arachidonic acid ("AA"; 500 μg / mL), with and without various concentrations of aspirin ("ASA"). [Figure 17] 1 is a bar graph of occlusion times for whole blood, whole blood treated with various concentrations of aspirin (ASA), and whole blood treated with various concentrations of aspirin and supplemented with FDPD (approximately 200,000-400,000 / μL) as measured by response to collagen-coated plastic under shear using T-TAS® technology. [Figure 18]Figure 1 shows the recovery of FDPD-promoted clot formation in whole blood in the presence of ASA (200 micromolar), cangrelor (1 micromolar), and AP2 6F1 (40 micrograms), as measured by occlusion time in a T-TAS AR chip coated with thromboplastin and collagen. [Figure 19] 1 shows the recovery of FDPD-promoted clot formation in whole blood in the presence of ASA (200 micromolar), cangrelor (1 micromolar), and 6F1 (40 micrograms / mL), as measured by occlusion (pressure) overtime. [Figure 20] 1 shows the effect of FDPD supplementation to aspirin (ASA)-inhibited whole blood (500 micromolar) on the interaction with plastic-immobilized porcine collagen under high shear as measured by AUC. [Figure 21] Figure 1 shows the effect of FDPD supplementation to aspirin (ASA)-inhibited whole blood (500 micromolar) on the interaction with plastic-immobilized porcine collagen under high shear, as measured by occlusion (pressure) over time. [Figure 22] 1 shows the effect of FDPD supplementation to aspirin (ASA)-inhibited whole blood (100 micromolar) on the interaction with plastic-immobilized porcine collagen under high shear as measured by AUC. [Figure 23] Figure 1 shows the effect of FDPD supplementation to aspirin (ASA)-inhibited whole blood (100 micromolar) on the interaction with plastic-immobilized porcine collagen under high shear, as measured by occlusion (pressure) over time. [Figure 24] 1 shows the effect of FDPD supplementation of normal and aspirin-inhibited plasma on peak thrombin. [Figure 25A] 1 shows the effect of cangrelor alone or cangrelor plus FDPD on platelet occlusion using T-TAS® technology. [Figure 25B] 25B is a bar graph of occlusion time for the data set from FIG. 25A. [Figure 26A]1 shows the effect of tirofiban alone or with random donor platelets (RDP) or FDPD on platelet occlusion using the T-TAS® technique. [Figure 26B] 26B is a bar graph of occlusion time for the dataset from FIG. 26A. [Figure 27A] 1 shows the effect of eptifibatide alone or with RDP or FDPD on platelet occlusion using the T-TAS® technique. [Figure 27B] 27B is a bar graph of occlusion time for the dataset from FIG. 27A. [Figure 28A] 1 shows the effect of AP2 alone or AP2 with RDP or FDPD on platelet occlusion using the T-TAS® technique. [Figure 28B] 28B is a bar graph of occlusion time for the data set from FIG. 28A. [Figure 29A] 1 shows the effect of FDPD on PRP collected from subjects on aspirin therapy using T-TAS® technology. [Figure 29B] 29B is a bar graph of occlusion time for the data set from FIG. 29A. [Figure 30A] 1 shows the effect of FDPD on PRP obtained from subjects undergoing aspirin therapy on thrombin generation. [Figure 30B] 1 is a bar graph of thrombin generation parameters for PRP taken from subjects on aspirin therapy with or without added FDPD. [Figure 31A] 1 shows aggregation measurements of PRP taken from subjects on ibuprofen therapy with added buffer, arachidonic acid, or collagen. [Figure 31B] 1 shows the effect of ADP on PRP taken from subjects on ibuprofen therapy with or without FDPD. [Figure 32] 1 shows the effect of FDPD dosing on bleeding time in mice treated with suprapharmacological doses of clopidogrel. [Figure 33A]1 shows the effect of cangrelor and ticagrelor on the occlusion time of platelet-rich plasma (PRP) with or without FDPD. [Figure 33B] 1 shows the effect of cangrelor and ticagrelor on the occlusion time of platelet-rich plasma (PRP) with or without FDPD. [Figure 34] 1 shows a tail snip assay to demonstrate the ability of FDPD to restore bleeding time in NOD-SCID mice treated with suprapharmacological clopidogrel. [Figure 35] 1 shows the ability of FDPD to restore bleeding time in New Zealand White rabbits treated with suprapharmacologic clopidogrel. Each line represents a different rabbit administered the same dose under identical conditions. [Figure 36A] FDPD is shown to be able to catalyze thrombin generation in the presence of 25 ng / ml of rivaroxaban. [Figure 36B] This shows that FDPD can partially restore endogenous thrombin generating capacity in the presence of 25 ng / ml of rivaroxaban. [Figure 37] This shows that the addition of FDPD to whole blood treated with rivaroxaban partially restored the occlusion time. [Figure 38A] 1 shows the ability of FDPD to generate activated factor thrombin in rivaroxaban-treated Octaplus. [Figure 38B] 1 shows the maximum thrombin concentration by FDPD in rivaroxaban-treated Octaplus. [Figure 38C] 1 shows the ATG lag time by FDPD in rivaroxaban-treated Octaplus. [Figure 39A] This shows the ability of FDPD to generate thrombin activators in rivaroxaban-treated fresh platelet-rich plasma (PRP). [Figure 39B] 1 shows the maximum thrombin concentration by FDPD in rivaroxaban-treated fresh platelet-rich plasma (PRP). [Figure 39C]1 shows the ATG lag time by FDPD in rivaroxaban-treated platelet-rich plasma (PRP). [Figure 40] 1 shows the effect of FDPD on the time to clot in the presence of an anticoagulant using the ACT test. [Figure 41A] 1 shows the effect of 0.1 U heparin on thrombin generation in pooled normal plasma comparing apheresis units (APU) with FDPD at 5K and 50K platelets per μL. [Figure 41B] The effect of 0.8 U / mL heparin antagonized by 4 μg / mL protamine (1 / 2 the recommended antagonizing dose) with FDPD at 10K and 50K platelets per μL is shown. [Figure 41C] The peak heights of thrombin generation for heparin and protamine treated samples are shown as listed on the x-axis. [Figure 42] 1 shows the occlusion time with aspirin treatment in the presence of FDPD versus PRP alone. [Figure 43] Figure 1 shows the occlusion time with both ticagrelor and aspirin in the presence of FDPD versus PRP alone. [Figure 44A] The aggregation response of FDPD in the presence of agonist but in the absence of fresh platelets is shown. [Figure 44B] 1 shows the aggregation response of platelet-rich plasma (PRP) in the presence of agonist but in the absence of fresh platelets. [Figure 44C] A comparison of the aggregation of FDPD and PRP in the presence of 20 μM ADP is shown. [Figure 44D] 1 shows a comparison of the aggregation of FDPD and PRP in the presence of 10 μg / ml collagen. [Figure 44E] 1 shows a comparison of the aggregation of FDPD and PRP in the presence of 300 μM epinephrine. [Figure 44F] 1 shows a comparison of the aggregation of FDPD and PRP in the presence of 1 mg / ml ristocetin. [Figure 44G] A comparison of the aggregation of FDPD and PRP in the presence of 10 μM TRAP-6 is shown. [Figure 44H] 1 shows a comparison of the aggregation of FDPD and PRP in the presence of 5 mg / ml arachidonic acid. [Figure 45A] By observing the expression of CD62P on apheresis platelets, we demonstrate that the TRAP-6 peptide can promote platelet activation. [Figure 45B] 1 shows that the TRAP-6 peptide cannot increase the expression of CD62P on FDPD. [Figure 46] FIG. 1 shows the measurement of thrombospondin (TSP-1) by flow cytometry in terms of mean fluorescence intensity (MFI) in resting fresh platelets, activated fresh platelets, and different lots of FDPD. [Figure 47] Measurement of von Willebrand factor (vWF) by flow cytometry in terms of mean fluorescence intensity (MFI) in resting fresh platelets, activated fresh platelets, and different lots of FDPD. [Figure 48] Forward scatter (FSC) measured by flow cytometry of apheresis platelets and FDPD is shown. [Figure 49] Exemplary flow cytometry data of FDPD unstained (dark data points) or stained (light data points) with anti-CD41 antibody are shown. [Figure 50] Exemplary histograms of FDPD incubated with Annexin V with (light data points) and without (dark data points) calcium are shown. [Figure 51] Exemplary histograms of FDPD incubated with anti-CD62 antibody (light data points) or isotype control (dark data points) are shown. [Figure 52] 1 shows plots of thrombin peak height for FDPD in the presence of PRP reagents containing tissue factor and phospholipids (solid and long dashed lines) and control cephalin (dots). [Figure 53A]1 is a plot of the occupancy of particles of different radii in human in-life stored platelets (Batch J) and platelet derivatives derived therefrom (before freeze-drying), as determined by dynamic light scattering (DLS). [Figure 53B] 1 is a plot of the occupancy of particles of different radii in human in-life stored platelets (batch K) and platelet derivatives derived therefrom (before freeze-drying), as determined by DLS. [Figure 53C] 1 is a plot of the occupancy of particles of different radii in human in-life stored platelets (batch L) and platelet derivatives derived therefrom (before freeze-drying), as determined by DLS. [Figure 54A] 1 is a plot of the occupancy of particles of different radii in human in-life stored platelets (batch D) and platelet derivatives derived therefrom (before freeze-drying), as determined by DLS. [Figure 54B] 1 is a plot of the occupancy of particles of different radii in human in-life stored platelets (batch E) and platelet derivatives derived therefrom (before freeze-drying), as determined by DLS. [Figure 54C] 1 is a plot of the occupancy of particles of different radii in human in-life stored platelets (batch F) and platelet derivatives derived therefrom (before freeze-drying), as determined by DLS. [Figure 55] Shown is a comparison of exemplary histograms of low plasma FDPD unstained (black), stained with an isotype control antibody (dark grey), or stained with FITC-labeled 9F9 antibody (light grey), and a table showing the mean fluorescence intensity of two replicates. [Figure 56] 1 shows a comparison of exemplary histograms of low plasma FDPD unstained (black) or stained with anti-PAC-1 antibody (light grey) and a table showing the mean fluorescence intensity of two replicates. DETAILED DESCRIPTION OF THE INVENTION

[0010] Detailed Description Before describing the embodiments of the present invention in detail, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. 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 the term belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials related to the cited publications. The present disclosure controls for any conflict with any incorporated publication.

[0011] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "sugar" includes a reference to one or more sugars and equivalents thereof known to those of skill in the art. Furthermore, use of terms that can be described using equivalent terms includes use of those equivalent terms. Thus, for example, use of the term "subject" should be understood to include "patient," "person," "animal," "human," and other terms used in the art to refer to a subject undergoing medical treatment. The use of plural terms to encompass a single concept should not be construed as limiting the concept to only those terms used.

[0012] It is to be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. Furthermore, when a range of values ​​is disclosed, one of ordinary skill in the art will understand that all other specific values ​​within the disclosed range are inherently disclosed by those values ​​and the ranges they represent, without the need to disclose each specific value or range herein. For example, a disclosed range of 1 to 10 includes 1 to 9, 1 to 5, 2 to 10, 3.1 to 6, 1, 2, 3, 4, 5, etc. Furthermore, each disclosed range includes values ​​up to 5% lower for the lower value in the range and up to 5% higher for the higher value in the range. For example, a disclosed range of 4 to 10 includes values ​​from 3.8 to 10.5. This concept is captured herein by the term "about."

[0013] It is understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments relating to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination were individually and expressly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such subcombination were individually and expressly disclosed herein.

[0014] As used herein, the term "coagulopathy" refers to any abnormality in hemostasis that results in either excessive bleeding or blood clotting. Coagulopathy caused by administering an antiplatelet or anticoagulant to a subject typically involves an increased likelihood of bleeding. Thus, the methods herein for treating coagulopathy in exemplary embodiments are methods for reducing the likelihood of bleeding in a subject.

[0015] As used herein, the term "platelets" may include whole platelets, fragmented platelets, platelet derivatives, or FDPDs. "Platelets" within the above definition may include, for example, platelets in whole blood, platelets in plasma, platelets in a buffer solution optionally supplemented with selected plasma proteins, cryopreserved platelets, dried platelets, cryopreserved platelets, thawed cryopreserved platelets, rehydrated dried platelets, rehydrated cryopreserved platelets, cryopreserved platelets, thawed cryopreserved platelets, or rehydrated cryopreserved platelets. "Platelets" may refer to "platelets" of a mammal, such as a human, or "platelets" of a non-human mammal, etc. As used herein, a "preparation agent" may include any suitable component. In some embodiments, the preparation agent may include a liquid medium. In some embodiments, the preparation may include one or more salts selected from phosphate salts, sodium salts, potassium salts, calcium salts, magnesium salts, and any other salts that may be found in blood or blood products or known to be useful for drying platelets, or any combination of two or more thereof. In some embodiments, the preparation includes one or more salts, such as phosphate salts, sodium salts, potassium salts, calcium salts, magnesium salts, and any other salts that may be found in blood or blood products. Exemplary salts include sodium chloride (NaCl), potassium chloride (KCl), and combinations thereof. In some embodiments, the preparation includes about 0.5 mM to about 100 mM of one or more salts. In some embodiments, the preparation includes about 0.5 mM to about 100 mM (e.g., about 0.5 to about 2 mM, about 2 mM to about 90 mM, about 2 mM to about 6 mM, about 50 mM to about 100 mM, about 60 mM to about 90 mM, about 70 mM to about 85 mM) of one or more salts. In some embodiments, the formulation comprises about 5 mM, about 75 mM, or about 80 mM of one or more salts.In some embodiments, the formulation comprises one or more salts selected from calcium salts, magnesium salts, and combinations of the two, at a concentration of about 0.5 mM to about 2 mM.

[0016] As used herein, a "thrombosome" (sometimes referred to herein, particularly in the Examples and Figures, as a "Tsome" or "T") is a platelet derivative that has been treated with an incubation substance (e.g., any of the incubation substances described herein) and cryopreserved (i.e., freeze-dried). Thus, the thrombosome is a freeze-dried platelet derivative (FDPD). In some cases, FDPD can be prepared from pooled platelets. FDPD can have a shelf life of 2-3 years in dried form at ambient temperature and can be rehydrated with sterile water within minutes for immediate infusion. One example of an FDPD is THROMBOSOMES®, which is in clinical trials for the treatment of acute bleeding in thrombocytopenic patients and is a product of Cellphire, Inc. In a non-limiting exemplary embodiment, the FDPD composition herein, or an exemplary freeze-dried platelet derivative (i.e., "FDPD") composition, e.g., a composition prepared according to Example 15 herein, comprises a population of platelet derivatives with a reduced tendency to aggregate, such that under aggregation conditions that include an agonist but no platelets, no more than 10% of the platelet derivatives in the population of platelet derivatives aggregate; 6 In a non-limiting exemplary embodiment, an FDPD composition, or an exemplary FDPD composition herein, e.g., a composition prepared according to Example 15 herein, is a composition comprising platelet derivatives, wherein at least 50% of the platelet derivatives are CD41-positive platelet derivatives, and wherein less than 15%, less than 10%, or in a further non-limiting exemplary embodiment, less than 5% of the CD41-positive platelet derivatives are microparticles having a diameter of less than 0.5 μm, and wherein the platelet derivatives are at least 10 6At least 0.5, 1.0, and in a non-limiting exemplary embodiment, 1.5 thrombin generating potency units (TGPU) per platelet derivative. In certain exemplary embodiments, including non-limiting examples of the preceding exemplary embodiment, the platelet derivatives have a diameter of 0.5-2.5 μm.

[0017] As used herein, an "anticoagulant" refers to an antithrombotic agent that does not include antiplatelet agents. Typically, agents that inhibit vitamin K-dependent synthesis of Factor IIa, Factor VIIa, Factor IX, Factor Xa, Factor XI, tissue factor, or clotting factors (e.g., Factor II, Factor VII, Factor IX, or Factor X), or agents that activate antithrombin (e.g., antithrombin III), are considered to be anticoagulants. Other mechanisms of anticoagulant action are known. Anticoagulants include dabigatran, argatroban, hirudin, rivaroxaban, apixaban, edoxaban, fondaparinux, warfarin, heparin, and low-molecular-weight heparins.

[0018] As used herein, an "antiplatelet agent" refers to an antithrombotic agent and does not include anticoagulants. Typically, agents that inhibit P2Y receptors (e.g., P2Y12), glycoprotein IIb / IIIa (i.e., CD41), or agents that antagonize thromboxane synthase or thromboxane receptors are considered to be antiplatelet agents. Other mechanisms of antiplatelet agents are known. As used herein, aspirin is considered to be an antiplatelet agent but not an anticoagulant. Examples of antiplatelet agents include aspirin (also known as acetylsalicylic acid or ASA), cangrelor (e.g., KENGREAL®), ticagrelor (e.g., BRILINTA®), clopidogrel (e.g., PLAVIX®), prasugrel (e.g., EFFIENT®), eptifibatide (e.g., INTEGRILIN®), tirofiban (e.g., AGGRASTAT®), and abciximab (e.g., REOPRO®). For purposes of this disclosure, an antiplatelet agent is defined as an agent that binds to P2Y receptors (e.g., P2Y 12), agents that inhibit glycoprotein IIb / IIIa, or agents that antagonize thromboxane synthase or thromboxane receptors. Non-limiting examples of thromboxane A2 antagonists are aspirin, terutroban, and picotamide. Non-limiting examples of P2Y receptor antagonists include cangrelor, ticagrelor, elinogrel, clopidogrel, prasugrel, and ticlopidine. Non-limiting examples of glycoprotein IIb / IIIa antagonists include abciximab, eptifibatide, and tirofiban. NSAIDS (e.g., ibuprofen) are also considered antiplatelet agents for purposes of this disclosure. Other mechanisms of antiplatelet agents are known. Antiplatelet agents also include PAR1 antagonists, PAR4 antagonists, GPVI antagonists, and α2β1 collagen receptor antagonists. Non-limiting examples of PAR-1 antagonists include vorapaxar and atopaxar. As used herein, aspirin is considered an antiplatelet agent, but not an anticoagulant. Further non-limiting examples of antiplatelet agents include cilostazol, prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, and sarpogrelate.

[0019] In some embodiments, the antiplatelet agent can be selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, and combinations thereof. In some embodiments, the antiplatelet agent can be selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, and combinations thereof. In some embodiments, the antiplatelet agent may be selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, cilostazol, prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, sarpogrelate, and combinations thereof. In some embodiments, the antiplatelet agent may include multiple antiplatelet agents, such as any two (or more) of the antiplatelet agents described herein. In some embodiments, the antiplatelet agent may be aspirin and clopidogrel.

[0020] Cangrelor, like clopidogrel, ticagrelor, and prasugrel, inhibits P2Y agonists on platelets. 12 (ADP) receptor blocking. Cangrelor can in some cases be used as a representative of this class of drugs. Unlike clopidogrel and prasugrel, cangrelor does not require hepatic metabolism to be biologically active.

[0021] Eptifibatide is a peptide therapeutic agent that blocks the fibrin-binding role of the GPIIb-IIIa receptor on platelets. The drug is typically administered intravenously as a 180 μg / kg bolus followed by a 2 μg / kg / min continuous infusion. Eptifibatide blood concentrations are typically approximately 1-2 μM. Bleeding times generally return to normal within approximately 1 hour of discontinuation of the drug.

[0022] Aspirin is an irreversible cyclooxygenase (COX) inhibitor. The COX enzyme in platelets is responsible for the synthesis of thromboxane A2, prostaglandin E2, and prostacyclin (PGI2). Because aspirin permanently inactivates the COX enzyme in platelets, and platelets lack the nuclear material to synthesize new enzymes, new platelets must be generated to overcome the effects of aspirin. Without thromboxane A2, prostaglandin E2, and prostacyclin (PGI2), platelets have limited proaggregatory activity. Many individuals are maintained on low doses of aspirin to prevent unwanted clotting events. Aspirin bioavailability varies significantly depending on the route of administration, with a peak of 500 μM following a single 500 mg IV dose and 44 μM orally at the same dose.

[0023] Antiplatelet class drugs are widely used to prevent unwanted clotting episodes that lead to heart failure, stroke, and the like. In many cases, antiplatelet drugs may need to be reversed or stopped, or the likelihood of bleeding may need to be reduced in some other way in subjects with antiplatelet drugs in their bloodstream, thus increasing the subject's likelihood of bleeding. With advance notice, such as in the context of a preplanned surgery, the dose of antiplatelet drugs may be stopped prior to surgery to prevent unwanted bleeding during surgery. When antiplatelet drugs need to be rapidly reversed, reversals are typically not readily available, expensive, or pose significant risks to the patient. When rapid antiplatelet reversal is required, platelet transfusions are typically administered, but the response is often only partial reversal. A caveat to this reversal process is that while newly infused platelets themselves are susceptible to the antiplatelet activity of circulating drugs, in some embodiments, compositions described herein (e.g., comprising FDPD) are not. In some embodiments, compositions described herein (e.g., comprising FDPD) are active reversals. In some embodiments, the hemostatic activity of a composition described herein (eg, comprising FDPD) is resistant to antiplatelet agents.

[0024] Some exemplary antiplatelet agents and potential methods of reversal are described below.

[0025] Acetylsalicylic acid (ASA; aspirin) - Aspirin functions as a COX-1 blocker on platelets, which irreversibly inhibits platelet-derived thromboxane formation, thereby rendering them inactive. Clinically, aspirin can be counteracted by platelet transfusion in emergency situations or by halting treatment prior to upcoming surgery.

[0026] Clopidogrel (e.g., PLAVIX®) - Clopidogrel acts to prevent ADP from binding to its receptor on platelets. ADP binding leads to platelet shape changes and aggregation. Clopidogrel is irreversible. Clinically, clopidogrel may be counteracted by platelet transfusion in emergency situations or by stopping treatment when upcoming surgery is planned.

[0027] Cangrelor (e.g., KENGREAL®) - Cangrelor acts to prevent ADP from binding to its receptor on platelets. ADP binding leads to platelet shape changes and aggregation. Clopidogrel is reversible, and platelet function returns approximately 1 hour after the infusion is stopped. Clinically, this is generally preferable when reversal is needed after a procedure.

[0028] Ticagrelor (e.g., BRILINTA®) - Ticagrelor prevents ADP from binding to its receptor and acts as an inverse agonist. Ticagrelor is reversible, and platelet function can return approximately 72 hours after the last dose. Reversal of ticagrelor's effects can be affected by the time since the last dose. If the last dose was more than 24 hours ago, platelet transfusion may be a therapeutic option to reverse the results.

[0029] Effient (e.g., PRASUGREL®) - Effient acts to prevent ADP from binding to its receptor and acts as an irreversible antagonist. As an irreversible antagonist, new platelets must be formed to overcome its effects. Clinically, Effient is antagonized by platelet transfusion in emergency situations or by stopping treatment when upcoming surgery is planned.

[0030] Eptifibatide (Integrilin) ​​- Eptifibatide acts to block GpIIb / IIIa and acts as a reversible antagonist. Clinically, Integrilin is antagonized by platelet transfusion in emergency situations or by stopping treatment when upcoming surgery is planned.

[0031] While platelet infusions are currently used as a treatment for antiplatelet drugs, platelet infusions only act to counteract the effects of these drugs. In some embodiments, FDPDs are not reactive to these drugs and maintain their ability to support blood clotting. This makes FDPD-mediated therapy entirely unique and introduces new uses for the product.

[0032] Platelet-derived products are not currently used as a treatment to counteract the activity of antithrombotic agents (i.e., anticoagulants or antiplatelet drugs), and such effects could result in adverse outcomes or pose unacceptable risks to subjects, for example, during surgical procedures or as a result of traumatic events. There are no currently approved reversals for antiplatelet agents that otherwise reduce a subject's likelihood of bleeding or restore hemostasis after treatment with anticoagulants and / or antiplatelet drugs. Therefore, emergency treatments (e.g., pre-surgery, trauma) typically require comprehensive preventative measures to avoid or mitigate bleeding. Non-limiting examples include infusions of plasma, red blood cells, and antifibrinolytic agents. Platelet derivatives, in exemplary embodiments, freeze-dried platelet derivatives (FDPDs) provided herein, overcome this long-standing need and are an effective alternative or complement to these common treatment or risk mitigation strategies.

[0033] The results provided in numerous examples in the Examples section herein demonstrate the effects of compositions containing FDPD products in in vitro models of subjects taking antiplatelet drugs. FDPD compositions and other freeze-dried platelet products are designed to be injected into a subject's bloodstream after trauma or a diagnosis of hemostatic failure. These antiplatelet drugs utilize multiple forms of platelet inhibitory mechanisms that inhibit platelet responses to adenosine diphosphate (ADP), arachidonic acid, fibrinogen, and von Willebrand factor binding, to name a few. These include drugs such as aspirin, clopidogrel, ticagrelor, Effient, cangrelor, and eptifibatide. Regardless of the mechanism, the FDPDs provided herein reduce the likelihood of bleeding in subjects taking such antiplatelet agents and, in some embodiments, can restore normal hemostasis in the subject.

[0034] Without being bound by any particular theory, it is believed that certain platelet derivatives, in exemplary embodiments, FDPDs provided herein, may act, at least in part, by providing a procoagulant, negatively charged surface to enhance thrombin generation beyond that inhibited by anticoagulants. Similarly, without being bound by any particular theory, it is believed that certain platelet derivatives, in exemplary embodiments, FDPDs provided herein, may act, at least in part, by binding to and coaggregating with circulating platelets. Thus, such FDPDs offer the surprising property of being able to reduce the likelihood of bleeding in subjects taking antithrombotic agents (i.e., anticoagulants or antiplatelet agents) despite having reduced aggregation capacity and retaining at least some, if not all, of the surface markers targeted by at least some, if not all, antiplatelet agents.

[0035] Described herein are products and methods for controlling bleeding and improving healing. The compositions, products, and methods described herein can also be used to counteract the activity of any of the antiplatelet agents disclosed herein (e.g., by way of non-limiting example, aspirin (also known as acetylsalicylic acid or ASA), cangrelor (e.g., KENGREAL®), ticagrelor (e.g., BRILINTA®), clopidogrel (e.g., PLAVIX®), prasugrel (e.g., EFFIENT®), eptifibatide (e.g., INTEGRILIN®), tirofiban (e.g., AGGRASTAT®), or abciximab (e.g., REOPRO®)). The products and methods disclosed herein are directed to embodiments that may, in certain embodiments, aid in wound closure and healing.

[0036] In certain aspects provided herein, a composition comprising platelets, or in an exemplary embodiment, a composition comprising a platelet derivative, which in a further exemplary embodiment is FDPD, can be delivered to a wound on the surface of a patient or within a patient. In various embodiments, the composition comprising platelets, or in an exemplary embodiment, a platelet derivative (which in a further exemplary embodiment is FDPD), or in an exemplary embodiment, a platelet derivative (which in a further exemplary embodiment is FDPD), can be applied in a selected form, including, but not limited to, an adhesive bandage, a compression bandage, a liquid solution, an aerosol, a matrix composition, and a coated suture or other medical closure. In embodiments, the composition comprising platelets, or in an exemplary embodiment, a platelet derivative (which in a further exemplary embodiment is FDPD), can be administered to all or only a portion of the affected area on the surface of a patient. In other embodiments, the composition comprising platelets, or in an exemplary embodiment, a platelet derivative (which in a further exemplary embodiment is FDPD), can be administered systemically, e.g., via the bloodstream. In embodiments, application of the platelet derivative can provide a hemostatic effect for 2 or 3 days, preferably 5 to 10 days, or most preferably up to 14 days.

[0037] Some aspects provide methods of treating a coagulation disorder in a subject, the methods comprising administering to a subject in need thereof an effective amount of a composition comprising platelets, or in exemplary embodiments, a composition comprising a platelet derivative (which in further exemplary embodiments is FDPD). In some embodiments, the FDPD-containing composition further comprises additional components, such as components that were present when such FDPD was freeze-dried. Such additional components may include one or more salts, buffers, and components of an incubation material, which in certain embodiments include a cryoprotectant (also called a lyophilizing agent) and / or an organic solvent. For example, such compositions may include one or more sugars, which in exemplary embodiments include trehalose and in further exemplary embodiments include polysucrose, as further provided herein.

[0038] Some embodiments provide methods of treating a coagulation disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0039] In some embodiments of any of the methods described herein, the coagulation disorder is the result of the presence of an antiplatelet agent in the subject's blood.

[0040] Some aspects provide a method of treating a coagulation disorder in a subject who has been or is being treated with an antiplatelet agent, the method comprising administering to a subject in need thereof an effective amount of a composition comprising platelets, or in an exemplary embodiment, a composition comprising a platelet derivative (which in a further exemplary embodiment is FDPD), wherein the composition comprises an incubation material comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0041] Some embodiments provide methods of treating a coagulation disorder in a subject who has been or is being treated with an antiplatelet agent, the method comprising administering to a subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0042] Some aspects provide methods for restoring normal hemostasis in a subject, the methods comprising administering to a subject in need thereof an effective amount of a composition comprising platelets, or in exemplary embodiments, a composition comprising a platelet derivative (which in further exemplary embodiments is FDPD). In some embodiments, the composition comprising FDPD further comprises additional components, such as components that were present when such FDPD was freeze-dried. Such additional components may include one or more salts, buffers, and components of an incubation material, which in certain embodiments includes a cryoprotectant (also called a lyophilizing agent) and / or an organic solvent. For example, such compositions may include one or more sugars, which in exemplary embodiments include trehalose and in further exemplary embodiments include polysucrose, as further provided herein.

[0043] Some embodiments provide a method of restoring normal hemostasis in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form a composition.

[0044] Some aspects provide methods of restoring normal hemostasis in a subject, the subject being treated or having been treated with an antiplatelet agent, the method comprising administering to a subject in need thereof an effective amount of a composition comprising platelets or, in exemplary embodiments, a composition comprising a platelet derivative (which in further exemplary embodiments is FDPD). In some embodiments, the composition comprising FDPD further comprises additional components, such as components that were present when such FDPD was freeze-dried. Such additional components may include one or more salts, buffers, and components of an incubation material, which in certain embodiments includes a cryoprotectant (also called a lyophilizing agent) and / or an organic solvent. For example, such compositions may include one or more sugars, which in exemplary embodiments include trehalose and, in further exemplary embodiments, polysucrose, as further provided herein.

[0045] Some embodiments provide a method of restoring normal hemostasis in a subject who has been or is being treated with an antiplatelet agent, the method comprising administering to a subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0046] The compositions described herein can also be administered in some cases to prepare patients for surgery.For some patients who take antiplatelet agents, it may be difficult or impossible to reduce the dosage of antiplatelet agents before surgery (for example, in the case of trauma or other emergency surgery).For some patients who take antiplatelet agents, it may be inadvisable to reduce the dosage of antiplatelet agents before surgery (for example, if the dosage of antiplatelet agents is reduced over time, the patient will be at risk of thrombotic events (for example, deep vein thrombosis, pulmonary embolism, or stroke)).

[0047] Accordingly, some embodiments provide methods for preparing a subject for surgery, the methods comprising administering to a subject in need thereof an effective amount of a composition comprising platelets, or in exemplary embodiments, a composition comprising a platelet derivative (which in further exemplary embodiments is FDPD). In some embodiments, the composition comprising FDPD further comprises additional components, such as components that were present when such FDPD was freeze-dried. Such additional components may include one or more salts, buffers, and components of an incubation material, which in certain embodiments include a cryoprotectant (also called a lyophilizing agent) and / or an organic solvent. For example, such compositions may include one or more sugars, which in exemplary embodiments include trehalose and in further exemplary embodiments include polysucrose, as further provided herein.

[0048] Some embodiments provide methods of preparing a subject for surgery, the methods comprising administering to a subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form the composition.

[0049] Some embodiments provide methods of preparing a subject for surgery, the subject being treated or having been treated with an antiplatelet agent, the method comprising administering to the subject in need thereof an effective amount of a composition comprising platelets, or in an exemplary embodiment, a composition comprising a platelet derivative (which in a further exemplary embodiment is FDPD). In some embodiments, the composition comprising FDPD further comprises additional components, such as components that were present when such FDPD was freeze-dried. Such additional components may include one or more salts, buffers, and components of an incubation material, which in certain embodiments include a cryoprotectant (also called a lyophilizer) and / or an organic solvent. For example, such compositions may include one or more sugars, which in an exemplary embodiment include trehalose and in a further exemplary embodiment include polysucrose, as further provided herein.

[0050] Some embodiments provide a method of preparing a subject for surgery, wherein the subject is or has been treated with an antiplatelet agent, the method comprising administering to a subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0051] In some embodiments, the surgery may be an emergency surgery (eg, in the case of trauma) or a scheduled surgery.

[0052] In some embodiments, anticoagulant treatment can be stopped (e.g., in preparation for surgery) before a composition comprising a platelet derivative is administered to a subject, in exemplary embodiments. In some embodiments, anticoagulant treatment can be continued for a period of time after a composition comprising a platelet derivative is administered to a subject, in exemplary embodiments. Such a period can include 1, 2, 3, 4, 5, 6, or 7 days, or 1, 2, 3, or 4 weeks, or 1, 2, or 3 months or more.

[0053] Some embodiments provide methods of improving the effectiveness of an antiplatelet agent in a subject, the methods comprising administering to a subject in need thereof an effective amount of a composition, which in an exemplary embodiment comprises an effective amount of a platelet derivative (which in a further exemplary embodiment is FDPD), the composition comprising an incubation material comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0054] Some embodiments provide a method of improving the effectiveness of an antiplatelet agent in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form the composition.

[0055] In some embodiments, the effectiveness of an antiplatelet agent may need to be improved due to an inaccurate dose of the antiplatelet agent. For example, in some embodiments, the effectiveness of an antiplatelet agent may be improved after an overdose of the antiplatelet agent. In some embodiments, the effectiveness of an antiplatelet agent may need to be improved due to a potential interaction with another drug (e.g., a second antiplatelet agent). For example, in some embodiments, the effectiveness of an antiplatelet agent may be improved after an incorrect dose of two or more drugs, at least one of which is an antiplatelet agent.

[0056] In some embodiments of any of the methods described herein, the composition may further comprise an active agent, such as an antifibrinolytic agent. Non-limiting examples of antifibrinolytic agents include ε-aminocaproic acid (EACA), tranexamic acid, aprotinin, aminomethylbenzoic acid, and fibrinogen. In some embodiments, the platelets or platelet derivatives may be loaded with an active agent, such as an antifibrinolytic agent.

[0057] Compositions comprising FDPD herein, in certain embodiments, can reduce the likelihood of bleeding, and in exemplary embodiments, have the surprising property of restoring hemostasis in subjects with increased likelihood of bleeding after administration of FDPD, regardless of whether the subject's laboratory test for bleeding likelihood is negative or positive. Such increased likelihood of bleeding in exemplary embodiments is typically due to an effective amount of antiplatelet agent being delivered to the subject and present in the subject's blood. Thus, in any of the aspects herein, in some embodiments, a composition comprising FDPD has the property of being able to reduce a subject's likelihood of bleeding, regardless of whether a post-administration assessment of bleeding likelihood, if performed, results in a normal or abnormal result. In some embodiments, such a post-administration assessment comprises an in vitro clinical test performed on a sample taken or collected a period of time, e.g., 1-4 hours, 1-3 hours, or 1-2 hours, after administration of a composition comprising FDPD to the subject. In other embodiments of any of the aspects herein, a composition comprising FDPD has the property of being able to reduce a subject's likelihood of bleeding, such that normal hemostasis is restored in subjects with increased likelihood of bleeding, regardless of whether a post-administration assessment of bleeding likelihood results in a normal or abnormal result. In some embodiments, such post-administration assessments, if performed, include in vitro clinical tests performed on samples taken or collected a period of time, e.g., 1-4 hours, 1-3 hours, or 1-2 hours, after administration of a composition comprising FDPD to a subject. The period of time can be, for example, within 0 minutes to 72 hours, or within 10 minutes to 72 hours, or within 10 minutes to 48 hours, or within 10 minutes to 24 hours, or within 10 minutes to 4 hours, or within 10 minutes to 1 hour, or within 10 minutes to 30 minutes, or within 30 minutes to 24 hours, or within 30 minutes to 4 hours, or within 30 minutes to 1 hour. In certain embodiments, the clinical tests are one or more, two or more, or three or more of the bleeding parameters disclosed herein.

[0058] In any of the aspects herein, in some embodiments, a composition comprising a platelet derivative (e.g., FDPD) has the property of being capable of reducing the likelihood of bleeding in a subject with an elevated likelihood of bleeding. Furthermore, a composition comprising FDPD typically has the additional surprising property that, after administration to a subject in an amount effective to, for example, reduce the subject's likelihood of bleeding, the subject may have abnormal values ​​for one or more coagulation parameters in one or more in vitro clinical tests, e.g., a post-administration assessment performed using, or an in vitro clinical test performed on, a blood sample taken 15 minutes to 4 hours, 30 minutes to 4 hours, 1 hour to 4 hours, or 15 minutes to 2 hours, 30 minutes to 2 hours, or 1 hour to 2 hours, or 15 minutes to 1 hour, or 30 minutes to 1 hour after administration of the composition comprising FDPD. In some subembodiments of this embodiment, the composition comprising FDPD has the property of being capable of reducing the subject's likelihood of bleeding to near-normal or normal hemostasis, or to near- or at a hemostasis level, in the absence of an antiplatelet agent. In these embodiments, however, the FDPD-containing composition, after administration to a subject in an effective amount, possesses the additional surprising property that such property is independent of post-administration clinical testing for bleeding potential. Thus, in some embodiments, the subject will have abnormal values ​​for one or more coagulation parameters in a post-administration assessment performed using a blood sample drawn 1-4 hours after administration of the FDPD-containing composition, or any of the time ranges recited immediately above, or in an in vitro clinical test performed on such a blood sample. It will be understood that for methods involving FDPD-containing compositions having such properties, or any property that includes evaluation or testing, it is not necessary to actually perform the testing to practice such a method, unless such testing step is actually recited as a step of the method.

[0059] Platelet derivatives, particularly FDPDs provided herein, are produced, for example, using the methods provided herein and, in certain embodiments, have certain specified properties as disclosed herein. For example, in some embodiments, FDPDs comprise (detectable amounts of) a biomolecule (e.g., a receptor) that is targeted by an antiplatelet antagonist. For example, FDPDs can comprise one or more biomolecules that are targeted by one or more antiplatelet antagonists that are administered or have been administered to the same subject to which the composition comprising FDPD is administered. In some embodiments, the receptor present on FDPD is selected from a P2Y receptor (e.g., P2Y12 receptor), glycoprotein IIb (i.e., CD41) / IIIa (i.e., CD61), thromboxane synthase or thromboxane receptor, PAR1, PAR4, VPVI, or a collagen receptor (e.g., α2β1 collagen receptor). In certain embodiments, at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the platelet derivatives, in exemplary embodiments, FDPDs, are positive for (i.e., have detectable levels of) a biomolecule targeted by an antiplatelet agent administered to a subject and / or are detectable in the subject's blood. As some notable, non-limiting examples, the biomarker present on FDPDs can be CD41 or the CD41 / CD61 complex. In some embodiments, the CD41 / CD61 complex on FDPDs is bound by fibrinogen (Factor 1).

[0060] In certain embodiments, the composition comprising FDPD comprises a population of FDPD with reduced aggregation propensity such that under aggregation conditions containing an agonist but not platelets, no more than 2%, 3%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, or 25% of the FDPD in the population aggregates. 6at least 1.2 (e.g., at least 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5) thrombin generating potency units (TGPU) per particle.

[0061] In certain embodiments, the FDPD has one or more characteristics of superactivated platelets. Such characteristics include the following: A) The presence of thrombospondin (TSP) on the surface of FDPD at higher levels than on the surface of resting platelets. B) The presence of von Willebrand factor (vWF) on the surface of FDPDs at levels higher than on the surface of resting platelets, and C) failure to increase the expression of platelet activation markers in the presence of an agonist compared to the expression of platelet activation markers in the absence of an agonist; may include one or more of:

[0062] In some embodiments, less than 5% of the population of FDPDs, and in exemplary embodiments, CD41-positive FDPDs, are microparticles having a diameter of less than 0.5 μm. The platelet derivatives herein have been observed to have a number of surprising properties, as disclosed in further detail herein. As shown in the examples provided herein, in some aspects and embodiments, the platelet derivatives are solid, such as in powder form; however, it will be understood that properties of such platelet derivatives may be identified, confirmed, and / or measured when compositions comprising such platelet derivatives are in liquid form.

[0063] In some embodiments, the platelets or platelet derivatives (e.g., FDPD) have a particle size (e.g., diameter, largest dimension) of at least about 0.5 μm (e.g., at least about at least about 0.6 μm, at least about 0.7 μm, at least about 0.8 μm, at least about 0.9 μm, at least about 1.0 μm, at least about 1.2 μm, at least about 1.5 μm, at least about 2.0 μm, at least about 2.5 μm, or at least about 5.0 μm). In some embodiments, the particle size is less than about 5.0 μm (e.g., less than about 2.5 μm, less than about 2.0 μm, less than about 1.5 μm, less than about 1.0 μm, less than about 0.9 μm, less than about 0.8 μm, less than about 0.7 μm, less than about 0.6 μm, less than about 0.5 μm, less than about 0.4 μm, or less than about 0.3 μm). In some embodiments, the particle size is about 0.5 μm to about 5.0 μm (e.g., about 0.5 μm to about 4.0 μm, about 0.5 μm to about 2.5 μm, about 0.6 μm to about 2.0 μm, about 0.7 μm to about 1.0 μm, about 0.5 μm to about 0.9 μm, or about 0.6 μm to about 0.8 μm).

[0064] In some embodiments, at least 50% (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%) of the platelets or platelet derivatives (e.g., FDPD) have a particle size in the range of about 0.5 μm to about 5.0 μm (e.g., about 0.5 μm to about 4.0 μm, about 0.5 μm to about 2.5 μm, about 0.6 μm to about 2.0 μm, about 0.7 μm to about 1.0 μm, about 0.5 μm to about 0.9 μm, or about 0.6 μm to about 0.8 μm). In some embodiments, up to 99% (e.g., up to about 95%, up to about 80%, up to about 75%, up to about 70%, up to about 65%, up to about 60%, up to about 55%, or up to about 50%) of the platelets or platelet derivatives (e.g., FDPD) are in the range of about 0.5 μm to about 5.0 μm (e.g., about 0.5 μm to about 4.0 μm, about 0.5 μm to about 2.5 μm, about 0.6 μm to about 2.0 μm, about 0.7 μm to about 1.0 μm, about 0.5 μm to about 0.9 μm, or about 0.6 μm to about 0.8 μm). In some embodiments, about 50% to about 99% (e.g., about 55% to about 95%, about 60% to about 90%, about 65% to about 85%, about 70% to about 80%) of the platelets or platelet derivatives (e.g., FDPD) are in the range of about 0.5 μm to about 5.0 μm (e.g., about 0.5 μm to about 4.0 μm, about 0.5 μm to about 2.5 μm, about 0.6 μm to about 2.0 μm, about 0.7 μm to about 1.0 μm, about 0.5 μm to about 0.9 μm, or about 0.6 μm to about 0.8 μm).

[0065] Platelets or platelet derivatives (e.g., FDPD) described herein can have cell surface markers. The presence of cell surface markers can be determined using any suitable method. In some embodiments, the presence of cell surface markers can be determined using binding proteins (e.g., antibodies) specific for one or more cell surface markers and flow cytometry (e.g., a positive rate of, e.g., about 2.7 x 10). 5and about 4.8 μL of anti-CD41 antibody, about 3.3 μL of anti-CD42 antibody, about 1.3 μL of annexin V, or about 2.4 μL of anti-CD62 antibody. Non-limiting examples of cell surface markers include CD41 (also known as glycoprotein IIb or GPIIb, which can be assayed, for example, using an anti-CD41 antibody), CD42 (which can be assayed, for example, using an anti-CD42 antibody), CD62 (also known as CD62P or P-selectin, which can be assayed, for example, using an anti-CD62 antibody), phosphatidylserine (which can be assayed, for example, using annexin V (AV)), and CD47 (used in autorecognition; the absence of this marker can sometimes result in phagocytosis). The positive rate of any cell surface marker can be any appropriate positive rate. For example, a population of platelet derivatives (e.g., FDPDs), such as those prepared by the methods described herein and included in the compositions herein, can have an average CD41 positivity of at least 55% (e.g., at least 60%, at least 65%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%). In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the platelet derivatives that are CD41 positive have a size range of 0.5-2.5 μm. In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the platelet derivatives that are CD41 positive have a size range of 0.4-2.8 μm. In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the platelet derivatives that are positive for CD41 have a size in the range of 0.3 to 3 μm.

[0066] As another example, platelets or platelet derivatives (e.g., FDPDs) such as those described herein can have an average CD42 positivity of at least 65% (e.g., at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%). In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the platelet derivatives that are positive for CD42 have a size range of 0.5-2.5 μm. In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the platelet derivatives that are positive for CD42 have a size range of 0.4-2.8 μm. In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the platelet derivatives that are positive for CD42 have a size in the range of 0.3 to 3 μm.

[0067] As another example, platelets or platelet derivatives (e.g., FDPDs), such as those prepared by the methods described herein, can have an average CD62 positivity of at least 10% (e.g., 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 82%, at least 83%, at least 84%, at least 85%, at least 90%, or at least 95%). In some embodiments, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the platelet derivatives that are positive for CD62 have a size in the range of 0.5 to 2.5 μm. In some embodiments, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the platelet derivatives that are positive for CD62 have a size in the range of 0.4 to 2.8 μm. In some embodiments, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the platelet derivatives that are positive for CD62 have a size in the range of 0.3 to 3 μm.

[0068] As yet another example, platelets or platelet derivatives (e.g., FDPDs), such as those prepared by the methods described herein, can have an average Annexin V positivity of at least 25% (e.g., 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%, at least 97%, or at least 99%). In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the platelet derivatives that are Annexin V positive have a size in the range of 0.5 to 2.5 μm. In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the platelet derivatives that are positive for Annexin V have a size range of 0.4 to 2.8 μm. In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the platelet derivatives that are positive for Annexin V have a size range of 0.3 to 3 μm.

[0069] As another example, platelets or platelet derivatives (e.g., FDPDs), such as those prepared by the methods described herein, can have an average CD47 positivity of at least about 8% (e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55%).

[0070] Platelets or platelet derivatives (e.g., FDPD) described herein can generate thrombin in the presence of a reagent including, for example, tissue factor and phospholipids. For example, in some cases, platelets or platelet derivatives (e.g., FDPD) described herein (e.g., about 4.8×10 3For example, in some cases, a platelet or platelet derivative (e.g., FDPD) described herein (e.g., at a concentration of about 4.8×10 particles / μL) may produce a thrombin peak height (TPH) of at least 25 nM (e.g., at least 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 52 nM, 54 nM, 55 nM, 56 nM, 58 nM, 60 nM, 65 nM, 70 nM, 75 nM, or 80 nM) in the presence of a reagent containing tissue factor (e.g., at 0.25 pM, 0.5 pM, 1 pM, 2 pM, 5 pM, or 10 pM), and optionally, a phospholipid. 3 In some cases, platelets or platelet derivatives (e.g., FDPD) described herein (e.g., at a concentration of about 4.8 x 10 particles / µL) can produce TPH of about 25 nM to about 100 nM (e.g., about 25 nM to about 50 nM, about 25 to about 75 nM, about 50 to about 100 nM, about 75 to about 100 nM, about 35 nM to about 95 nM, about 45 to about 85 nM, about 55 to about 75 nM, or about 60 to about 70 nM) in the presence of a reagent containing tissue factor (e.g., at 0.25 pM, 0.5 pM, 1 pM, 2 pM, 5 pM, or 10 pM) and, optionally, a phospholipid. 3 (at a concentration of 10 particles / µL) would be, for example, 20 µL of PRP reagent and approximately 4.8 x 10 3 and 80 μL of a composition containing platelets or platelet derivatives (e.g., FDPD) at 1000 particles / μL, in the presence of a PRP reagent (catalog number TS30.00 from Thrombinoscope), can produce a TPH of, for example, at least 25 nM (e.g., at least 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 52 nM, 54 nM, 55 nM, 56 nM, 58 nM, 60 nM, 65 nM, 70 nM, 75 nM, or 80 nM). In some cases, the platelets or platelet derivatives (e.g., FDPD) described herein (e.g., about 4.8×10 3 (at a concentration of 10 particles / µL) would be, for example, 20 µL of PRP reagent and approximately 4.8 x 10 3and 80 μL of a composition containing platelets or a platelet derivative (e.g., FDPD), in the presence of a PRP reagent (catalog number TS30.00 from Thrombinoscope) can generate TPH of about 25 nM to about 100 nM (e.g., about 25 nM to about 50 nM, about 25 to about 75 nM, about 50 to about 100 nM, about 75 to about 100 nM, about 35 nM to about 95 nM, about 45 to about 85 nM, about 55 to about 75 nM, or about 60 to about 70 nM).

[0071] The platelets or platelet derivatives (e.g., FDPD) described herein can generate thrombin in the presence of a reagent including, for example, tissue factor and phospholipids. For example, in some cases, the platelets or platelet derivatives (e.g., FDPD) can generate thrombin in the presence of a reagent including, for example, tissue factor and phospholipids. 6 For example, in some cases, the platelets or platelet derivatives (e.g., FDPD) may have at least 1.2 (e.g., at least 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5) thrombin generating potency units (TGPU) per particle. 6 1.2 to 2.5 TPGU per particle (e.g., 10 6 The calibrator may have a potency of 1.2-2.0, 1.3-1.5, 1.5-2.25, 1.5-2.0, 1.5-1.75, 1.75-2.5, 2.0-2.5, or 2.25-2.5 TPGU per 1,000,000 particles. TPGU can be calculated as follows: TGPU / 1,000,000 particles = [TPH in nM] * [potency factor in IU / (nM)] / [576,000 particles in well]. Similarly, the potency factor for a thrombin sample can be calculated as follows: potency factor = calculated calibrator activity (IU) / effective calibrator activity (nM). In some cases, the calibrator activity can be based on the WHO International Thrombin Standard.

[0072] The platelets or platelet derivatives described herein (e.g., FDPD) may be capable of clotting, as determined, for example, by using a Total Thrombus Analysis System (T-TAS®). In some cases, the platelets or platelet derivatives described herein may be capable of clotting at least 70×10 3 particles / µL (e.g., at least 73 x 10 3 pieces, 100×10 3 pieces, 150×10 3 pieces, 173×10 3 pieces, 200×10 3 pieces, 250×10 3 pieces, or 255 x 10 3 At a concentration of at least 70×10 particles / μL, for example, in platelet-reduced citrated whole blood, the platelets or platelet derivatives described herein may result in a T-TAS occlusion time (e.g., time to reach 80 kPa) of less than 14 minutes (e.g., less than 13.5, 13, 12.5, 12, 11.5, or 11 minutes). 3 particles / µL (e.g., at least 73 x 10 3 pieces, 100×10 3 pieces, 150×10 3 pieces, 173×10 3 pieces, 200×10 3 pieces, 250×10 3 pieces, or 255 x 10 3 At a concentration of 0.015 particles / μL, for example, in platelet-depleted citrated whole blood, the antibody may provide an area under the curve (AUC) of at least 1300 (e.g., at least 1380, 1400, 1500, 1600, or 1700).

[0073] A platelet or platelet derivative (e.g., FDPD) described herein may be capable of thrombin-induced capture in the presence of thrombin. In some cases, a platelet or platelet derivative (e.g., FDPD) described herein may have at least 5% (e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 67%, 70%, 75%, 85%, 90%, or 99%) thrombin-induced capture in the presence of thrombin. In some cases, platelets or platelet derivatives (e.g., FDPDs) described herein may have a thrombin-induced capture percentage of about 25% to about 100% (e.g., about 25% to about 50%, about 25% to about 75%, about 50% to about 100%, about 75% to about 100%, about 40% to about 95%, about 55% to about 80%, or about 65% to about 75%) in the presence of thrombin. Thrombin-induced capture may be determined by any suitable method, for example, light transmission aggregometry. Without being bound by any particular theory, it is believed that thrombin-induced capture is the result of an interaction between fibrinogen present on the surface of the platelet derivative and thrombin.

[0074] The platelet derivatives (e.g., FDPD) described herein may be capable of coaggregating in the presence of, for example, an aggregation agonist and fresh platelets. Non-limiting examples of aggregation agonists include collagen, epinephrine, ristocetin, arachidonic acid, adenosine diphosphate, and thrombin receptor-associated protein (TRAP). In some cases, the platelets or platelet derivatives (e.g., FDPD) described herein may have a percent coaggregation of at least 5% (e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 67%, 70%, 75%, 85%, 90%, or 99%) in the presence of an aggregation agonist and fresh platelets. In some cases, platelets or platelet derivatives (e.g., FDPD) described herein may have a percent coaggregation of about 25% to about 100% (e.g., about 25% to about 50%, about 25% to about 75%, about 50% to about 100%, about 75% to about 100%, about 40% to about 95%, about 55% to about 80%, or about 65% to about 75%) in the presence of an aggregation agonist. Percent coaggregation may be determined by any suitable method, such as light transmission aggregometry.

[0075] In certain exemplary embodiments herein, the platelet derivative composition, an FDPD composition, comprises a population of platelet derivatives (e.g., FDPDs) that have a reduced tendency to aggregate under aggregation conditions that include an agonist but do not include fresh platelets, compared to the tendency of fresh platelets, and / or are activated to aggregate under these conditions. The platelet derivatives (e.g., FDPDs) described herein in exemplary embodiments exhibit a reduced tendency to aggregate under aggregation conditions that include an agonist but do not include fresh platelets, compared to the tendency of fresh platelets and / or activated platelets to aggregate under these conditions. Surprisingly, such FDPDs have the ability to increase platelet coagulation and aggregation in in vitro and in vivo assays in the presence of antithrombotic agents, such as anticoagulants and antiplatelet agents, under conditions in which such antithrombotic agents reduce coagulation and / or aggregation, including in the presence of two of such agents. It is notable that platelet derivative aggregation differs from coaggregation in that aggregation conditions typically do not include fresh platelets, whereas coaggregation conditions include fresh platelets. Exemplary aggregation and coaggregation conditions are provided in the Examples herein. Thus, in some embodiments, the platelet derivatives described herein have an increased tendency to coaggregate in the presence of fresh platelets and an agonist, while exhibiting a decreased tendency to aggregate in the absence of fresh platelets and an agonist compared to the tendency of fresh platelets to aggregate under these conditions. In some embodiments, the platelet derivative compositions comprise a population of platelet derivatives with a decreased tendency to aggregate, such that no more than 2%, 3%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, or 25% of the platelet derivatives in the population aggregate under aggregation conditions that include an agonist but not platelets, in exemplary embodiments, not including fresh platelets. In some embodiments, the population of platelet derivatives aggregates in the range of 2-30%, 5-25%, 10-30%, 10-25%, or 12.5-25% of the platelet derivatives under aggregation conditions that include an agonist but not platelets, in exemplary embodiments, not including fresh platelets.

[0076] As provided in the Examples herein, exemplary aggregation conditions and related methods include treating an FDPD sample preparation with an agonist at room temperature, for example, 20 μM ADP, 0.5 mg / mL arachidonic acid, 10 μg / mL collagen, 200 μM epinephrine, 1 mg / mL ristocetin, and a final agonist concentration of 10 μM TRAP-6, and measuring by LTA, for example, 5 minutes after adding the agonist to the FDPD sample, which can be compared to the LTA measurement of the sample before adding the agonist.

[0077] In some embodiments, the platelet derivatives described herein are maximally activated such that in the presence of an agonist, the platelet derivatives fail to exhibit an increase in the level of platelet activation markers present thereon compared to the level of the platelet activation marker present before exposure to the agonist. In some embodiments, the platelet derivatives described herein fail to increase the expression of platelet activation markers in the presence of an agonist compared to the expression of the platelet activation marker in the absence of the agonist. In some embodiments, the agonist is selected from the group consisting of collagen, epinephrine, ristocetin, arachidonic acid, adenosine diphosphate, and thrombin receptor-associated protein (TRAP). In some embodiments, the platelet activation marker is selected from the group consisting of annexin V and CD62. In some embodiments, the platelet derivatives described herein fail to increase the expression of annexin V in the presence of TRAP. An increase in the amount of a platelet activation marker on a platelet indicates a state of platelet activation. However, in some embodiments, the platelet derivatives described herein fail to increase the amount of a platelet activation marker on their surface, even in the presence of an agonist. This characteristic indicates that the platelet derivatives described herein are maximally activated. In some embodiments, the platelet derivatives described herein can exhibit a state of maximal activation in the absence of an agonist, and thus the properties can be beneficial when maximal activation of platelets is required.

[0078] Thrombospondin is a glycoprotein secreted from the alpha granules of platelets upon activation. In the presence of divalent cations, the secreted protein binds to the surface of activated platelets and is responsible for the intrinsic lectin-like activity associated with activated platelets. In some embodiments, platelet derivatives have thrombospondin (TSP-1) present on their surface at a level greater than that present on the surface of resting, activated, or freeze-dried, fixed platelets. In some embodiments, platelet derivatives have thrombospondin (TSP-1) present on their surface at a level at least 10%, 20%, 25%, 30%, 50%, 60%, 70%, 80%, 90%, or 100% greater than that present on the surface of resting or freeze-dried, fixed platelets. In some embodiments, platelet derivatives have thrombospondin (TSP-1) present on their surface at a level greater than or equal to 100% greater than that present on the surface of resting or freeze-dried, fixed platelets. In some embodiments, when analyzed for binding of an anti-thrombospondin (TSP) antibody to a platelet derivative using flow cytometry, the platelet derivative exhibits a mean fluorescence intensity (MFI) that is at least 2-fold, 5-fold, 7-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold greater than the MFI for binding of the anti-TSP antibody to resting platelets in the absence of an agonist. In some embodiments, when analyzed for binding of an anti-thrombospondin (TSP) antibody to a platelet derivative using flow cytometry, the platelet derivative exhibits a mean fluorescence intensity (MFI) that is at least 2-fold, 5-fold, 7-fold, 10-fold, 20-fold, 30-fold, or 40-fold greater than the MFI for binding of the anti-TSP antibody to lyophilized, fixed platelets in the absence of an agonist. In some embodiments, when analyzed for binding of an anti-thrombospondin (TSP) antibody to a platelet derivative using flow cytometry, the platelet derivative exhibits a mean fluorescence intensity (MFI) that is 10-800 times, 20-800 times, 100-700 times, 150-700 times, 200-700 times, or 250-500 times higher compared to the MFI of binding of the anti-TSP antibody to resting platelets in the absence of agonist.In some embodiments, when analyzed for binding of an anti-thrombospondin (TSP) antibody to a platelet derivative using flow cytometry, the platelet derivative exhibits a mean fluorescence intensity (MFI) that is at least 2-fold, 5-fold, 7-fold, 10-fold, 20-fold, 30-fold, or 40-fold higher than the MFI of binding of the anti-TSP antibody to activated platelets in the absence of an agonist. In some embodiments, when analyzed for binding of an anti-thrombospondin (TSP) antibody to a platelet derivative using flow cytometry, the platelet derivative exhibits a mean fluorescence intensity (MFI) that is 2-40-fold, 5-40-fold, 5-35-fold, 10-35-fold, or 10-30-fold higher than the MFI of binding of the anti-TSP antibody to activated platelets in the absence of an agonist.

[0079] Von Willebrand factor (vWF) is a multimeric glycoprotein that plays a major role in blood coagulation. vWF functions as a bridging molecule, promoting platelet binding to the subendothelium and to other platelets, thereby facilitating platelet adhesion and aggregation. vWF also binds to collagen, promoting clot formation at the site of injury. In some embodiments, the platelet derivatives described herein have von Willebrand factor (vWF) present on their surface at a level greater than that present on the surface of resting, activated, or freeze-dried, fixed platelets. In some embodiments, the platelet derivatives have von Willebrand factor (vWF) present on their surface at a level at least 10%, 20%, 25%, 30%, 50%, 60%, 70%, 80%, 90%, or 100% greater than that present on the surface of resting or freeze-dried, fixed platelets. In some embodiments, when analyzed for binding of an anti-von Willebrand factor (vWF) antibody to the platelet derivative using flow cytometry, the platelet derivative exhibits a mean fluorescence intensity (MFI) that is at least 1.5-fold, 2-fold, 3-fold, or 4-fold higher than the MFI of binding of the anti-vWF antibody to resting platelets or freeze-dried, fixed platelets in the absence of an agonist. In some embodiments, when analyzed for binding of an anti-von Willebrand factor (vWF) antibody to the platelet derivative using flow cytometry, the platelet derivative exhibits a mean fluorescence intensity (MFI) that is 2-4-fold, or 2.5-3.5-fold higher than the MFI of binding of the anti-vWF antibody to resting platelets or freeze-dried, fixed platelets in the absence of an agonist.

[0080] In further exemplary aspects and embodiments herein, platelet derivatives, in exemplary embodiments, FDPD, are surrounded by a compromised plasma membrane. In these further exemplary aspects and embodiments, the platelet derivatives lack an integral membrane around them. Instead, the membrane surrounding such platelet derivatives (e.g., FDPD) contains pores larger than those observed in living cells. Without being limited by theory, in embodiments in which the platelet derivative has a compromised membrane, it is believed that such platelet derivatives have a reduced ability or are unable to transduce signals from the external environment into intraparticle responses typically transduced in living platelets. Furthermore, such platelet derivatives (e.g., FDPD) are not believed to be capable of mitochondrial activation or glycolysis.

[0081] A compromised membrane can be identified by the platelet derivative's inability to retain more than 50% of the lactate dehydrogenase (LDH) compared to fresh, cryopreserved, or cryopreserved platelets. In some embodiments, the platelet derivative cannot retain more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the lactate dehydrogenase retained in fresh, cryopreserved, or cryopreserved platelets. In some embodiments, the platelet derivative exhibits increased permeability to antibodies. In some embodiments, the antibody can be an IgG antibody. Increased permeability can be identified by targeting of the IgG antibody to a stable intracellular antigen. One non-limiting type of stable intracellular antigen is beta-tubulin. A compromised membrane in a platelet derivative can also be determined by flow cytometry studies.

[0082] Platelets or platelet derivatives (e.g., FDPD) described herein may retain some metabolic activity, as evidenced, for example, by lactate dehydrogenase (LDH) activity. In some cases, platelets or platelet derivatives (e.g., FDPD) described herein may retain at least about 10% (e.g., at least about 12%, 15%, 20%, 25%, 30%, 35%, 40%, or 45%) of the LDH activity of donor apheresis platelets. Without being bound by any particular theory, it is believed that adding increasing amounts of polysucrose increases the amount of remaining LDH activity (e.g., products of formulations with 8% polysucrose have more retained LDH activity than products of formulations with 4% polysucrose). Also without being bound by any particular theory, it is believed that heat treatment of lyophilized compositions containing platelets or platelet derivatives (e.g., FDPD) increases the amount of retained LDH activity. As another example, metabolic activity can be evidenced by retained esterase activity, such as the ability of cells to cleave the acetate group on carboxyfluorescein diacetate succinimidyl ester (CFDASE) to unmask the fluorophore.

[0083] Coagulation parameters of blood (e.g., a subject's blood) can be assessed at any appropriate time during the methods described herein. For example, one or more coagulation parameters of blood can be assessed prior to administration of a composition comprising platelets, or in an exemplary embodiment, a composition comprising a platelet derivative (which in a further exemplary embodiment is an FDPD as described herein), e.g., to determine the need for administration of a composition comprising platelets or a platelet derivative as described herein. For example, such coagulation parameters can be assessed using a pre-administration evaluation or test, such as an in vitro clinical test. Such a test can be performed on a fluid sample, e.g., a blood sample, collected within 7, 5, 3, 2, or 1 day, or within 12, 8, 6, 4, 2, or 1 hour, prior to administering a composition comprising a platelet derivative to the subject. As another example, one or more coagulation parameters of blood can be assessed after administration of a composition comprising platelets or a platelet derivative as described herein, e.g., to determine the effectiveness of the administered composition, to determine whether an additional dose of the composition is warranted, or to determine whether it is safe to perform a surgical procedure. Such post-administration evaluations or tests can be performed on a fluid sample, e.g., a blood sample, taken within 7, 5, 3, 2, or 1 day, or within 12, 8, 6, 4, 2, or 1 hour, after administering a composition comprising a platelet derivative to a subject.

[0084] Thus, any of the methods described herein can include assessing one or more coagulation parameters of the blood prior to administration of a composition comprising platelets or platelet derivatives described herein, assessing one or more coagulation parameters of the blood after administration of a composition comprising platelets or platelet derivatives described herein, or both.

[0085] Any suitable method can be used to estimate (evaluate) blood coagulation parameters. Non-limiting examples of methods include the World Health Organization (WHO) bleeding scale, prothrombin time (PT) assay, thrombin generation (TGA; for example, can be used to generate parameters such as peak thrombin, endogenous thrombin potential (ETP), and lag time), thromboelastography (TEG), multielectrode aggregometry, light transmittance aggregometry (LTA), activated clotting time (ACT), P2Y12 response unit (PRU) or aspirin response unit (ARU) test, and partial thromboplastin time (PTT or aPTT).

[0086] The WHO bleeding scale was developed to help clinicians and researchers estimate bleeding, particularly in the context of toxicity reporting in cancer care, but it is also used in other contexts.

[0087] Prothrombin time (PT) is a measure of the time it takes for blood to clot, typically in the presence of tissue factor. In some cases, PT can be affected by laboratory reagents, so the normalized interstitial pressure ratio (INR) is more often used.

[0088] The activated partial thromboplastin time (aPTT) is a measure of the time it takes for blood to clot, typically in the presence of an activator such as silica, celite, kaolin, or ellagic acid. In some cases, the INR may be used instead of or in addition to the aPTT, as the aPTT can be affected by laboratory reagents.

[0089] The thrombin generation assay measured thrombin generation after procoagulant-mediated sample activation, which results from thrombin enzymatic cleavage of a fluorescent peptide and release of a fluorescent molecule. Peak thrombin is a measure of the maximum thrombin generated, lag time is the time to the onset of thrombin generation, and ETP is the total thrombin potentially generated.

[0090] In some embodiments, the patient may have a peak thrombin of about 60 nM to about 170 nM, e.g., about 65 nM to about 170 nM, e.g., about 65 nM to about 120 nM, e.g., about 80 nM, prior to administration of a composition comprising platelets or platelet derivatives described herein.

[0091] Thrombin clotting time (TCT) is a measure of the time it takes for blood to clot after excess thrombin is added.

[0092] TEG assesses intrinsic hemostasis via a plot of clot strength over time. Calcium chloride (CaCl) is typically used as the starting reagent. The TEG waveform (see, e.g., Figure 16) has multiple parameters that can provide information about clotting. R time = reaction time - the waiting time from the start of the test to initial fibrin formation. K = kinetics - rate of initial fibrin formation, time taken to achieve a certain level of clot strength (e.g., 20 mm amplitude) Alpha angle = slope of the line between R and K - measures the rate of clot formation. MA = Maximum amplitude (mm) - represents the final strength of the fibrin clot. A 30 = Amplitude after 30 min of reaching maximum amplitude - represents the rate of dissolution phase.

[0093] In hypocoagulable blood, the R time increases and the MA decreases. The R time typically provides a wider range of response than the MA.

[0094] Multielectrode aggregometry (MEA) can be used to assess blood coagulation parameters. MEA measures the change in electrical impedance as platelets aggregate on a metal electrode. Typically, aggregation agonists such as ADP, epinephrine, collagen, or ristocetin are used to initiate aggregation.

[0095] Light transmittance assays (LTA) may be used to assess blood clotting parameters; unagglutinated blood allows very little light to pass through, while aggregation (typically initiated by an agonist) results in increased aggregation.

[0096] In the Total Thrombus Formation Analysis System (T-TAS®, FUJIMORI KOGYO CO., LTD.), samples are passed through a collagen-coated microchannel using mineral oil. The change in pressure is used to estimate clot formation. The occlusion onset time is the time it takes to reach 10 kPa, and the occlusion time is the time it takes to reach a Δ80 kPa using an AR chip (e.g., Zacros item number, TC0101). According to the manufacturer, the AR chip can be used to analyze the formation of mixed white clots, which are primarily composed of fibrin and activated platelets. It has a channel (300 μm wide x 50 μm high) coated with collagen and tissue factor and can be used to analyze coagulation function and platelet function. In comparison, the PL chip can be used to analyze the formation of platelet clots, which are primarily composed of activated platelets. The PL chip has a channel coated only with collagen and can be used to analyze platelet function.

[0097] The ACT assay measures the clotting time (t ACT This is the most basic, yet perhaps most reliable, method for measuring t, which is determined by the resistance to gravity of a magnet as a clot forms around it. A typical donor's blood is measured using only CaCl2, with a t of approximately 200-300 seconds. ACT It has.

[0098] VerifyNow measures platelet aggregation in P2Y12 inhibitors (PRU) in PRU units (P2Y12 response units) or platelet dysfunction in aspirin in ARU units (aspirin response units). The VerifyNow system is a turbidimetric-based optical detection system utilizing microbeads that measure platelet-induced aggregation as an increase in light transmittance, available from Werfren (https: / / www.werfen.com).

[0099] The VerifyNow-P2Y12 Assay / VerifyNow PRU Test is a rapid test that stimulates platelets with ADP in the presence of PGE1 [prostaglandin E1] to inhibit downstream activation of the second ADP receptor, P2Y1, making the assay more sensitive and specific for the P2Y12 receptor and the activity of drugs that bind to the P2Y12 receptor. The assay system reagents are designed to specifically measure P2Y12-mediated platelet aggregation.

[0100] The methodology of the VerifyNow Aspirin Assay is very similar to the VerifyNow-P2Y12 Assay / VerifyNow PRU Test, which incorporates arachidonic acid to measure platelet response to aspirin. In individuals taking aspirin, aspirin irreversibly inhibits COX-1, an enzyme that converts arachidonic acid to thromboxane A2 [TxA2] and ultimately activates the GPIIb / IIIa receptor, which is involved in platelet aggregation. In the presence of aspirin, aggregation does not occur.

[0101] Exemplary normal ranges for some of these coagulation parameters are shown below in Table E1. Typically, values ​​outside the ranges shown below are considered abnormal.

[0102] (Table E1) TIFF2024507362000002.tif157151TIFF2024507362000003.tif107144

[0103] Some embodiments provide methods for increasing thrombin generation in a subject, the methods comprising administering to a subject in need thereof an effective amount of a composition, which in an exemplary embodiment comprises an effective amount of a platelet derivative (which in a further exemplary embodiment is FDPD), the composition comprising an incubation material comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0104] Some embodiments provide a method of increasing thrombin generation in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form a composition.

[0105] Some embodiments provide methods for increasing peak thrombin in a subject, the methods comprising administering to a subject in need thereof an effective amount of a composition, which in an exemplary embodiment comprises an effective amount of a platelet derivative (which in a further exemplary embodiment is FDPD), the composition comprising an incubation material comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0106] Some embodiments provide a method of increasing peak thrombin in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0107] In some embodiments, prior to administration, the subject's peak thrombin was less than 66 nM (e.g., less than 64 nM, 62 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, or 5 nM). In some embodiments, after administration, the subject's peak thrombin is greater than 66 nM (e.g., greater than 68 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, or 150 nM). In some embodiments, after administration, the subject's peak thrombin is between 66 and 166 nM. Peak thrombin can be measured by any suitable method.

[0108] In some embodiments, a composition provided herein, or a composition produced by a method described herein, can be administered to a subject to indicate an abnormal result in the assessment of one or more coagulation parameters, e.g., that the subject is in a hypocoagulable state.

[0109] Some embodiments include a method of treating a coagulation disorder in a subject who is or has been administered an antiplatelet agent, the method comprising: (a) determining that the subject has an abnormal result on an assessment of one or more coagulation parameters; and (b) after (a), administering to the subject in need thereof an effective amount of a composition comprising platelets or a platelet derivative and an incubation material comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0110] Some embodiments include a method of treating a coagulation disorder in a subject who is or has been administered an antiplatelet agent, the method comprising: (a) determining that the subject has an abnormal result on an assessment of one or more coagulation parameters; and (b) after (a), administering to the subject in need thereof an effective amount of a composition, the composition having been prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0111] Some embodiments include a method of treating a coagulation disorder in a subject who is receiving or has been administered an antiplatelet agent, the method comprising administering to a subject in need thereof an effective amount of a composition comprising platelets or platelet derivatives and an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, wherein prior to administration, e.g., moments before or immediately before administration, the subject has been determined to have an abnormal result on an assessment of one or more coagulation parameters.

[0112] Some embodiments include a method of treating a coagulation disorder in a subject who is or has been administered an antiplatelet agent, the method comprising administering to a subject in need thereof an effective amount of a composition prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form a composition, and prior to administration, e.g., moments before or immediately before administration, the subject has been determined to have an abnormal result on an assessment of one or more coagulation parameters.

[0113] In some embodiments of any of the methods herein, the subject has been administered an antiplatelet agent or has been administered an antiplatelet agent within any suitable time frame. For example, in some cases, the subject has been administered a composition comprising an antiplatelet agent and / or a platelet derivative, in exemplary embodiments comprising FDPD, before the effects of a previous dose of antiplatelet agent have worn off. For example, in some cases, the subject has been administered an antiplatelet agent and the effects of the antiplatelet agent have not worn off. As another example, in some cases, the subject has been administered an antiplatelet agent (e.g., the most recent dose) within about 1 week, about 5 days, about 3 days, about 36 hours, about 24 hours, about 18 hours, about 12 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, or about 1 hour. As another example, in some cases, the subject has been administered an antiplatelet agent and the last dose (e.g., the most recent dose prescribed by a medical professional or self-administered by the subject) was within about 1 week, within about 5 days, within about 3 days, within about 36 hours, within about 24 hours, within about 18 hours, within about 12 hours, within about 8 hours, within about 6 hours, within about 4 hours, within about 2 hours, or within about 1 hour.

[0114] In some embodiments of any of the methods herein, the determination of an abnormal result for the assessment of one or more clotting parameters may be at any suitable time point. For example, the determination of an abnormal result for the assessment of one or more clotting parameters may be before the abnormal result reverts to a normal result. As another example, the determination of an abnormal result for the assessment of one or more clotting parameters may be within about 1 week, about 5 days, about 3 days, about 36 hours, about 24 hours, about 18 hours, about 12 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, or about 1 hour of administration.

[0115] In some embodiments, the method may further include determining a result of the evaluation of the one or more coagulation parameters after administration. For example, in some embodiments, the evaluation of the one or more coagulation parameters after administration indicates a normal result for at least one of the one or more coagulation parameters. In some embodiments, the result of the evaluation of the one or more coagulation parameters after administration is improved from the result of the evaluation of the one or more parameters before administration.

[0116] In some cases, a subject may be administered an antiplatelet agent that should not have been administered, for example, if the subject is confused or if a medical error has occurred. In some such cases, the subject may be administered any of the compositions provided herein or a composition produced by any of the methods described herein.

[0117] Some embodiments include a method of treating a coagulation disorder in a subject, the method comprising: (a) determining that an antiplatelet agent has been administered to the subject contrary to medical indication; and (b) administering to the subject in need thereof an effective amount of a composition comprising platelets or a platelet derivative and an incubation material comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0118] Some embodiments include a method of treating a coagulation disorder in a subject, the method comprising: (a) determining that the subject has been administered an antiplatelet agent contrary to medical indication; and (b) administering to the subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition.

[0119] Some embodiments include a method of treating a coagulation disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition comprising platelets or a platelet derivative and an incubation material comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, wherein the subject is determined to have been administered an antiplatelet agent against medical indication.

[0120] Some embodiments include a method of treating a coagulation disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form a composition, wherein the subject is determined to have been administered an antiplatelet agent against medical indication.

[0121] Some embodiments include a method of restoring normal hemostasis in a subject, the method comprising: (a) determining that an antiplatelet agent has been administered to the subject contrary to medical indication; and (b) administering to the subject in need thereof an effective amount of a composition comprising platelets or a platelet derivative and an incubation material comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0122] Some embodiments include a method of restoring normal hemostasis in a subject, the method comprising: (a) determining that an antiplatelet agent has been administered to the subject contrary to medical indication; and (b) administering to the subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition.

[0123] Some embodiments include a method of restoring normal hemostasis in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition comprising platelets or a platelet derivative and an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, wherein the subject is determined to have been administered an antiplatelet agent against medical indication.

[0124] Some embodiments include a method of restoring normal hemostasis in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form a composition, wherein the subject is determined to have been administered an antiplatelet agent contrary to medical indications.

[0125] In some embodiments of any of the methods herein, determining that a subject has been administered an antiplatelet agent contrary to medical indication can be at any appropriate time. For example, determining that a subject has been administered an antiplatelet agent contrary to medical indication can be before the antiplatelet agent has disappeared. As another example, determining that a subject has been administered an antiplatelet agent contrary to medical indication can be within about 1 week, within about 5 days, within about 3 days, within about 36 hours, within about 24 hours, within about 18 hours, within about 12 hours, within about 8 hours, within about 6 hours, within about 4 hours, within about 2 hours, or within about 1 hour of administering a composition provided herein or a composition produced by a method described herein.

[0126] Administration of the antiplatelet agent can involve any suitable method, including self-administration by the subject or administration by a medical professional.

[0127] The medical instructions may be in any suitable manner, including oral instructions, written instructions, or both oral and written instructions.

[0128] In some cases, the subject may have been or is being administered a second agent that affects (e.g., decreases) platelet function, e.g., such administration may render the subject hypercoagulable.

[0129] Some embodiments include a method of treating a coagulation disorder in a subject, the method comprising: (a) determining that the subject has been administered an antiplatelet agent and a second agent that reduces platelet function; and (b) administering to the subject in need thereof an effective amount of a composition comprising platelets or platelet derivatives and an incubation material comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0130] Some embodiments include a method of treating a coagulation disorder in a subject, the method comprising: (a) determining that the subject has been administered an antiplatelet agent and a second agent that reduces platelet function; and (b) administering to the subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0131] Some embodiments include a method of treating a coagulation disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition comprising platelets or platelet derivatives and an incubation material comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, wherein the subject is determined to have been administered an antiplatelet agent and a second agent that reduces platelet function.

[0132] Some embodiments include a method of treating a coagulation disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form a composition, wherein the subject is determined to have been administered an antiplatelet agent and a second agent that reduces platelet function.

[0133] Some embodiments include a method of restoring normal hemostasis in a subject, the method comprising: (a) determining that an antiplatelet agent and a second agent that reduces platelet function have been administered to the subject; and (b) administering to the subject in need thereof an effective amount of a composition comprising platelets or platelet derivatives and an incubation material comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0134] Some embodiments include a method of restoring normal hemostasis in a subject, the method comprising: (a) determining that the subject has been administered an antiplatelet agent and a second agent that reduces platelet function; and (b) administering to the subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0135] Some embodiments include a method of restoring normal hemostasis in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition comprising platelets or platelet derivatives and an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, wherein the subject is identified as having been administered an antiplatelet agent and a second agent that reduces platelet function.

[0136] Some embodiments include a method of restoring normal hemostasis in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form a composition, wherein the subject is identified as having been administered an antiplatelet agent and a second agent that reduces platelet function.

[0137] In some embodiments, administration of the second agent is stopped (e.g., if the benefits of stopping the second agent outweigh the costs of stopping the second agent), while in some embodiments, administration of the second agent is continued (e.g., if removal of the second agent would be harmful to the subject, as can be the case with certain medications, such as antidepressants).

[0138] The second agent can be any suitable second agent that affects (e.g., decreases) platelet function. For example, the second agent can include (or can be selected from the group consisting of) an antihypertensive agent, a proton pump inhibitor, or a combination thereof. As another example, the second agent can include (or can be selected from the group consisting of) a chemotherapeutic agent, an antibiotic, a cardiovascular agent, an H2 antagonist, a neuropsychiatric agent, or a combination thereof. In some embodiments, the second agent can include (or can be) an antidepressant (e.g., a selective serotonin reuptake inhibitor (SSRI), a serotonin antagonist and reuptake inhibitor (SARI), a serotonin and norepinephrine reuptake inhibitor (SNRI), or a combination thereof). In some embodiments, the second agent is not an anticoagulant.

[0139] In some embodiments of any of the methods provided herein, administration of the antiplatelet agent is stopped. In some embodiments of any of the methods provided herein, administration of the antiplatelet agent is continued.

[0140] In certain emergency situations, it may be impossible to determine in a timely manner whether a subject is receiving an antiplatelet agent. In some such cases, a composition provided herein or a composition produced by a method provided herein may be administered to a subject to prevent coagulation disorders. In some embodiments, a composition provided herein or a composition produced by a method provided herein may be administered to a subject to reduce the likelihood of coagulation disorders in the subject.

[0141] Some embodiments include a method of preventing or reducing the likelihood of a coagulation disorder in a subject, the method comprising: (a) determining that information regarding whether the subject has been administered an antiplatelet agent is unavailable; and (b) administering to the subject an effective amount of a composition comprising platelets or platelet derivatives and an incubation material comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0142] Some embodiments include a method of preventing or reducing the likelihood of a coagulation disorder in a subject, the method comprising: (a) determining that information regarding whether the subject has been administered an antiplatelet agent is unavailable; and (b) administering to the subject an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0143] Some embodiments include a method of preventing or reducing the likelihood of coagulation disorders in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition comprising platelets or platelet derivatives and an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, wherein the subject has been determined to be a subject for whom information regarding whether the subject has been administered an antiplatelet agent is unavailable.

[0144] Some embodiments include a method of preventing or reducing the likelihood of a coagulation disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form a composition, wherein the subject has been determined to be a subject for which information regarding whether the subject has been administered an antiplatelet agent is unavailable.

[0145] In some embodiments of any of the methods herein, determining that information regarding whether the subject has been administered an antiplatelet agent is unavailable can be at any suitable time point. For example, determining that information regarding whether the subject has been administered an antiplatelet agent can be within about 1 week, within about 5 days, within about 3 days, within about 36 hours, within about 24 hours, within about 18 hours, within about 12 hours, within about 8 hours, within about 6 hours, within about 4 hours, within about 2 hours, or within about 1 hour of administering a composition provided herein or a composition produced by a method described herein.

[0146] There are several reasons why information about whether a subject has received an antiplatelet agent may not be available, including, for example, that the subject cannot be identified, that the subject's medical history is unavailable, that the subject requires emergency treatment, that the subject requires emergency surgery, that the subject is undergoing emergency surgery, or a combination thereof.

[0147] In some embodiments of any of the methods provided herein, the method may further include determining that the subject has an abnormal result for the assessment of one or more of the coagulation parameters. In some embodiments of any of the methods provided herein, the subject has been determined to have an abnormal result for the assessment of one or more of the coagulation parameters.

[0148] In some cases, before the abnormal result is determined, the subject was previously identified as having a normal result for at least one of the one or more coagulation parameters.

[0149] In some embodiments of any of the methods provided herein, the method may further include determining the results of an assessment of one or more coagulation parameters after administration of a composition provided herein or a composition produced by a method provided herein. In some such cases, the assessment of the one or more coagulation parameters after administration indicates a normal result as defined in Table E1 for at least one of the one or more coagulation parameters. In some embodiments, the result of the assessment of the one or more coagulation parameters after administration is improved from the result of the assessment of the one or more parameters before administration.

[0150] In some embodiments, the subject is identified as having an abnormal result for one or more assessments of coagulation parameters during surgery (eg, emergency surgery or planned surgery).

[0151] The assessment of the one or more coagulation parameters can be any suitable assessment of coagulation parameters, such as any of the assessments of coagulation parameters provided herein. In some embodiments, the assessment of coagulation parameters can be selected from the group consisting of the World Health Organization (WHO) bleeding scale, prothrombin time (PT) assay, international normalized ratio (INR), thrombin generation (TGA), platelet elasticity (TEG), multielectrode aggregometry (MEA), light transmittance aggregometry (LTA), activated clotting time (ACT), VerifyNow, and partial thromboplastin time (e.g., PTT or aPTT), subparameters thereof, and any combination thereof.

[0152] In some embodiments, the one or more coagulation parameters include an assessment of bleeding (e.g., based on the World Health Organization (WHO) bleeding scale). In some embodiments, prior to administration, the subject has bleeding grade 2, 3, or 4 based on the WHO bleeding scale. In some embodiments, after administration, the subject has bleeding grade 0 or 1 based on the WHO bleeding scale. In some embodiments, after administration, the subject has bleeding one grade lower based on the WHO bleeding scale than before administration. In some embodiments, after administration, the subject has bleeding two grades lower based on the WHO bleeding scale than before administration. In some embodiments, after administration, the subject has bleeding three grades lower based on the WHO bleeding scale than before administration.

[0153] In some embodiments, the one or more coagulation parameters include an assessment of prothrombin time (PT). In some embodiments, an abnormal PT result includes a PT of greater than about 14 seconds (e.g., greater than about 15 seconds, greater than 18 seconds, greater than 20 seconds, greater than 25 seconds, or more). In some embodiments, after administration, the subject has a decrease in PT of at least 1 second (e.g., at least 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, or more). In some embodiments, after administration, the subject has a normal PT as defined in Table E1.

[0154] In some embodiments, the one or more clotting parameters include an assessment of activated partial thromboplastin time (aPTT). In some embodiments, an abnormal aPTT result includes an aPTT of greater than about 40 seconds (e.g., greater than about 43 seconds, 45 seconds, 50 seconds, 55 seconds, 60 seconds, 65 seconds, 70 seconds, or more). In some embodiments, after administration, the subject has a decrease in aPTT of at least 5 seconds (e.g., at least 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, or more). In some embodiments, after administration, the subject has a normal aPTT as defined in Table E1.

[0155] In some embodiments, the one or more coagulation parameters include an assessment of thrombin clotting time (TCT). In some embodiments, an abnormal TCT result includes a TCT of greater than about 35 seconds (e.g., greater than about 38 seconds, greater than 40 seconds, greater than 45 seconds, greater than 50 seconds, greater than 55 seconds, greater than 60 seconds, or more). In some embodiments, after administration, the subject has a decrease in TCT of at least 5 seconds (e.g., at least 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, or more). In some embodiments, after administration, the subject has a normal TCT as defined in Table E1.

[0156] In some embodiments, the assessment of one or more coagulation parameters includes thromboelastography (TEG). In some embodiments, an abnormal TEG result includes a maximum amplitude (MA) of less than about 50 mm (e.g., less than about 48 mm, less than 45 mm, less than 40 mm, less than 35 mm, or less). In some embodiments, after administration, the subject has an increase in MA of at least 5 mm (e.g., at least 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, or more). In some embodiments, after administration, the subject has normal MA as defined in Table E1. In some embodiments, an abnormal TEG result includes a percent aggregation of less than about 85% (e.g., less than about 83%, less than 80%, less than 75%, less than 70%, or less) (in the presence of 1 mmol / L arachidonic acid). In some embodiments, after administration, the subject has an increase in percent aggregation (in the presence of 1 mmol / L arachidonic acid) of at least 2 percentage points (e.g., at least 3 percentage points, 5 percentage points, 8 percentage points, 10 percentage points, 12 percentage points, 15 percentage points, 18 percentage points, 20 percentage points, or more). In some embodiments, after administration, the subject has normal percent aggregation (in the presence of 1 mmol / L arachidonic acid) as defined in Table E1. In some embodiments, TEG is used to assess adenosine diphosphate-induced platelet-fibrin clot strength. In some embodiments, TEG is used to assess arachidonic acid-induced platelet-fibrin clot strength.

[0157] In some embodiments, the assessment of one or more coagulation parameters includes VerifyNow. In some embodiments, an abnormal VerifyNow result includes a P2Y12 response unit (PRU) of less than about 195 (e.g., less than about 190, less than 185, less than 180, less than 170, less than 165, less than 160, less than 155, or less). In some embodiments, after administration, the subject has an increase in PRU of at least 25 (e.g., at least 30, 35, 40, 45, 50, 75, 100, or more). In some embodiments, after administration, the subject has a normal PRU as defined in Table E1. In some embodiments, an abnormal VerifyNow result includes an aspirin response unit (ARU) of less than about 550 (e.g., less than about 540, less than 530, less than 520, less than 510, less than 500, less than 490, less than 480, less than 470, or less). In some embodiments, after administration, the subject has an increase in ARU of at least 25 (e.g., at least 30, 35, 40, 45, 50, 75, 100, or more). In some embodiments, after administration, the subject has a normal ARU as defined in Table E1.

[0158] In some embodiments, the assessment of one or more coagulation parameters comprises multielectrode aggregometry (MEA). In some embodiments, the abnormal MEA result comprises an abnormal ADP-induced platelet activity result. In some embodiments, the abnormal MEA result comprises an ADP-induced platelet activity result of less than about 50 units (U) (e.g., less than about 48 U, less than 45 U, less than 40 U, ​​less than 35 U, or less). In some embodiments, after administration, the subject has an increase in ADP-induced platelet activity of at least 5 U (e.g., at least 8 U, 10 U, 15 U, 20 U, or more). In some embodiments, after administration, the subject has a normal value for ADP-induced platelet activity, as defined in Table E1. In some embodiments, the abnormal MEA result comprises an abnormal arachidonic acid-induced platelet activity result. In some embodiments, an abnormal MEA result includes an arachidonic acid-induced platelet activity result of less than about 70 units (U) (e.g., less than about 68 U, less than 65 U, less than 60 U, less than 55 U, less than 50 U, less than 45 U, or less). In some embodiments, after administration, the subject has an increase in arachidonic acid-induced platelet activity of at least 5 units (e.g., at least 8 units, 10 units, 15 units, 20 units, or more). In some embodiments, after administration, the subject has a normal value for arachidonic acid-induced platelet activity, as defined in Table E1.

[0159] In some embodiments, the assessment of one or more coagulation parameters comprises light transmittance aggregometry (LTA). In some embodiments, an abnormal LTA result comprises a percent aggregation of less than about 60% (e.g., less than about 58%, 55%, 50%, 45%, or less) in the presence of 5 μmol / L adenosine diphosphate. In some embodiments, an abnormal LTA result comprises a percent aggregation of less than about 65% (e.g., less than about 63%, 60%, 55%, 50%, 45%, or less) in the presence of 2 μg / mL collagen. In some embodiments, an abnormal LTA result comprises a percent aggregation of less than about 65% (e.g., less than about 63%, 60%, 55%, 50%, 45%, or less) in the presence of 1 mmol / L arachidonic acid. In some embodiments, abnormal LTA results include percent aggregation of less than about 69% (e.g., less than about 67%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, or less) in the presence of 2 mmol / L arachidonic acid. In some embodiments, abnormal LTA results include percent aggregation of less than about 73% (e.g., less than about 70%, less than 65%, less than 60%, less than 55%, less than 50%, or less) in the presence of 5 mmol / L arachidonic acid. In some embodiments, after administration, the subject has an increase in percent aggregation (in the presence of 5 μmol / L adenosine diphosphate) of at least 2 percentage points (e.g., at least 3 percentage points, 5 percentage points, 8 percentage points, 10 percentage points, 12 percentage points, or more). In some embodiments, after administration, the subject has normal percent aggregation as defined in Table E1 (in the presence of 5 μmol / L adenosine diphosphate). In some embodiments, after administration, the subject has an increase in percent aggregation (in the presence of 2 μg / mL collagen) of at least 2 percentage points (e.g., at least 3 percentage points, 5 percentage points, 8 percentage points, 10 percentage points, 12 percentage points, or more).In some embodiments, after administration, the subject has normal percent aggregation as defined in Table E1 (in the presence of 2 μg / mL collagen). In some embodiments, after administration, the subject has an increase in percent aggregation (in the presence of 1 mmol / L arachidonic acid) of at least 2 percentage points (e.g., at least 3 percentage points, 5 percentage points, 8 percentage points, 10 percentage points, 12 percentage points, or more). In some embodiments, after administration, the subject has normal percent aggregation as defined in Table E1 (in the presence of 1 mmol / L arachidonic acid). In some embodiments, after administration, the subject has an increase in percent aggregation (in the presence of 2 mmol / L arachidonic acid) of at least 2 percentage points (e.g., at least 3 percentage points, 5 percentage points, 8 percentage points, 10 percentage points, 12 percentage points, or more). In some embodiments, after administration, the subject has normal percent aggregation as defined in Table E1 (in the presence of 2 mmol / L arachidonic acid). In some embodiments, after administration, the subject has an increase in percent aggregation (in the presence of 5 mmol / L arachidonic acid) of at least 2 percentage points (e.g., at least 3 percentage points, 5 percentage points, 8 percentage points, 10 percentage points, 12 percentage points, or more). In some embodiments, after administration, the subject has normal percent aggregation as defined in Table E1 (in the presence of 5 mmol / L arachidonic acid).

[0160] In some embodiments, an additional antiplatelet agent antagonist may be administered to a subject in addition to a composition provided herein or a composition produced by a method described herein. The additional antiplatelet agent antagonist may be administered in any order with a composition provided herein or a composition produced by a method provided herein. In some embodiments, administration of the composition occurs simultaneously with administration of the additional antiplatelet agent antagonist. In some embodiments, administration of the composition occurs after administration of the additional antiplatelet agent antagonist. In some embodiments, administration of the composition occurs before administration of the additional antiplatelet agent antagonist.

[0161] In one aspect of any of the embodiments herein, the subject does not have cancer.

[0162] As used herein, an "effective amount" refers to an amount of a composition comprising an amount of platelets, typically a platelet derivative (which in exemplary embodiments is FDPD), effective to treat a subject. Such treatment reduces the subject's likelihood of bleeding, for example, with respect to the methods herein for treating a coagulation disorder in a subject or for counteracting the effects of an antithrombotic agent (i.e., an antiplatelet or anticoagulant). In some embodiments, the likelihood of bleeding can be reduced to an extent such that normal hemostasis is restored in the subject, for at least a period of time, e.g., to the level of a subject without an antiplatelet agent in their body. Thus, in some embodiments, an effective amount of a composition comprising a platelet derivative, e.g., FDPD, is an amount that results in a reduction in the subject's likelihood of bleeding, and in some embodiments, normal hemostasis, over any period of time. In some embodiments, the likelihood of bleeding is reduced for at least 10, 20, 30, 40, 50, or 60 minutes after administration of an effective amount of a composition comprising a platelet derivative, e.g., FDPD, or a second, third, fourth, fifth, or sixth dose of a platelet derivative, each of which, or two or more of which, or all, cumulatively constitute an effective dose.

[0163] Such amounts of platelets, or typically platelet derivatives (e.g., FDPD), comprise any suitable dosage of a composition comprising a platelet derivative described herein that may be administered to a subject, which, in exemplary embodiments, results in a reduced likelihood of bleeding in the subject. For example, in some embodiments, the dose of a composition comprising platelets or a platelet derivative (e.g., FDPD) is about or exactly 1.0 x 10 7 ~1.0×10 11 particles (e.g., FDPD) / kg of subject, 1.0 x 10 7 ~1.0×10 10 Particles (e.g., FDPD) / Target kg , 1.6×10 7 ~1.0×10 10 particles (e.g., FDPD) / kg of subject, 1.6 x 10 7 ~5.1×10 9 particles (e.g., FDPD) / kg of subject, 1.6 x 10 7 ~3.0×10 9 particles (e.g., FDPD) / kg of subject, 1.6 x 10 7 ~1.0×10 9 particles (e.g., FDPD) / kg of subject, 1.6 x 10 7 ~5.0×10 8 particles (e.g., FDPD) / kg of subject, 1.6 x 10 7 ~1.0×10 8 particles (e.g., FDPD) / kg of subject, 1.6 x 10 7 ~5.0×10 7 particles (e.g., FDPD) / kg of subject, 5.0 x 10 7 ~1.0×10 8 particles (e.g., FDPD) / kg of subject, 1.0 x 10 8 ~5.0×10 8 particles (e.g., FDPD) / kg of subject, 5.0 x 10 8 ~1.0×10 9 particles (e.g., FDPD) / kg of subject, 1.0 x 10 9 ~5.0×10 9 particles (e.g., FDPD) / kg of subject, or 5.0 x 10 9 ~1.0×10 10In some embodiments, an effective amount of a composition comprising FDPD is an amount on an active basis having a potency of 250 to 5000 TGPU per kg of subject. Such amounts on an active basis can be combined with any of the particle count ranges / kg in this paragraph.

[0164] In certain embodiments, any of the dose ranges provided above, in exemplary embodiments, 1×10 11 Dose ranges including less than 1.0 x 10 particles / kg may be administered to a subject more than once. 7 particles ~ approx. 1.0 x 10 10 The dose range of the particles can be administered 2-10 times, or 2-8 times, or 2-6 times, or 3-8 times, or 3-6 times, or 4-6 times within a time frame of 1, 2, 3, 4, 5, or 7 days after the first dose.

[0165] In some embodiments of the methods herein, the composition is administered topically. In some embodiments, topical administration can include administration via a solution, cream, gel, suspension, putty, particle, or powder. In some embodiments, as described in PCT Publication No. 2017 / 040238 (e.g., paragraphs

[0013] -

[0069] ), which corresponds to U.S. Patent Application No. 15 / 776,255 (incorporated herein by reference in its entirety), topical administration can include administration via a bandage (e.g., an adhesive bandage or compression bandage) or medical closure (e.g., suture, staple), in which, for example, a platelet derivative (e.g., cryopreserved platelets (e.g., FDPD)) can be embedded or coated thereon.

[0166] In some embodiments of the methods herein, the composition is administered parenterally. In some exemplary embodiments of the methods herein, the composition is administered intravenously. In some embodiments of the methods herein, the composition is administered intramuscularly. In some embodiments of the methods herein, the composition is administered intrathecally. In some embodiments of the methods herein, the composition is administered subcutaneously. In some embodiments of the methods herein, the composition is administered intraperitoneally.

[0167] In some embodiments of the methods herein, the composition is dried before the administering step. In exemplary embodiments of the methods herein, the composition is freeze-dried before the administering step. In exemplary embodiments, such FDPD compositions are prepared according to the methods provided in the Examples herein. In exemplary embodiments of the methods herein, the composition is rehydrated after the drying or freeze-drying step, for example, within 24, 12, 8, 6, 4, 3, 2, or 1 hour, or within 30, 20, 15, 10, or 5 minutes before administration to a subject.

[0168] In some embodiments, the antiplatelet agent is selected from the group consisting of aspirin (also known as acetylsalicylic acid or ASA); a P2Y12 inhibitor such as cangrelor (e.g., KENGREAL®), ticagrelor (e.g., BRILINTA®), clopidogrel (e.g., PLAVIX®), or prasugrel (e.g., EFFIENT®); a glycoprotein IIb / IIIa inhibitor such as eptifibatide (e.g., INTEGRILIN®), tirofiban (e.g., AGGRASTAT®), or abciximab (e.g., REOPRO®); and a supplement, such as an herbal supplement; or any combination thereof. Examples of supplements include ginger, ginseng, ginkgo, green tea, kava, saw palmetto, boldo (Peumus boldus), danshen (Salvia miltiorrhiza), dong quai (Angelica sinensis), papaya (Carica papaya), fish oil, and vitamin E. Examples of herbal supplements include ginger, ginseng, and ginkgo. The prescribing information for each of the FDA-approved anticoagulants provided herein is incorporated by reference in its entirety. Such prescribing information includes, for example:

[0169] DURLAZA® (aspirin) Prescribing Information Highlights, Updated December 2019.

[0170] KENGREAL® (cangrelor) Prescribing Information Highlights, 6 / 2015.

[0171] BRILINTA® (ticagrelor) Prescribing Information Highlights, 09 / 2016.

[0172] Highlights of PLAVIX® (clopidogrel bisulfate) prescribing information, August 2010 update.

[0173] Highlights of EFFIENT® (prasugrel) prescribing information, updated September 2011.

[0174] Highlights of Prescribing Information for INTEGRILIN® (eptifibatide), Updated March 2013.

[0175] AGGRASTAT® (tirofiban) Prescribing Information Highlights, August 2016 Update.

[0176] REOPRO® (abciximab) Prescribing Information, Revision Date: 11 / 1997.

[0177] TICLID® (ticlopidine hydrochloride) Approval Package, Revised March 2001.

[0178] Description - MOTRIN® (ibuprofen), effective January 2007.

[0179] ZONTIVITY™ (vorapaxar) Prescribing Information Highlights, Updated May 2014.

[0180] PLETAL® (cilostazol) Prescribing Information Highlights, Updated May 2017.

[0181] Highlights of VELETRI® (epoprostenol) prescribing information, updated June 2012.

[0182] PERSANTINE® (dipyridamole) prescribing information, updated December 2019.

[0183] REMODULIN® (acenocoumarol) prescribing information highlights, December 2014.

[0184] In some embodiments, the antiplatelet agent is aspirin. In some embodiments, the antiplatelet agent is cangrelor (e.g., KENGREAL®). In some embodiments, the antiplatelet agent is ticagrelor (e.g., BRILINTA®). In some embodiments, the antiplatelet agent is clopidogrel (e.g., PLAVIX®). In some embodiments, the antiplatelet agent is prasugrel (e.g., EFFIENT®). In some embodiments, the antiplatelet agent is eptifibatide (e.g., INTEGRILIN®). In some embodiments, the antiplatelet agent is tirofiban (e.g., AGGRASTAT®). In some embodiments, the antiplatelet agent is abciximab (e.g., REOPRO®). In some embodiments, the antiplatelet agent is terutroban. In some embodiments, the antiplatelet agent is picotamide. In some embodiments, the antiplatelet agent is elinogrel. In some embodiments, the antiplatelet agent is ticlopidine. In some embodiments, the antiplatelet agent is ibuprofen. In some embodiments, the antiplatelet agent is vorapaxar. In some embodiments, the antiplatelet agent is atopaxar. In some embodiments, the antiplatelet agent is cilostazol. In some embodiments, the antiplatelet agent is prostaglandin E1. In some embodiments, the antiplatelet agent is epoprostenol. In some embodiments, the antiplatelet agent is dipyridamole. In some embodiments, the antiplatelet agent is treprostinil sodium. In some embodiments, the antiplatelet agent is sarpogrelate. In some embodiments, the antiplatelet agent is a supplement. In some embodiments, the antiplatelet agent is an herbal supplement.

[0185] In some embodiments, the antiplatelet agent was last administered at a dosage and time relative to the time the platelet derivative-containing composition is administered to the subject, such that the subject's blood contains, in exemplary embodiments, a detectable amount of the antiplatelet agent. Typically, such an agent is present in the subject's blood at the time the platelet derivative-containing composition is administered to the subject, and in an amount sufficient to increase the subject's likelihood of bleeding. For example, the antiplatelet agent is present at a concentration sufficient to produce an abnormal value of one or more coagulation parameters, e.g., in an in vitro test. In some embodiments, the antiplatelet agent is present in the subject at the time the FDPD-containing composition is administered at a level that increases the subject's likelihood of bleeding. In some embodiments, the antiplatelet agent is present at Cmax within 15, 30, or 45 minutes, or within 1, 2, 3, 4, 6, or 8 hours after the FDPD-containing composition is administered or the first or last dose of the FDPD-containing composition is administered.

[0186] In some embodiments, the antiplatelet agent comprises aspirin at a dosage of about 80 mg to about 700 mg (e.g., 80 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, or 700 mg) once, twice, three times, or four times daily. In some embodiments, the antiplatelet agent comprises aspirin, and the subject has a C of about 3 to about 25 mg / L. max(e.g., about 3 to about 5 mg / L at a dose of about 100 mg, about 10 to about 15 mg / L at a dose of about 300 mg, or about 20 to about 25 mg / L at a dose of about 500 mg. See also, e.g., Nagelschmitz, J et al., "Pharmacokinetics and pharmacodynamics of acetylsalicylic acid after intravenous and oral administration to healthy volunteers," Clinical Pharmacology: Advances and Applications, vol. 6, pp. 51-9, 19 Mar. 2014, doi:10.2147 / CPAA.S47895.) In some embodiments, the antiplatelet agent comprises aspirin, and prior to administration of a composition provided herein or a composition produced by a method described herein, the subject had an ARU of about 350 to about 549 (e.g., about 400 to about 500) ARU. In some embodiments, the antiplatelet agent comprises aspirin, and after administration of a composition provided herein or a composition produced by a method described herein, the subject had an ARU of about 550 to about 700 ARU (e.g., about 600 to about 700 ARU). In some embodiments, the antiplatelet agent comprises aspirin, and before administration of a composition provided herein or a composition produced by a method described herein, the subject had a high on-treatment platelet reactivity (HPR) using more than about 30 units (e.g., more than about 33 units, more than 35 units, more than 40 units, more than 45 units, more than 50 units, or more) of MEA. See, e.g., Kruger, Jan-Christopher, et al., "Monitoring ASA and P2Y12-specific platelet inhibition—comparison of conventional (single) and multiple electrode aggregometry," Scandinavian Journal of Clinical and Laboratory Investigation 74.7 (2014):568-574.

[0187] In some embodiments, the antiplatelet agent comprises cangrelor at an initial dosage of about 25 to about 35 μg / kg body weight of the subject (e.g., about 30 μg / kg body weight of the subject) or a subsequent dosage of about 3 to about 5 μg / kg body weight of the subject / minute (e.g., about 4 μg / kg body weight of the subject / minute). In some embodiments, the antiplatelet agent comprises cangrelor, and the subject has a C of about 400 to about 500 ng / mL. max (See, for example, the KENGREAL® manufacturer's fact sheet at https: / / resources.chiesiusa.com / Kengreal / KENGREAL_Dosing_and_Administration_Fact_Sheet.pdf. In some embodiments, the antiplatelet agent comprises cangrelor, and prior to administration of a composition provided herein or a composition produced by a method described herein, the subject had a maximum amplitude (TEG-ADP) of less than about 50 mm (e.g., less than about 48 mm, less than 45 mm, less than 40 mm, or less). In some embodiments, the antiplatelet agent comprises cangrelor, and after administration of a composition provided herein or a composition produced by a method described herein, the subject had a maximum amplitude (TEG-ADP) of at least about 50 mm (e.g., at least 53 mm, 55 mm, 50 mm, 60 mm, 65 mm, 70 mm, or more). For example, Corliss BM,Polifka AJ,Harris NS,Hoh BL,Fox WC.Laboratory assessments of therapeutic platelet inhibition in endovascular neurosurgery:comparing results of the VerifyNow P2Y12 assay to thromboelastography with platelet mapping.J Neurosurg.2018 Nov 1;129(5):1160-1165.doi:10.3171 / 2017.6.JNS17535.PMID:29271717.

[0188] In some embodiments, the antiplatelet agent comprises ticagrelor at an initial dosage of about 170 to about 190 mg (e.g., about 180 mg), or at a subsequent dosage in the first year of treatment of about 80 to about 100 mg (e.g., about 90 mg) twice daily, or at a subsequent dosage in the second year of treatment of about 50 to about 70 mg (e.g., about 60 mg) twice daily, optionally in combination with aspirin. In some embodiments, the antiplatelet agent comprises ticagrelor, and the subject has a C of about 550 to about 650 ng / mL (e.g., about 600 mg / L). maxFor example, Teng R, Muldowney S, Zhao Y, Berg JK, Lu J, Khan ND. Pharmacokinetics and pharmacodynamics of ticagrelor in subjects on hemodialysis and subjects with normal renal function. Eur J Clin Pharmacol. 2018 Sep;74(9):1141-1148. doi:10.1007 / s00228-018-2484-7. Epub 2018 May 30. PMID:29850937; PMCID:PMC6096709. In some embodiments, the antiplatelet agent comprises ticagrelor, and prior to administration of a composition provided herein or a composition produced by a method described herein, the subject had a PRU of less than about 180 (e.g., less than about 175, less than 170, less than 165, less than 160, less than 155, less than 150, or less). In some embodiments, the antiplatelet agent comprises ticagrelor, and after administration of a composition provided herein or a composition produced by a method described herein, the subject had a PRU of at least 180 (e.g., at least 180, 185, 190, 195, 200, 225, 250, 275, 300, 350, or more). In some embodiments, the antiplatelet agent comprises ticagrelor, and after administration of a composition provided herein or a composition produced by a method described herein, the subject had a PRU of about 180 to about 376 PRU (e.g., about 200 to about 300 PRU). In some embodiments, the antiplatelet agent comprises ticagrelor, and prior to administration of a composition provided herein or a composition produced by a method described herein, the subject had a maximum amplitude (TEG-ADP) of less than about 50 mm (e.g., less than about 48 mm, less than 45 mm, less than 40 mm, or less).In some embodiments, the antiplatelet agent comprises ticagrelor, and after administering a composition provided herein or a composition produced by a method described herein, the subject has a maximum amplitude (TEG-ADP) of at least about 50 mm (e.g., at least 53 mm, 55 mm, 50 mm, 60 mm, 65 mm, 70 mm, or more). See, e.g., Corliss BM, Polifka AJ, Harris NS, Hoh BL, Fox WC. Laboratory assessments of therapeutic platelet inhibition in endovascular neurosurgery: comparing results of the VerifyNow P2Y12 assay to thromboelastography with platelet mapping. J Neurosurg. 2018 Nov 1;129(5):1160-1165. doi:10.3171 / 2017.6.JNS17535. PMID:29271717.

[0189] In some embodiments, the antiplatelet agent comprises clopidogrel at an initial dosage of about 275 to about 325 mg (e.g., about 300 mg) or at subsequent dosages of about 70 to about 80 mg (e.g., about 75 mg) once daily, optionally in combination with aspirin. In some embodiments, the antiplatelet agent comprises clopidogrel, and the subject has a C of about 1 to about 40 mg / L. max(e.g., about 1 to about 15 ng / mL at a dose of about 75 mg, or about 1 to about 40 ng / mL at a dose of about 300 mg). See, for example, Karazniewicz-Lada, Marta et al. "Clinical pharmacokinetics of clopidogrel and its metabolites in patients with cardiovascular diseases." Clinical Pharmacokinetics vol. 53, 2 (2014): 155-64. doi:10.1007 / s40262-013-0105-2. In some embodiments, the antiplatelet agent comprises clopidogrel, and prior to administration of a composition provided herein or a composition produced by a method described herein, the subject had a PRU of less than about 180 (e.g., less than about 175, less than 170, less than 165, less than 160, less than 155, less than 150, or less). In some embodiments, the antiplatelet agent comprises clopidogrel, and after administration of a composition provided herein or a composition produced by a method described herein, the subject had a PRU of at least 180 (e.g., at least 180, 185, 190, 195, 200, 225, 250, 275, 300, 350, or more). In some embodiments, the antiplatelet agent comprises clopidogrel, and after administration of a composition provided herein or a composition produced by a method described herein, the subject had a PRU of about 180 to about 376 PRU (e.g., about 200 to about 300 PRU). In some embodiments, the antiplatelet agent comprises clopidogrel, and prior to administration of a composition provided herein or a composition produced by a method described herein, the subject had a high on-treatment platelet reactivity (HPR) using greater than about 47 units (e.g., greater than about 48 units, greater than 50 units, greater than 55 units, or more) of MEA.For example, Kruger, Jan-Christopher, et al. "Monitoring ASA and P2Y12-specific platelet inhibition - comparison of conventional (single) and multiple electrode aggregometry." Scandinavian journal of clinical and laboratory investigation 74.7 (2014): 568-574. In some embodiments, the antiplatelet agent comprises clopidogrel, and prior to administration of a composition provided herein or a composition produced by a method described herein, the subject had a maximum amplitude (TEG-ADP) of less than about 50 mm (e.g., less than about 48 mm, less than 45 mm, less than 40 mm, or less). In some embodiments, the antiplatelet agent comprises clopidogrel, and after administration of a composition provided herein or a composition produced by a method described herein, the subject had a maximum amplitude (TEG-ADP) of at least about 50 mm (e.g., at least 53 mm, 55 mm, 50 mm, 60 mm, 65 mm, 70 mm, or more). For example, Corliss BM,Polifka AJ,Harris NS,Hoh BL,Fox WC.Laboratory assessments of therapeutic platelet inhibition in endovascular neurosurgery:comparing results of the VerifyNow P2Y12 assay to thromboelastography with platelet mapping.J Neurosurg.2018 Nov 1;129(5):1160-1165.doi:10.3171 / 2017.6.JNS17535.PMID:29271717.

[0190] In some embodiments, the antiplatelet agent comprises prasugrel, at an initial dosage of about 50 to about 70 mg (e.g., about 60 mg), or at subsequent dosages of about 3 to about 12 mg (e.g., about 5 mg or about 10 mg) once daily, optionally in combination with aspirin. In some embodiments, the antiplatelet agent comprises prasugrel, and the subject has a C of about 200 to about 525 ng / mL (e.g., about 330 to about 350 ng / mL at a dose of about 20 mg). maxachieved. See, for example, Umemura, Kazuo, and Takayuki Iwaki. "The Pharmacokinetics and Pharmacodynamics of Prasugrel and Clopidogrel in Healthy Japanese Volunteers." Clinical Pharmacology in Drug Development vol. 5, 6 (2016): 480-487. doi:10.1002 / cpdd.259. In some embodiments, the antiplatelet agent comprises prasugrel, and prior to administration of a composition provided herein or a composition produced by a method described herein, the subject had a PRU of less than about 180 (e.g., less than about 175, less than 170, less than 165, less than 160, less than 155, less than 150, or less). In some embodiments, the antiplatelet agent comprises prasugrel, and after administration of a composition provided herein or a composition produced by a method described herein, the subject had a PRU of at least 180 (e.g., at least 180, 185, 190, 195, 200, 225, 250, 275, 300, 350, or more). In some embodiments, the antiplatelet agent comprises prasugrel, and after administration of a composition provided herein or a composition produced by a method described herein, the subject had a PRU of about 180 to about 376 PRU (e.g., about 200 to about 300 PRU). In some embodiments, the antiplatelet agent comprises prasugrel, and prior to administration of a composition provided herein or a composition produced by a method described herein, the subject had a maximum amplitude (TEG-ADP) of less than about 50 mm (e.g., less than about 48 mm, less than 45 mm, less than 40 mm, or less). In some embodiments, the antiplatelet agent comprises prasugrel, and after administering a composition provided herein or a composition produced by a method described herein, the subject had a maximum amplitude (TEG-ADP) of at least about 50 mm (e.g., at least 53 mm, 55 mm, 50 mm, 60 mm, 65 mm, 70 mm, or more).See, e.g., Corliss BM, Polifka AJ, Harris NS, Hoh BL, Fox WC. Laboratory assessments of therapeutic platelet inhibition in endovascular neurosurgery: comparing results of the VerifyNow P2Y12 assay to thromboelastography with platelet mapping. J Neurosurg. 2018 Nov 1;129(5):1160-1165. doi:10.3171 / 2017.6.JNS17535. PMID:29271717. In some embodiments, the antiplatelet agent comprises prasugrel, and prior to administration of a composition provided herein or a composition produced by a method described herein, the subject had a high on-treatment platelet reactivity (HPR) using greater than about 47 units (e.g., greater than about 48 units, greater than 50 units, greater than 55 units, or more) of MEA. For example, Kruger, Jan-Christopher, et al. “Monitoring ASA and P2Y12-specific platelet inhibition-comparison of conventional(single) and multiple electrode aggregometry.” Scandinavian journal of clinical and laboratory investigation 74.7(2014):568-574.

[0191] In some embodiments, the antiplatelet agent comprises eptifibatide at an initial dosage of about 170 to about 190 mcg / kg of subject's body weight (e.g., about 180 mcg / kg of subject's body weight), optionally at a second initial dosage of about 170 to about 190 mcg / kg of subject's body weight (e.g., about 180 mcg / kg of subject's body weight), or at subsequent doses of about 1 to about 2 mcg / kg of subject's body weight / minute (e.g., about 1.5 mcg / kg of subject's body weight / minute).

[0192] In some embodiments, the antiplatelet agent comprises tirofiban at an initial dosage of about 0.3 to about 0.5 μg / kg body weight of subject / minute (e.g., about 0.5 μg / kg body weight of subject / minute) for about 30 minutes, or at subsequent dosages of about 0.1 μg / kg body weight of subject / minute.

[0193] In some embodiments, the antiplatelet agent comprises abciximab at an initial dosage of about 0.2 to about 0.3 mg / kg of subject's body weight (e.g., about 0.25 mg / kg of subject's body weight), or at subsequent dosages of about 0.10 to about 0.15 μg / kg of subject's body weight / minute (e.g., about 0.125 μg / kg of subject's body weight / minute). In some embodiments, the antiplatelet agent comprises abciximab at an initial dosage of about 0.2 to about 0.3 mg / kg of subject's body weight (e.g., about 0.25 mg / kg of subject's body weight), or at subsequent dosages of about 8 to about 10 μg / minute (e.g., about 9 μg / minute).

[0194] In some embodiments, the antiplatelet agent comprises ticlopidine at a dosage of about 240 to about 260 mg (eg, about 250 mg) twice daily.

[0195] In some embodiments, the antiplatelet agent comprises ibuprofen in a dosage of about 100 to about 600 mg (e.g., about 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, or 600 mg) once, twice, three times, or four times daily.

[0196] In some embodiments, the antiplatelet agent comprises vorapaxar, optionally with aspirin or clopidogrel, at a dose of about 2 to about 3 mg (eg, about 2.5 mg) once daily.

[0197] In some embodiments, the antiplatelet agent comprises cilostazol in a dosage of about 40 to about 110 mg (eg, about 50 mg, 75 mg, or 100 mg) twice daily.

[0198] In some embodiments, the antiplatelet agent comprises epoprostenol at an initial dosage of about 2 ng / kg body weight of the subject / min, or subsequent dosages of about 4, 6, 8, 10, 12, 14, 16, 18, or 20 ng / kg body weight of the subject / min.

[0199] In some embodiments, the antiplatelet agent comprises dipyridamole in a dosage of about 60 to about 110 mg (eg, about 75 mg or 100 mg) four times daily.

[0200] In some embodiments, the antiplatelet agent comprises treprostinil sodium at a dosage of about 0.5 to about 3.0 ng / kg body weight of the subject / minute (e.g., about 0.625 ng / kg body weight of the subject / minute, about 1.25 ng / kg body weight of the subject / minute, or about 2.5 ng / kg body weight of the subject / minute).

[0201] In some embodiments, rehydrating the composition comprising a platelet derivative comprises adding an aqueous liquid to the platelet derivative (e.g., FDPD). In some embodiments, the aqueous liquid is water. In some embodiments, the aqueous liquid is an aqueous solution (e.g., a buffer solution). In some embodiments, the aqueous liquid is saline. In some embodiments, the aqueous liquid is a suspension.

[0202] In some embodiments, the rehydrated platelet derivative (e.g., FDPD) has coagulation factor levels representing all individual factors involved in clotting (e.g., Factor VII, Factor VIII, and Factor IX) at or above 40 International Units (IU).

[0203] In some embodiments, the platelet derivative (e.g., FDPD) has less than about 10%, e.g., less than about 8%, e.g., less than about 6%, e.g., less than about 4%, e.g., less than about 2%, e.g., less than about 0.5% platelet membrane cross-linking via proteins and / or lipids present on the membrane. In some embodiments, the rehydrated platelet derivative (e.g., FDPD) has less than about 10%, e.g., less than about 8%, e.g., less than about 6%, e.g., less than about 4%, e.g., less than about 2%, e.g., less than about 0.5% platelet membrane cross-linking via proteins and / or lipids present on the membrane.

[0204] In some embodiments, the platelets, typically platelet derivatives, and in exemplary embodiments, FDPD, have a particle size (e.g., diameter, largest dimension) of at least about 0.2 μm (e.g., at least about 0.3 μm, at least about 0.4 μm, at least about 0.5 μm, at least about 0.6 μm, at least about 0.7 μm, at least about 0.8 μm, at least about 0.9 μm, at least about 1.0 μm, at least about 1.2 μm, at least about 1.5 μm, at least about 2.0 μm, at least about 2.5 μm, or at least about 5.0 μm). In some embodiments, the particle size is less than about 5.0 μm (e.g., less than about 2.5 μm, less than about 2.0 μm, less than about 1.5 μm, less than about 1.0 μm, less than about 0.9 μm, less than about 0.8 μm, less than about 0.7 μm, less than about 0.6 μm, less than about 0.5 μm, less than about 0.4 μm, or less than about 0.3 μm). In some embodiments, the particle size is from about 0.3 μm to about 5.0 μm (e.g., from about 0.4 μm to about 4.0 μm, from about 0.5 μm to about 2.5 μm, from about 0.6 μm to about 2.0 μm, from about 0.7 μm to about 1.0 μm, from about 0.5 μm to about 0.9 μm, or from about 0.6 μm to about 0.8 μm).

[0205] In some embodiments, at least 50% (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%) of the platelets, or in exemplary embodiments, platelet derivatives (e.g., FDPD), have a particle size in the range of about 0.3 μm to about 5.0 μm (e.g., about 0.4 μm to about 4.0 μm, about 0.5 μm to about 2.5 μm, about 0.6 μm to about 2.0 μm, about 0.7 μm to about 1.0 μm, about 0.5 μm to about 0.9 μm, or about 0.6 μm to about 0.8 μm). In some embodiments, up to 99% (e.g., up to about 95%, up to about 80%, up to about 75%, up to about 70%, up to about 65%, up to about 60%, up to about 55%, or up to about 50%) of the platelets, or in exemplary embodiments, platelet derivatives (e.g., FDPD), are in the range of about 0.3 μm to about 5.0 μm (e.g., about 0.4 μm to about 4.0 μm, about 0.5 μm to about 2.5 μm, about 0.6 μm to about 2.0 μm, about 0.7 μm to about 1.0 μm, about 0.5 μm to about 0.9 μm, or about 0.6 μm to about 0.8 μm). In some embodiments, about 50% to about 99% (e.g., about 55% to about 95%, about 60% to about 90%, about 65% to about 85%, about 70% to about 80%) of the platelets, or in exemplary embodiments, platelet derivatives (e.g., FDPD), are in the range of about 0.3 μm to about 5.0 μm (e.g., about 0.4 μm to about 4.0 μm, about 0.5 μm to about 2.5 μm, about 0.6 μm to about 2.0 μm, about 0.7 μm to about 1.0 μm, about 0.5 μm to about 0.9 μm, or about 0.6 μm to about 0.8 μm).

[0206] In some exemplary embodiments, the microparticles can be particles having a particle size (e.g., diameter, largest dimension) of less than about 0.5 μm (e.g., less than about 0.45 μm or less than 0.4 μm). In some cases, the microparticles can be particles having a particle size of about 0.01 μm to about 0.5 μm (e.g., about 0.02 μm to about 0.5 μm).

[0207] Compositions comprising platelets or platelet derivatives (e.g., FDPD), such as those prepared according to the methods described herein, can have a particulate content that contributes less than about 5.0% (e.g., less than about 4.5%, less than 4.0%, less than 3.5%, less than 3.0%, less than 2.5%, less than 2.0%, less than 1.5%, less than 1.0%, or less than 0.5%) of the total scattering intensity of all particles between about 1 nm and about 60,000 nm in the composition. In some embodiments, the platelet derivative composition comprises a population of platelet derivatives comprising CD41-positive platelet derivatives, wherein less than 15%, less than 10%, less than 7.5%, less than 5%, less than 4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, less than 0.5%, or less than 0.1% of the CD41-positive platelet derivatives are microparticles having a diameter of less than 1 μm, less than 0.9 μm, less than 0.8 μm, less than 0.7 μm, less than 0.6 μm, less than 0.5 μm, less than 0.4 μm, less than 0.3 μm, less than 0.2 μm, or less than 0.1 μm, and in certain exemplary embodiments, less than 0.5 μm. In some embodiments, the platelet derivative composition comprises a population of platelet derivatives comprising CD42-positive platelet derivatives, wherein less than 15%, less than 10%, less than 7.5%, less than 5%, less than 4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, less than 0.5%, or less than 0.1% of the CD42-positive platelet derivatives are microparticles having a diameter of less than 1 μm, less than 0.9 μm, less than 0.8 μm, less than 0.7 μm, less than 0.6 μm, less than 0.5 μm, less than 0.4 μm, less than 0.3 μm, less than 0.2 μm, or less than 0.1 μm, and in certain exemplary embodiments, less than 0.5 μm. In some embodiments, the platelet derivative composition comprises a population of platelet derivatives comprising CD61-positive platelet derivatives, wherein less than 15%, less than 10%, less than 7.5%, less than 5%, less than 4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, less than 0.5%, or less than 0.1% of the CD61-positive platelet derivatives are microparticles having a diameter of less than 1 μm, less than 0.9 μm, less than 0.8 μm, less than 0.7 μm, less than 0.6 μm, less than 0.5 μm, less than 0.4 μm, less than 0.3 μm, less than 0.2 μm, or less than 0.1 μm, and in certain exemplary embodiments, less than 0.5 μm.In some exemplary embodiments, the microparticles have a diameter of less than 0.5 μm. In some embodiments of any of the aspects and embodiments herein that include a platelet derivative composition in powder form, the diameter of the microparticles is determined after rehydrating the platelet derivative composition with an appropriate solution. In some embodiments, the amount of solution used to rehydrate the platelet derivative composition is equal to the amount of buffer or modifier present in the freeze-drying process. As used herein, the microparticle content "by scattering intensity" refers to the microparticle content based on the scattering intensity of all particles in the composition with a radius of about 1 nm to about 60,000 nm. The microparticle content can be measured by any suitable method, for example, dynamic light scattering (DLS). In some cases, this can be (or can be adjusted to be) about 1.060 cP, since the viscosity of the sample used for DLS is approximately the viscosity of plasma. In some embodiments, the platelet derivative composition according to any aspect or embodiment comprises a population of platelet derivatives and microparticles, wherein the numerical ratio of platelet derivatives to microparticles is at least 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, or 99:1. In some embodiments, the platelet derivatives have a diameter in the range of 0.5 to 2.5 μm, and the microparticles have a diameter of less than 0.5 μm.

[0208] In some embodiments, platelets are isolated, eg, in a liquid medium, eg, prior to treatment to form a platelet derivative, or prior to direct administration to a subject.

[0209] In some embodiments, the platelets are donor-derived platelets. In some embodiments, the platelets are obtained by a process that includes an apheresis step. In some embodiments, the platelets are pooled platelets.

[0210] In some embodiments, platelets are pooled from multiple donors. Such platelets pooled from multiple donors may also be referred to herein as pooled platelets. In some embodiments, the donors are more than 5, e.g., more than 10, e.g., more than 20, e.g., more than 50, e.g., up to about 100 donors. In some embodiments, the donors are about 5 to about 100, e.g., about 10 to about 50, e.g., about 20 to about 40, e.g., about 25 to about 35. Pooled platelets can be used to make any of the compositions described herein.

[0211] In some embodiments, the platelets are derived in vitro. In some embodiments, the platelets are derived or prepared in culture. In some embodiments, the preparation of platelets comprises deriving or expanding platelets from a culture of megakaryocytes. In some embodiments, the preparation of platelets comprises deriving or expanding platelets (or megakaryocytes) from a culture of human pluripotent stem cells (PCS), including embryonic stem cells (ESCs) and / or induced pluripotent stem cells (iPSCs).

[0212] Thus, in some embodiments, the platelets are prepared prior to treating a subject as described herein. In some embodiments, the platelets are freeze-dried. In some embodiments, the platelets are cryopreserved.

[0213] In some embodiments, the platelets or pooled platelets may be acidified to a pH of about 6.0 to about 7.4 prior to incubation with the incubation agent. In some embodiments, the method comprises acidifying the platelets to a pH of about 6.5 to about 6.9. In some embodiments, the method comprises acidifying the platelets to a pH of about 6.6 to about 6.8. In some embodiments, the acidification comprises adding a solution comprising acid citrate dextrose (ACD) to the pooled platelets.

[0214] In some embodiments, tangential flow filtration (TFF) is used in processing platelets to make platelet derivatives, and in exemplary embodiments, FDPDs, for use in aspects herein. For example, TFF can be used for concentration and / or buffer or other solution exchange so that the platelets are suspended in an appropriate concentration range in an appropriate medium, e.g., an incubation substance and / or lyophilizing agent, or an incubation substance that is or includes a lyophilizing agent, e.g., before the composition is dried to form a platelet derivative, or in exemplary embodiments, before a platelet composition is freeze-dried to form an FDPD.

[0215] In some embodiments, the method may include an initial dilution step, e.g., diluting a starting material (e.g., an unprocessed blood product (e.g., donor apheresis material (e.g., pooled donor apheresis material)) with a preparative agent (e.g., any of the preparative agents described herein) to form a diluted starting material. In some cases, the initial dilution step may include diluting with a preparative agent in a mass of preparative agent equal to at least about 10% of the mass of the starting material (e.g., at least about 15%, 25%, 50%, 75%, 100%, 150%, or 200% of the mass of the starting material). In some embodiments, the initial dilution step may be performed using a TFF device.

[0216] In some embodiments, the method can include concentrating (e.g., concentrating platelets) (e.g., concentrating the starting material or diluted starting material) to form an enriched platelet composition. For example, the concentration can be about 1000×10 3 ~About 4000×10 3 platelets / µL (e.g., approximately 1000 x 10 3 ~About 2000×10 3 pieces, approximately 2000×10 3 pieces~approx.3000×10 3 pieces, or approximately 4000 x 10 3 In some embodiments, the concentration step may be performed using a TFF device.

[0217] The concentration of platelets or platelet derivatives (e.g., FDPD) can be determined by any suitable method, for example, a counter can be used to quantify the concentration of blood cells in suspension using impedance (e.g., Beckman Coulter AcT 10 or AcT diff 2).

[0218] In some embodiments, TFF can include diafiltration (sometimes referred to as "washing") of the starting material, diluted starting material, concentrated platelet composition, or a combination thereof. In some embodiments, diafiltration can include washing with at least two (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, or more) diafiltration volumes. In some embodiments, TFF can include buffer exchange. In some embodiments, a buffer can be used in TFF. The buffer can be any suitable buffer. In some embodiments, the buffer can be a preparation (e.g., any of the preparations described herein). In some embodiments, the buffer can be the same preparation as used for dilution. In some embodiments, the buffer can be a different preparation than used for dilution. In some embodiments, the buffer solution may comprise a lyophilization agent comprising a buffering agent, a base, a bulking agent, optionally a salt, and optionally at least one organic solvent, such as an organic solvent selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methylpyrrolidone, dimethylacetamide (DMAC), or a combination thereof. The buffer solution may be any suitable buffering agent. In some embodiments, the buffering agent can be HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). The base can be any suitable base. In some embodiments, the base can be sodium bicarbonate. In some embodiments, the sugar can be a monosaccharide. In some embodiments, the bulking agent can be a sugar. In some embodiments, the sugar can include sucrose, maltose, trehalose, glucose (e.g., dextrose), mannose, or xylose. In some embodiments, the monosaccharide can be trehalose. In some embodiments, the bulking agent can include polysucrose. The salt can be any suitable salt. In some embodiments, the salt can be selected from the group consisting of sodium chloride (NaCl), potassium chloride (KCl), or a combination thereof.

[0219] In some embodiments, membranes having pore sizes of about 0.1 μm to about 1 μm (e.g., about 0.1 μm to about 1 μm, about 0.1 μm to about 0.5 μm, about 0.2 to about 0.45 μm, about 0.45 to about 1 μm, about 0.1 μm, about 0.2 μm, about 0.45 μm, about 0.65 μm, or about 1 μm) can be used for TFF. The membrane can be made of any suitable material. In some cases, the membrane can be a hydrophilic membrane. In some embodiments, the membrane can be a hydrophobic membrane. In some embodiments, membranes having a nominal molecular weight cutoff (NMWCO) of at least about 100 kDa (e.g., at least about 200, 300 kDa, 500 kDa, or 1000 kDa) can be used for TFF. TFF can be performed with any suitable pore size in the range of 0.1 μm to 1.0 μm, with the goal of reducing the particulate content in the composition and increasing the content of platelet derivatives in the composition. Those skilled in the art will recognize the necessary optimization of pore size to retain platelet derivatives and allow particulates to pass through the membrane. In exemplary embodiments, the pore size is such that particulates pass through the membrane and the TFF-processed composition has less than 5% particulates. In exemplary embodiments, the pore size is such that the TFF-processed composition has less than 100×10 3 ~20,000×10 3The pore size is such that the maximum amount of platelet derivative is retained in the process, allowing for a platelet derivative concentration in the range of 0.01 to 0.1. The pore size during the TFF process can be utilized to obtain a higher concentration of platelet derivative in the platelet derivative composition, such that the person administering the platelet derivative to a subject in need must rehydrate / reconstitute fewer vials, thus being more efficient in terms of time and labor during the process of preparing such platelet derivatives for downstream procedures, such as the therapeutic methods provided herein. TFF can be performed at any suitable temperature. In some embodiments, TFF can be performed at a temperature of about 20°C to about 37°C (e.g., about 20°C to about 25°C, about 20°C to about 30°C, about 25°C to about 30°C, about 30°C to about 35°C, about 30°C to about 37°C, about 25°C to about 35°C, or about 25°C to about 37°C). In some embodiments, TFF may be performed at a flow rate (e.g., circulation flow rate) of about 100 mL / min to about 800 mL / min (e.g., about 100 to about 200 mL / min, about 100 to about 400 mL / min, about 100 to about 600 mL / min, about 200 to about 400 mL / min, about 200 to about 600 mL / min, about 200 to about 800 mL / min, about 400 to about 600 mL / min, about 400 to about 800 mL / min, about 600 to about 800 mL / min, about 100 mL / min, about 200 mL / min, about 300 mL / min, about 400 mL / min, about 500 mL / min, about 600 mL / min, about 700 mL / min, or about 800 mL / min).

[0220] In some embodiments, TFF can be performed until a specific endpoint is reached to form a TFF treatment composition. The endpoint can be any suitable endpoint. In some embodiments, the endpoint can be a percentage of residual plasma (e.g., about 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less residual plasma). In some embodiments, the endpoint can be relative absorbance at 280 nm (A280). For example, the endpoint can be an A280 (e.g., using a 0.5 cm path length) of about 50% or less (e.g., about 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less) of the A280 before TFF (e.g., starting material or diluted starting material). In some embodiments, the A280 can be relative to a system measuring 7.5% plasma = 1.66 AU. In some embodiments, an instrument for measuring A280 can be configured as follows: a 0.5 cm gap flow cell can be attached to the filtrate line of a TFF system. The flow cell can be connected to a photometer using fiber optic cables (a light source cable and a photodetector cable) attached to each side of the flow cell. The flow cell can be made with silica glass lenses on both sides of the fiber optic cables. Apart from the relative protein concentration of a protein in an aqueous medium, the protein concentration in an aqueous medium can also be measured in absolute units.In some embodiments, the protein concentration in the aqueous medium is 15% or less, or 14% or less, or 13% or less, or 12% or less, or 11% or less, or 10% or less, or 9% or less, or 8% or less, or 7% or less, or 6% or less, or 5% or less, or 4% or less, or 3% or less, or 2% or less, or 1% or less, or 0.1% or less, or 0.01% or less. In some exemplary embodiments, the protein concentration is less than 3% or less than 4%. In some embodiments, the protein concentration is in the range of 0.01-15%, or 0.1-15%, or 1-15%, or 1-10%, or 0.01-10%, or 3-12%, or 5-10% in the TFF processing composition. In some embodiments, the endpoint can be absolute A280 (e.g., using a 0.5 cm path length). For example, the endpoint can be an A280 of 2.50 AU or less, 2.40 AU or less, 2.30 AU or less, 2.20 AU or less, 2.10 AU or less, 2.0 AU or less, 1.90 AU or less, 1.80 AU or less, or 1.70 AU or less (e.g., 1.66 AU or less, 1.6 AU or less, 1.5 AU or less, 1.4 AU or less, 1.3 AU or less, 1.2 AU or less, 1.1 AU or less, 1.0 AU or less, 0.9 AU or less, 0.8 AU or less, 0.7 AU or less, 0.6 AU or less, 0.5 AU or less, 0.4 AU or less, 0.3 AU or less, 0.2 AU or less, or 0.1 AU or less) (e.g., using a 0.5 cm path length). In some embodiments, the percentage of plasma remaining, relative A280, or A280 can be determined based on the aqueous medium of a composition comprising platelets and an aqueous medium. In some embodiments, the percentage of remaining plasma can be determined based on a known correlation to A280. In some embodiments, the TFF can include concentration or dilution of the sample (e.g., using a preparation agent), so that the endpoint can be platelet concentration. For example, the endpoint can be at least about 2000 x 10. 3 platelets / µL (e.g., at least approximately 2050 x 10 3 , 2100×10 3 , 2150×10 3 , 2200×10 3 , 2250×10 3 , 2300×10 3, 2350×10 3 , 2400×10 3 , 2450×10 3 , or 2500 x 10 3 As another example, the endpoint may be a platelet concentration of about 1000 x 10 3 ~approximately 2500 platelets / µL (e.g., approximately 1000 x 10 3 ~About 2000×10 3 , about 1500×10 3 ~Approx. 2300×10 3 , or approximately 1700 × 10 3 ~Approx. 2300×10 3 In some embodiments, the endpoint is a platelet concentration in the TFF treatment composition of at least 100×10 3 Platelets / µL, 200 x 10 3 Platelets / µL, 400 x 10 3 Platelets / µL, 1000 x 10 3 Platelets / µL, 1250 x 10 3 Platelets / µL, 1500 x 10 3 Platelets / µL, 1750 x 10 3 Platelets / µL: 2000 x 10 3 Platelets / µL, 2250 x 10 3 Platelets / µL: 2500 x 10 3 Platelets / µL, 2750 x 10 3 Platelets / µL, 3000 x 10 3 Platelets / µL, 3250 x 10 3 Platelets / µL, 3500 x 10 3 Platelets / µL, 3750 x 10 3 Platelets / µL: 4000 x 10 3 Platelets / µL, 4250 x 10 3 Platelets / µL: 4500 x 10 3 Platelets / µL, 4750 x 10 3 Platelets / µL: 5000 x 10 3 Platelets / µL, 5250 x 10 3 Platelets / µL, 5500 x 10 3Platelets / µL, 5750 x 10 3 Platelets / µL, 6000 x 10 3 Platelets / µL: 7000 x 10 3 Platelets / µL, 8000 x 10 3 Platelets / µL: 9000 x 10 3 Platelets / µL: 10,000 x 10 3 Platelets / µL: 11,000 x 10 3 Platelets / µL: 12,000 x 10 3 Platelets / µL: 13,000 x 10 3 Platelets / µL: 14,000 x 10 3 Platelets / µL: 15,000 x 10 3 Platelets / µL: 16,000 x 10 3 Platelets / µL: 17,000 x 10 3 Platelets / µL: 18,000 x 10 3 Platelets / µL: 19,000 x 10 3 Platelets / µL: 20,000 x 10 3 In some embodiments, the platelets or platelet derivatives in the TFF treatment composition may be 100×10 platelets / μL. 3 ~20,000×10 3 platelets / µL, or 1000 x 10 3 ~20,000×10 3 platelets / µL, or 1000 x 10 3 ~10,000×10 3 platelets / µL, or 500 x 10 3 ~5,000×10 3 platelets / µL, or 1000 x 10 3 ~5,000×10 3 platelets / µL, or 2000 x 10 3 ~8,000×10 3 platelets / µL, or 10,000 x 10 3 ~20,000×10 3 platelets / µL, or 15,000 x 10 3 ~20,000×10 3 The range is 100 platelets / µL.

[0221] In some embodiments, the endpoint may include two or more criteria (eg, percentage of residual plasma and platelet concentration, relative A280 and platelet concentration, or absolute A280 and platelet concentration).

[0222] Typically, the TFF-processed composition is then lyophilized, optionally with a heat treatment step, to form the final blood product (e.g., platelets, cryopreserved platelets, FDPD). However, in some cases, the TFF-processed composition may be considered the final blood product.

[0223] In some embodiments, blood products may be prepared using centrifugation of a blood product (e.g., an unprocessed blood product (e.g., donor apheresis material (e.g., pooled donor apheresis material)) or a partially processed blood product (e.g., a blood product that has undergone TFF)). In some embodiments, blood products may be prepared without centrifuging a blood product (e.g., an unprocessed blood product (e.g., donor apheresis material) or a partially processed blood product (e.g., a blood product that has undergone TFF)). Centrifugation may include any suitable step. In some embodiments, centrifugation may include slow acceleration, slow deceleration, or a combination thereof. In some embodiments, centrifugation can include centrifugation at about 1400 × g to about 1550 × g (e.g., about 1400 to about 1450 × g, about 1450 to about 1500 × g, or 1500 to about 1550 × g, about 1400 × g, about 1410 × g, about 1430 × g, about 1450 × g, about 1470 × g, about 1490 × g, about 1500 × g, about 1510 × g, about 1530 × g, or about 1550 × g). In some embodiments, the duration of centrifugation can be about 10 minutes to about 30 minutes (e.g., about 10 to about 20 minutes, about 20 to about 30 minutes, about 10 minutes, about 20 minutes, or about 30 minutes).

[0224] In some embodiments, the final blood product may be prepared using both TFF and centrifugation (eg, TFF followed by centrifugation, or centrifugation followed by TFF).

[0225] Also provided herein are compositions prepared by any of the methods described herein.

[0226] In some embodiments, the compositions described herein may be analyzed at multiple points during processing. In some embodiments, the starting material (e.g., donor apheresis material (e.g., pooled donor apheresis material)) may be analyzed for antibody content (e.g., HLA or HNA antibody content). In some embodiments, the starting material (e.g., donor apheresis material (e.g., pooled donor apheresis material)) may be analyzed for protein concentration (e.g., by absorbance at 280 nm (e.g., using a 0.5 cm pathlength)). In some embodiments, compositions at intermediate stages of processing (e.g., when the protein concentration has decreased to 75% or less (e.g., 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or less) of the protein concentration of the unprocessed blood product) may be analyzed for antibody content (e.g., HLA or HNA antibody content). In some embodiments, the antibody content (e.g., HLA or HNA antibody content) of a blood product at an intermediate step in processing may be reduced by at least 5% compared to the antibody content of the starting material (e.g., may be reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the final blood product (e.g., platelets (e.g., cryopreserved platelets, FDPD)) may be analyzed for antibody content (e.g., HLA or HNA antibody content). In some embodiments described herein, the final blood product may be a composition comprising platelets and an aqueous medium. In some embodiments, the antibody content (e.g., HLA or HNA antibody content) of the final blood product (e.g., platelets (e.g., cryopreserved platelets, FDPD)) may be reduced by at least 5% (e.g., may be reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) compared to the antibody content of the starting material.In some embodiments, the final blood product may have no detectable levels of antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies. In some embodiments, the aqueous medium of the compositions described herein may be analyzed as described herein.

[0227] In some embodiments, the platelets are isolated prior to incubation with an incubation substance and / or a lyophilizing agent. In some embodiments, the incubation substance is or includes a lyophilizing agent, as disclosed in more detail herein. In some embodiments, the method further includes isolating the platelets by using centrifugation. In some embodiments, the centrifugation is performed at a relative centrifugal force (RCF) of about 1000×g to about 2000×g. In some embodiments, the centrifugation is performed at a relative centrifugal force (RCF) of about 1300×g to about 1800×g. In some embodiments, the centrifugation is performed at a relative centrifugal force (RCF) of about 1500×g. In some embodiments, the centrifugation is performed for about 1 minute to about 60 minutes. In some embodiments, the centrifugation is performed for about 10 minutes to about 30 minutes. In some embodiments, the centrifugation is performed for about 30 minutes.

[0228] The incubation substance can include any suitable components. In some embodiments, the incubation substance can include a liquid medium. In some embodiments, the incubation substance can include one or more salts selected from phosphate salts, sodium salts, potassium salts, calcium salts, magnesium salts, and any other salt that can be found in blood or blood products or that is known to be useful for drying platelets, or any combination of two or more thereof.

[0229] In some embodiments, the incubation substance comprises one or more salts, such as phosphate salts, sodium salts, potassium salts, calcium salts, magnesium salts, and any other salts that may be found in blood or blood products. Exemplary salts include sodium chloride (NaCl), potassium chloride (KCl), and combinations thereof. In some embodiments, the incubation substance comprises about 0.5 mM to about 100 mM of one or more salts. In some embodiments, the incubation substance comprises about 0.5 mM to about 100 mM (e.g., about 0.5 to about 2 mM, about 2 mM to about 90 mM, about 2 mM to about 6 mM, about 50 mM to about 100 mM, about 60 mM to about 90 mM, about 70 mM to about 85 mM) of one or more salts. In some embodiments, the incubation substance comprises about 5 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM of one or more salts. In some embodiments, the incubation substance comprises one or more salts selected from calcium salts, magnesium salts, and combinations of the two, at a concentration of about 0.5 mM to about 2 mM.

[0230] Preferably, these salts are present in compositions containing platelets or platelet derivatives, such as freeze-dried platelets, in amounts about the same as those found in whole blood.

[0231] In some embodiments, the incubation substance further comprises a carrier protein. In some embodiments, the carrier protein comprises human serum albumin, bovine serum albumin, or a combination thereof. In some embodiments, the carrier protein is present in an amount of about 0.05% to about 1.0% (w / v).

[0232] The incubation agent can be any buffer that is non-toxic to platelets and provides adequate buffering capacity for the solution at the temperatures to which the solution will be exposed during the processes provided herein. Thus, the buffer can include any of the commercially available known biocompatible buffers, such as phosphate buffers, e.g., phosphate-buffered saline (PBS), bicarbonate / carbonate buffers, e.g., sodium bicarbonate buffer, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and Tris-based buffers, e.g., Tris-buffered saline (TBS). Similarly, it can be used with the following buffers: propane-1,2,3-tricarboxylic acid (tricarvallic acid), benzenepentacarboxylic acid, maleic acid, 2,2-dimethylsuccinic acid, EDTA, 3,3-dimethylglutaric acid, bis(2-hydroxyethyl)imino-tris(hydroxymethyl)-methane (BIS-TRIS), benzenehexacarboxylic acid (mellitic acid), N-(2-acetamido)imino-diacetic acid (ADA), butane-1,2,3,4-tetracarboxylic acid, pyrophosphoric acid, 1,1-cyclopentanediacetic acid (3,3-tetramethylene-glutaric acid), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), N-(2-acetamido)imino-diacetic acid (ADA). )-2-aminoethanesulfonic acid (ACES), 1,1-cyclohexanediacetic acid, 3,6-endomethylene-1,2,3,6-tetrahydrophthalic acid (EMTA, ENDCA), imidazole, 2-(aminoethyl)trimethylammonium chloride (CHOLAMINE), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 2-methylpropane-1,2,3-triscarboxylic acid (beta-methyltricarvallyic acid), 2-(N-morpholino)propane-sulfonic acid (MOPS), phosphoric acid, and N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES). In some embodiments, the incubation substance comprises one or more buffers, such as N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), or sodium bicarbonate (NaHCO). In some embodiments, the incubation medium comprises from about 5 to about 100 mM of one or more buffers.In some embodiments, the incubation substance comprises about 5 to about 50 mM (e.g., about 5 mM to about 40 mM, about 8 mM to about 30 mM, about 10 mM to about 25 mM) of one or more buffers. In some embodiments, the incubation substance comprises about 10 mM, about 20 mM, about 25 mM, or about 30 mM of one or more buffers.

[0233] In some embodiments, the incubation substance comprises one or more sugars, such as monosaccharides and disaccharides, including sucrose, maltose, trehalose, glucose, mannose, dextrose, and xylose. In some embodiments, the sugar is a monosaccharide. In some embodiments, the sugar is a disaccharide. In some embodiments, the sugar comprises a monosaccharide, a disaccharide, or a combination thereof. In some embodiments, the sugar is a non-reducing disaccharide. In some embodiments, the sugar comprises sucrose, maltose, trehalose, glucose (e.g., dextrose), mannose, or xylose. In some embodiments, the sugar comprises trehalose. In some embodiments, the incubation substance comprises starch. In some embodiments, the incubation substance comprises polysucrose, which is a polymer of sucrose and epichlorohydrin. In some embodiments, the incubation substance comprises about 10 mM to about 1,000 mM of one or more sugars. In some embodiments, the incubation substance comprises about 50 to about 500 mM of one or more sugars. In some embodiments, the one or more sugars are present in an amount of 10 mM to 500 mM. In some embodiments, the one or more sugars are present in an amount of 50 mM to 200 mM. In some embodiments, the one or more sugars are present in an amount of 100 mM to 150 mM. In some embodiments, the one or more sugars are a lyophilizing agent, for example, in some embodiments, the lyophilizing agent comprises trehalose, polysucrose, or a combination thereof.

[0234] In some embodiments, compositions comprising platelets or platelet derivatives (e.g., FDPD) can include one or more of water or saline. In some embodiments, compositions comprising platelets or platelet derivatives, such as freeze-dried platelets, can include DMSO.

[0235] In some embodiments, the incubation substance includes an organic solvent, such as an alcohol (e.g., ethanol). In such an incubation substance, the amount of solvent can range from 0.1% to 5.0% (v / v). In some embodiments, the organic solvent can range from about 0.1% (v / v) to about 5.0% (v / v), e.g., from about 0.3% (v / v) to about 3.0% (v / v), or from about 0.5% (v / v) to about 2% (v / v).

[0236] In some embodiments, suitable organic solvents include, but are not limited to, alcohols, esters, ketones, ethers, halogenated solvents, hydrocarbons, nitriles, glycols, alkyl nitrates, water, or mixtures thereof. In some embodiments, suitable organic solvents include, but are not limited to, methanol, ethanol, n-propanol, isopropanol, acetic acid, acetone, methyl ethyl ketone, methyl isobutyl ketone, methyl acetate, ethyl acetate, isopropyl acetate, tetrahydrofuran, isopropyl ether (IPE), tert-butyl methyl ether, dioxane (e.g., 1,4-dioxane), acetonitrile, propionitrile, methylene chloride, chloroform, toluene, anisole, cyclohexane, hexane, heptane, ethylene glycol, nitromethane, dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, dimethylamide, and combinations thereof. In some embodiments, the organic solvent is selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide (DMSO), dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methylpyrrolidone, dimethylacetamide (DMAC), or a combination thereof. In some embodiments, the organic solvent comprises ethanol, DMSO, or a combination thereof. The presence of an organic solvent, such as ethanol, can be beneficial in the processing of platelets, platelet derivatives, or FDPD (e.g., freeze-dried platelet derivatives).

[0237] In some embodiments, the incubation substance is incubated with the platelets in the presence of an aqueous medium, hi some embodiments, the incubation substance is incubated in the presence of a medium containing DMSO.

[0238] In some embodiments, one or more other ingredients may be incubated with the platelets. Exemplary ingredients may include prostaglandin E1 or prostacyclin, and / or EDTA / EGTA to prevent platelet aggregation and activation during the incubation process.

[0239] Non-limiting examples of incubation substance compositions that can be used are shown in Tables 1-5.

[0240] (Table 1) TIFF2024507362000004.tif107128

[0241] (Table 2) TIFF2024507362000005.tif104128

[0242] (Table 3) TIFF2024507362000006.tif83128

[0243] (Table 4) TIFF2024507362000007.tif69128

[0244] Table 4 is another exemplary incubation material. The pH can be adjusted to 7.4 with NaOH. Albumin is an optional component of Buffer B.

[0245] (Table 5) TIFF2024507362000008.tif79128

[0246] Table 5 provides additional exemplary incubation materials.

[0247] In some embodiments, platelets (e.g., apheresis platelets, platelets isolated from whole blood, pooled platelets, or a combination thereof) are incubated with an incubation substance at different temperatures between 15 and 45°C, or at about 37°C, for different durations.

[0248] In some embodiments, platelets (e.g., apheresis platelets, platelets isolated from whole blood, pooled platelets, or a combination thereof) form a suspension in an incubation substance and / or lyophilizing agent comprising a liquid medium, or an incubation substance comprising a lyophilizing agent, at a concentration of 10,000 platelets / μL to 10,000,000 platelets / μL, e.g., 50,000 platelets / μL to 2,000,000 platelets / μL, e.g., 100,000 platelets / μL to 500,000 platelets / μL, e.g., 150,000 platelets / μL to 300,000 platelets / μL, e.g., 200,000 platelets / μL.

[0249] Platelets (e.g., apheresis platelets, platelets isolated from whole blood, pooled platelets, or a combination thereof) can be incubated with an incubation substance that is or includes, e.g., a lyophilizing agent, for different durations, such as, for at least about 5 minutes (e.g., at least about 20 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, or at least about 48 hours). In some embodiments, platelets may be incubated with the incubation substance for about 48 hours or less (e.g., about 20 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, or about 42 hours or less). In some embodiments, platelets may be incubated with the incubation substance for about 10 minutes to about 48 hours (e.g., about 20 minutes to about 36 hours, about 30 minutes to about 24 hours, about 1 hour to about 20 hours, about 2 hours to about 16 hours, about 10 minutes to about 24 hours, about 20 minutes to about 12 hours, about 30 minutes to about 10 hours, or about 1 hour to about 6 hours). In some embodiments, the platelets, platelet derivatives, or FDPD are incubated with the incubation agent for a period of 5 minutes to 48 hours, such as 10 minutes to 24 hours, such as 20 minutes to 12 hours, such as 30 minutes to 6 hours, such as 1 hour to 3 hours, for example, about 2 hours.

[0250] In some embodiments, platelets (e.g., apheresis platelets, platelets isolated from whole blood, pooled platelets, or a combination thereof) are incubated with an incubation substance at different temperatures. In embodiments, the incubation is performed at 37°C. In certain embodiments, the incubation is performed at 4°C to 45°C, e.g., 15°C to 42°C. For example, in embodiments, the incubation is performed at 35°C to 40°C (e.g., 37°C) for 110 to 130 (e.g., 120) minutes, and for as long as 24 to 48 hours. In some embodiments, platelets are incubated with an incubation substance for different durations as disclosed herein and at temperatures between 15 to 45°C, or about 37°C.

[0251] In some embodiments, platelets (e.g., apheresis platelets, platelets isolated from whole blood, pooled platelets, or a combination thereof) are loaded with one or more active agents. In some embodiments, platelets may be loaded with an antifibrinolytic agent. Non-limiting examples of antifibrinolytic agents include ε-aminocaproic acid (EACA), tranexamic acid, aprotinin, aminomethylbenzoic acid, and fibrinogen.

[0252] Loading platelets (e.g., apheresis platelets, platelets isolated from whole blood, pooled platelets, or a combination thereof) with an active agent (e.g., an antifibrinolytic agent) can be carried out by any suitable method. See, e.g., PCT Publication Nos. 2020 / 113090A1, 2020 / 113101A1, 2020 / 113035A1, and 2020 / 112963A1. Generally, loading involves contacting the platelets with an antifibrinolytic agent. In some embodiments, loading can be carried out by combining the active agent with an incubation substance. In some embodiments, loading can be carried out in a step separate from the incubation step. For example, loading can be carried out in a step prior to the incubation step. In some such embodiments, the active agent can be provided to the platelets as a solution or suspension in any of the incubation substances described herein, which can be the same as or different from the incubation substance used in the incubation step. In some embodiments, the loading step may be performed during the incubation step. In some such embodiments, the active agent may be added to the incubation substance (e.g., as a solid or in a solution or suspension during incubation). In some embodiments, the loading step may be performed at a step subsequent to the incubation step. In some such embodiments, the platelets may be provided as a solution or suspension in any of the incubation substances described herein, which may be the same or different from the incubation substance used in the incubation step.

[0253] The active agent may be applied to the platelets at any suitable concentration. In some embodiments, the active agent is applied at a concentration of about 1 μM to about 100 mM (e.g., about 1 μM to about 10 μM, about 1 μM to about 50 μM, about 1 μM to about 100 μM, about 1 μM to about 500 μM, about 1 μM to about 1 mM, about 1 μM to about 10 mM, about 1 μM to about 25 mM, about 1 μM to about 50 mM, about 1 μM to about 75 mM, about 10 μM to about 100 mM, about 50 μM to about 100 mM, about 100 μM to about 100 mM, about 500 μM to about 100 mM, about 1 mM to about 100 mM, about 10 mM to about 100 mM, about 25 mM to about 100 mM, about 50 mM to about 100 mM, about 75 mM). to about 100 mM, about 10 μM to about 100 mM, about 200 μM to about 1 mM, about 800 μM to about 900 μM, about 400 μM to about 800 μM, about 500 μM to about 700 μM, about 600 μM, about 5 mM to about 85 mM, about 20 mM to about 90 mM, about 25 mM to about 75 mM, about 30 mM to about 90 mM, about 35 mM to about 65 mM, about 40 mM to about 60 mM, about 50 mM to about 60 mM, about 40 mM to about 70 mM, about 45 mM to about 55 mM, or about 50 mM).

[0254] In some embodiments, the method further comprises drying the platelets. In some embodiments, the drying step comprises lyophilizing the platelets. In some embodiments, the drying step comprises freeze-drying the platelets. In some embodiments, the method further comprises rehydrating the platelets obtained from the drying step.

[0255] In some embodiments, platelets are cryopreserved, cryopreserved, or lyophilized (eg, to produce FDPD) prior to use in therapy or functional assays.

[0256] Any known technique for drying platelets can be used in accordance with the present disclosure, so long as it can achieve a final residual moisture content of less than 5%. Preferably, the technique achieves a final residual moisture content of less than 2%, e.g., 1%, 0.5%, or 0.1%. Non-limiting examples of suitable techniques are freeze-drying (lyophilization) and spray-drying. Suitable lyophilization methods are provided in Table A. Additional exemplary lyophilization methods can be found in U.S. Patent No. 7,811,558, U.S. Patent No. 8,486,617, and U.S. Patent No. 8,097,403. An exemplary spray-drying method involves combining nitrogen as the drying gas with an incubation material according to the present disclosure, then introducing the mixture into a GEA Mobile Minor spray dryer manufactured by GEA Processing Engineering, Inc. (Columbia, MD, USA) with a two-fluid nozzle configuration, and spraying the mixture at an inlet temperature ranging from 150°C to 190°C, an outlet temperature ranging from 65°C to 100°C, an atomization rate ranging from 0.5 to 2.0 bar, an atomization rate ranging from 5 to 13 kg / hr, a nitrogen usage ranging from 60 to 100 kg / hr, and a run time of 10 to 35 minutes. The final step in spray-drying is the preferential recovery of the dried mixture. In some embodiments, the dried composition is stable for at least 6 months at temperatures ranging from -20°C or below to 90°C or above.

[0257] Table A. Exemplary freeze-drying protocol TIFF2024507362000009.tif86151

[0258] In some embodiments, drying platelets obtained as disclosed herein to produce a platelet derivative for use in any of the aspects or embodiments herein, e.g., freeze-drying platelets obtained as disclosed herein to produce a FDPD for use in any of the aspects or embodiments herein, comprises incubating the platelets with a lyophilizing agent (e.g., a non-reducing disaccharide). Thus, in some embodiments, the method for preparing platelets further comprises incubating the platelets with a lyophilizing agent. In some embodiments, the lyophilizing agent is a sugar. In some embodiments, the sugar is a disaccharide, such as a non-reducing disaccharide.

[0259] In some embodiments, platelets are incubated with a lyophilizing agent for a time sufficient to incubate the platelets with the lyophilizing agent and at a suitable temperature. In some embodiments, the incubation substance is a lyophilizing agent. Non-limiting examples of suitable lyophilizing agents are sugars, such as monosaccharides and disaccharides, including sucrose, maltose, trehalose, glucose (e.g., dextrose), mannose, and xylose. In some embodiments, non-limiting examples of lyophilizing agents include serum albumin, dextran, polyvinylpyrrolidone (PVP), starch, and hydroxyethyl starch (HES). In some embodiments, exemplary lyophilizing agents may include high molecular weight polymers. "High molecular weight" refers to polymers having an average molecular weight of about 70 kDa or greater and up to 1,000,000 kDa. Non-limiting examples are polymers of sucrose and epichlorohydrin (e.g., polysucrose). In some embodiments, the lyophilizing agent is polysucrose. Any amount of high molecular weight polymer can be used as a lyophilization agent, but it is preferred that an amount be used that achieves a final concentration of about 3% to 10% (w / v), e.g., 3 to 7%, e.g., 6%.

[0260] An exemplary sugar for use in the compositions disclosed herein is trehalose. Regardless of the identity of the sugar, the sugar can be present in any suitable amount in compositions that are dried to form platelet derivatives or freeze-dried to form FDPDs, as well as in rehydration compositions for such dried platelet derivatives and FDPDs. For example, the sugar can be present in an amount of 1 mM to 1 M. In embodiments, the sugar is present in an amount of 10 mM to 500 mM. In some embodiments, the sugar is present in an amount of 20 mM to 200 mM. In embodiments, the sugar is present in an amount of 40 mM to 100 mM. In various embodiments, the sugar is present at different specific concentrations within the ranges recited above, and one of skill in the art will readily appreciate the various concentrations without the need to specifically recite each one herein. When multiple sugars are present in the composition, each sugar can be present in an amount according to the ranges and specific concentrations recited above.

[0261] Within the processes provided herein for making the compositions provided herein, the addition of the lyophilizing agent can be the last step before drying. However, in some embodiments, the lyophilizing agent is added simultaneously with or before other components of the composition, such as salts, buffers, and optionally cryoprotectants or other components. In some embodiments, the lyophilizing agent is added to the incubation material and thoroughly mixed to form a dry solution, which is dispensed into drying containers (e.g., glass or plastic serum vials, lyophilization bags), and subjected to conditions that allow the solution to dry and form a dry composition.

[0262] The step of incubating the platelets with a cryoprotectant can include incubating the platelets for a time suitable for loading, which is long enough, along with the temperature, for the cryoprotectant to contact the platelets, preferably long enough for the cryoprotectant to be incorporated into the platelets to at least some extent. In embodiments, the incubation is from about 1 minute to about 180 minutes or more.

[0263] The step of incubating the platelets with the cryoprotectant can include incubating the platelets and cryoprotectant at a suitable temperature, if selected along with the amount of time allotted. Generally, the composition is incubated at a temperature above freezing for at least a time sufficient for the cryoprotectant to contact the platelets. In embodiments, the incubation is carried out at 37°C. In certain embodiments, the incubation is carried out at 20°C to 42°C. For example, in embodiments, the incubation is carried out at 35°C to 40°C (e.g., 37°C) for 110 to 130 (e.g., 120) minutes.

[0264] In various embodiments, the freeze-drying bag is a gas-permeable bag configured to allow gas to pass through at least a portion or all of the bag during processing. The gas-permeable bag may allow gas within the interior of the bag to be exchanged with atmospheric gas present in the surrounding environment. The gas-permeable bag may be permeable to gases such as oxygen, nitrogen, water, air, hydrogen, and carbon dioxide, allowing gas exchange to occur in the compositions provided herein. In some embodiments, the gas-permeable bag allows carbon dioxide to permeate through the wall of the bag, thereby allowing removal of a portion of the carbon dioxide present within the interior of the bag. In some embodiments, the release of carbon dioxide from the bag may be advantageous for maintaining a desired pH level of the composition contained within the bag.

[0265] In some embodiments, the container of the process herein is a closed or sealed gas-permeable container. In some embodiments, the container is a container that is closed or sealed and a portion of which is gas-permeable. In some embodiments, the surface area of ​​the gas-permeable portion of the closed or sealed container (e.g., bag) relative to the volume of the product contained in the container (hereinafter referred to as the "SA / V ratio") can be adjusted to improve the pH maintenance of the compositions provided herein. For example, in some embodiments, the SA / V ratio of the container is at least about 2.0 cm 2 / mL (e.g., at least about 2.1 cm2 / mL, at least approximately 2.2 cm 2 / mL, at least approximately 2.3 cm 2 / mL, at least approximately 2.4 cm 2 / mL, at least approximately 2.5 cm 2 / mL, at least approximately 2.6 cm 2 / mL, at least approximately 2.7 cm 2 / mL, at least approximately 2.8 cm 2 / mL, at least approximately 2.9 cm 2 / mL, at least approximately 3.0 cm 2 / mL, at least approximately 3.1 cm 2 / mL, at least approximately 3.2 cm 2 / mL, at least approximately 3.3 cm 2 / mL, at least approximately 3.4 cm 2 / mL, at least approximately 3.5 cm 2 / mL, at least approximately 3.6 cm 2 / mL, at least approximately 3.7 cm 2 / mL, at least approximately 3.8 cm 2 / mL, at least approximately 3.9 cm 2 / mL, at least approximately 4.0 cm 2 / mL, at least approximately 4.1 cm 2 / mL, at least approximately 4.2 cm 2 / mL, at least approximately 4.3 cm 2 / mL, at least approximately 4.4 cm 2 / mL, at least about 4.5 cm 2 / mL, at least approximately 4.6 cm 2 / mL, at least approximately 4.7 cm 2 / mL, at least approximately 4.8 cm 2 / mL, at least approximately 4.9 cm 2 / mL, or at least about 5.0 cm 2 In some embodiments, the SA / V ratio of the container can be up to about 10.0 cm 2 / mL (e.g., up to approximately 9.9 cm 2 / mL, maximum approximately 9.8cm 2 / mL, maximum approximately 9.7cm 2 / mL, maximum approximately 9.6cm 2 / mL, maximum approximately 9.5cm2 / mL, maximum about 9.4cm 2 / mL, maximum about 9.3cm 2 / mL, maximum about 9.2cm 2 / mL, maximum about 9.1cm 2 / mL, maximum about 9.0cm 2 / mL, maximum about 8.9cm 2 / mL, maximum about 8.8cm 2 / mL, maximum about 8.7cm 2 / mL, maximum about 8.6, cm 2 / mL Maximum about 8.5cm 2 / mL, maximum of about 8.4cm 2 / mL, maximum about 8.3cm 2 / mL, maximum about 8.2cm 2 / mL, maximum about 8.1cm 2 / mL, maximum about 8.0cm 2 / mL, maximum of about 7.9cm 2 / mL, maximum of about 7.8cm 2 / mL, maximum of about 7.7cm 2 / mL, maximum of about 7.6cm 2 / mL, maximum of about 7.5cm 2 / mL, maximum of about 7.4cm 2 / mL, maximum of about 7.3cm 2 / mL, maximum of about 7.2cm 2 / mL, maximum of about 7.1cm 2 / mL, maximum about 6.9cm 2 / mL, maximum about 6.8cm 2 / mL, maximum about 6.7cm 2 / mL, maximum about 6.6cm 2 / mL, maximum of about 6.5cm 2 / mL, maximum of about 6.4cm 2 / mL, maximum of about 6.3cm 2 / mL, maximum of about 6.2cm 2 / mL, maximum about 6.1cm 2 / mL, maximum about 6.0cm 2 / mL, maximum about 5.9cm 2 / mL, maximum about 5.8cm 2 / mL, maximum of about 5.7cm 2 / mL, maximum about 5.6cm 2 / mL, maximum about 5.5cm 2 / mL, maximum about 5.4cm2 / mL, maximum approximately 5.3cm 2 / mL, maximum approximately 5.2cm 2 / mL, maximum approximately 5.1cm 2 / mL, maximum approximately 5.0cm 2 / mL, maximum approximately 4.9cm 2 / mL, maximum approximately 4.8cm 2 / mL, maximum approximately 4.7cm 2 / mL, maximum approximately 4.6cm 2 / mL, maximum approximately 4.5cm 2 / mL, maximum approximately 4.4cm 2 / mL, maximum approximately 4.3cm 2 / mL, maximum about 4. cm 2cm 2 / mL, maximum approximately 4.1cm 2 / mL, or up to approximately 4.0 cm 2 In some embodiments, the SA / V ratio of the container can be about 2.0 to about 10.0 cm 2 / mL (e.g., approximately 2.1 cm 2 / mL ~ approx. 9.9cm 2 / mL, approx. 2.2cm 2 / mL ~ approx. 9.8cm 2 / mL, approx. 2.3cm 2 / mL ~ approx. 9.7cm 2 / mL, approx. 2.4cm 2 / mL ~ approx. 9.6cm 2 / mL, approx. 2.5cm 2 / mL ~ approx. 9.5cm 2 / mL, approx. 2.6cm 2 / mL ~ approx. 9.4cm 2 / mL, approx. 2.7cm 2 / mL ~ approx. 9.3cm 2 / mL, approx. 2.8cm 2 / mL ~ approx. 9.2cm 2 / mL, approx. 2.9cm 2 / mL ~ approx. 9.1cm 2 / mL, approx. 3.0cm 2 / mL ~ approx. 9.0cm 2 / mL, approx. 3.1cm 2 / mL ~ approx. 8.9cm 2 / mL, approx. 3.2cm 2 / mL ~ approx. 8.8cm 2 / mL, approx. 3.3cm2 / mL~about 8.7cm 2 / mL, about 3.4cm 2 / mL~about 8.6cm 2 / mL, about 3.5cm 2 / mL~about 8.5cm 2 / mL, about 3.6cm 2 / mL~about 8.4cm 2 / mL, about 3.7cm 2 / mL~about 8.3cm 2 / mL, about 3.8cm 2 / mL~about 8.2cm 2 / mL, about 3.9cm 2 / mL~about 8.1cm 2 / mL, about 4.0cm 2 / mL~about 8.0cm 2 / mL, about 4.1cm 2 / mL~about 7.9cm 2 / mL, about 4.2cm 2 / mL~about 7.8cm 2 / mL, about 4.3cm 2 / mL~about 7.7cm 2 / mL, about 4.4cm 2 / mL~about 7.6cm 2 / mL, about 4.5cm 2 / mL~about 7.5cm 2 / mL, about 4.6cm 2 / mL~about 7.4cm 2 / mL, about 4.7cm 2 / mL~about 7.3cm 2 / mL, about 4.8cm 2 / mL~about 7.2cm 2 / mL, about 4.9cm 2 / mL~about 7.1cm 2 / mL, about 5.0cm 2 / mL~about 6.9cm 2 / mL, about 5.1cm 2 / mL~about 6.8cm 2 / mL, about 5.2cm 2 / mL~about 6.7cm 2 / mL, about 5.3cm 2 / mL~about 6.6cm 2 / mL, about 5.4cm 2 / mL~about 6.5cm 2 / mL, approx. 5.5cm 2 / mL ~ approx. 6.4cm 2 / mL, approx. 5.6cm 2 / mL ~ approx. 6.3cm 2 / mL, approx. 5.7cm 2 / mL ~ approx. 6.2cm 2 / mL, or approximately 5.8 cm 2 / mL ~ approx. 6.1cm 2 / mL range.

[0266] The gas-permeable closed container (e.g., bag), or portions thereof, can be made of one or more of a variety of gas-permeable materials. In some embodiments, the gas-permeable bag can be made from one or more polymers, including fluoropolymers (such as polytetrafluoroethylene (PTFE) and perfluoroalkoxy (PFA) polymers), polyolefins (such as low-density polyethylene (LDPE), high-density polyethylene (HDPE)), fluorinated ethylene propylene (FEP), polystyrene, polyvinyl chloride (PVC), silicone, and any combination thereof.

[0267] In some embodiments, dried platelets or platelet derivatives (e.g., FDPD) can be subjected to heat treatment. Heating can be performed at a temperature greater than about 25°C (e.g., greater than about 40°C, greater than 50°C, greater than 60°C, greater than 70°C, greater than 80°C, or higher). In some embodiments, heating is performed at about 70°C to about 85°C (e.g., about 75°C to about 85°C, or about 75°C or 80°C). The temperature of heating can be selected along with the length of time the heating is performed. Typically, freeze-dried platelets are heated for at least 1 hour, but not more than 36 hours, although any suitable time can be used. Thus, in embodiments, heating is performed for at least 2 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least 20 hours, at least 24 hours, or at least 30 hours. For example, freeze-dried platelets can be heated for 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, or 30 hours. Non-limiting exemplary combinations include heating dried platelets or platelet derivatives (e.g., FDPD) at a temperature above 30°C for at least 30 minutes, heating dried platelets or platelet derivatives (e.g., FDPD) at a temperature above 50°C for at least 10 hours, heating dried platelets or platelet derivatives (e.g., FDPD) at a temperature above 75°C for at least 18 hours, and heating dried platelets or platelet derivatives (e.g., FDPD) at 80°C for 24 hours. In some embodiments, heating can be performed in a sealed container such as a capped vial. In some embodiments, the sealed container is subjected to a vacuum before heating. Heat treatment, particularly in the presence of a cryoprotectant such as albumin or polysucrose, has been found to improve the stability and shelf life of freeze-dried platelets. Indeed, certain combinations of serum albumin or polysucrose with a post-lyophilization heat treatment step have been shown to provide advantageous results compared to cryoprotectants without a heat treatment step. The cryoprotectant (e.g., sucrose) can be present in any suitable amount (e.g., about 3% to about 10% by mass or volume of platelets or platelet derivatives (e.g., FDPD)).

[0268] In some embodiments, platelets or platelet derivatives (e.g., FDPD) prepared as disclosed herein by a process that includes incubation with an incubation agent have a storage stability that is at least about equal to the storage stability of the platelets prior to incubation.

[0269] In some embodiments, the method further comprises cryopreserving the platelets or platelet derivatives (e.g., in an incubation substance, e.g., an incubation substance described herein) prior to administering the platelets or platelet derivatives.

[0270] In some embodiments, the method further comprises drying the composition comprising platelets or platelet derivatives (e.g., with an incubation substance, e.g., an incubation substance described herein) prior to administering the platelets or platelet derivatives (e.g., FDPD). In some embodiments, the method can further comprise heating the composition after the drying step. In some embodiments, the method can further comprise rehydrating the composition after the freeze-drying or heating step.

[0271] In some embodiments, the method further comprises freeze-drying the composition comprising platelets or platelet derivatives (e.g., with an incubation substance, e.g., an incubation substance described herein) prior to administering the platelets or platelet derivatives (e.g., FDPD). In some embodiments, the method can further comprise heating the composition after the freeze-drying step. In some embodiments, the method can further comprise rehydrating the composition after the freeze-drying or heating step.

[0272] In some embodiments, the method further includes cold storing the platelets, platelet derivatives, or FDPD (e.g., in an incubation substance, e.g., an incubation substance described herein) prior to administering the platelets, platelet derivatives, or FDPD.

[0273] Storage conditions include, for example, standard room temperature storage (e.g., storage at a temperature ranging from about 20 to about 30°C) or cryogenic storage (e.g., storage at a temperature ranging from about 1 to about 10°C). In some embodiments, the method further includes cryopreserving (e.g., with an incubation substance, e.g., an incubation substance described herein), freeze-drying, thawing, rehydrating, and combinations thereof, a composition comprising platelets or platelet derivatives (e.g., FDPD) prior to administering the platelets or platelet derivatives (e.g., FDPD). For example, in some embodiments, the method further includes drying (e.g., freeze-drying) (e.g., with an incubation substance, e.g., an incubation substance described herein) a composition comprising platelets or platelet derivatives (e.g., FDPD) (e.g., to form FDPD) prior to administering the platelets or platelet derivatives (e.g., FDPD). In some embodiments, the method can further include rehydrating the composition resulting from the drying step.

[0274] In some embodiments, provided herein is a composition comprising platelets or a platelet derivative (e.g., FDPD), polysucrose, and trehalose, the composition being made by the process of obtaining fresh platelets, optionally incubating the platelets in DMSO, isolating the platelets by centrifugation, resuspending the platelets in an incubation substance comprising trehalose and ethanol, thereby forming a first mixture, incubating the first mixture, mixing polysucrose with the first mixture, thereby forming a second mixture, and lyophilizing the second mixture to form a freeze-dried composition comprising platelets or a platelet derivative (e.g., FDPD), polysucrose, and trehalose.

[0275] In some embodiments, provided herein is a method of making a freeze-dried platelet composition comprising platelets or a platelet derivative (e.g., FDPD), polysucrose, and trehalose, the method comprising obtaining fresh platelets, optionally incubating the platelets in DMSO, isolating the platelets by centrifugation, resuspending the platelets in an incubation substance comprising trehalose and ethanol to thereby form a first mixture, incubating the first mixture, mixing polysucrose with the first mixture to thereby form a second mixture, and lyophilizing the second mixture to form a freeze-dried composition comprising platelets or a platelet derivative (e.g., FDPD), polysucrose, and trehalose.

[0276] In some embodiments, a process for producing freeze-dried platelets is provided herein, the process comprising: incubating isolated platelets in the presence of at least one sugar under the following conditions: a temperature of 20°C to 42°C for about 10 minutes to about 180 minutes; adding at least one cryoprotectant to the platelets; and freeze-drying the platelets, wherein the process optionally does not include isolating the platelets between the incubation and adding steps, and optionally does not include exposing the platelets to a platelet activation inhibitor. The cryoprotectant can be a polysaccharide (e.g., polysucrose). The process can further comprise heating the freeze-dried platelets at a temperature of 70°C to 80°C for 8 to 24 hours. The step of adding at least one cryoprotectant to the platelets can further comprise exposing the platelets to ethanol. The step of incubating the isolated platelets in the presence of at least one sugar can comprise incubating in the presence of at least one sugar. The step of incubating isolated platelets in the presence of at least one sugar may include incubating in the presence of at least one sugar. The incubating conditions may include incubating for about 100 minutes to about 150 minutes. The incubating conditions may include incubating for about 110 minutes to about 130 minutes. The incubating conditions may include incubating for about 120 minutes. The incubating conditions may include incubating at 35°C to 40°C. The incubating conditions may include incubating at 37°C. The incubating conditions may include incubating at 35°C to 40°C for 110 minutes to 130 minutes. The incubating conditions may include incubating at 37°C for 120 minutes. The at least one sugar may be trehalose, sucrose, or both trehalose and sucrose. The at least one sugar may be trehalose. The at least one sugar may be sucrose.

[0277] In some embodiments, provided herein is a method of preparing freeze-dried platelets, the method comprising: providing platelets; suspending the platelets in a salt buffer comprising about 100 mM trehalose and about 1% (v / v) ethanol to form a first composition; incubating the first composition at about 37°C for about 2 hours; adding polysucrose (e.g., polysucrose 400) to a final concentration of about 6% (w / v) to form a second composition; lyophilizing the second composition to form freeze-dried platelets; and heating the freeze-dried platelets at 80°C for 24 hours.

[0278] Certain embodiments disclosed herein may be further limited in the claims using the language "consisting of" or "consisting essentially of."

[0279] Illustrative Embodiments In this exemplary embodiments section, non-limiting exemplary aspects and embodiments are provided herein and are further discussed throughout the specification. For the sake of brevity and convenience, not all aspects and embodiments disclosed herein, nor all possible combinations of the disclosed aspects and embodiments, are listed in this section. Additional embodiments and aspects are provided in other sections of the specification. Furthermore, it will be understood that embodiments are provided that are specific embodiments for many aspects and can be combined with any other embodiments, for example, as discussed throughout this disclosure. Given the full disclosure of the specification, it is intended that any individual embodiment described below or in this full disclosure can be combined with any aspect described below or in this full disclosure, whether as an additional element that may be added to an aspect or as a narrower element of an element already present in an aspect. Such combinations may be provided as non-limiting exemplary combinations and / or are more specifically discussed in other sections of this detailed description.

[0280] In one aspect, provided herein is a method of treating a coagulation disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition comprising platelets, or in an exemplary embodiment, a platelet derivative and, in a further exemplary embodiment, FDPD, thereby treating the coagulation disorder. In an exemplary embodiment, the composition comprising a platelet derivative is administered such that the subject's likelihood of bleeding is reduced, and in an exemplary embodiment, such that normal hemostasis is restored in the subject.

[0281] In one aspect, provided herein is a method of treating a coagulation disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition, the composition having been prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, and in an exemplary embodiment, freeze-drying the incubated platelets to form a composition, the composition comprising a platelet derivative, and in an exemplary embodiment, FDPD, thereby treating the coagulation disorder. In an exemplary embodiment, the composition comprising the platelet derivative is administered to reduce the likelihood of bleeding in the subject, and in an exemplary embodiment, is administered to restore normal hemostasis in the subject.

[0282] In one aspect, provided herein is a method of restoring normal hemostasis in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition comprising platelets, or in an exemplary embodiment, a platelet derivative and, in a further exemplary embodiment, FDPD, thereby treating a coagulation disorder. In an exemplary embodiment, the composition comprising a platelet derivative is administered such that the subject's likelihood of bleeding is reduced, and in an exemplary embodiment, such that normal hemostasis is restored in the subject.

[0283] In one aspect, provided herein is a method of restoring normal hemostasis in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition, the composition having been prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, and in an exemplary embodiment, freeze-drying the incubated platelets to form a composition, the composition comprising a platelet derivative, and in a further exemplary embodiment, FDPD, thereby treating a coagulation disorder. In an exemplary embodiment, the composition comprising the platelet derivative is administered such that the subject's likelihood of bleeding is reduced, and in an exemplary embodiment, such that normal hemostasis is restored in the subject.

[0284] In one aspect, provided herein is a method of preparing a subject for surgery, the method comprising administering to a subject in need thereof an effective amount of a composition comprising platelets, or in an exemplary embodiment, a platelet derivative and, in a further exemplary embodiment, FDPD. Various characteristics of such exemplary embodiments of FDPD are provided herein, thereby treating a coagulation disorder. In exemplary embodiments, the composition comprising a platelet derivative is administered to reduce the likelihood of bleeding in the subject, and in exemplary embodiments, is administered to restore normal hemostasis in the subject. Implementations may include one or more of the following features: The surgery may be an emergency surgery. The surgery may be a planned surgery.

[0285] In one aspect, provided herein is a method of preparing a subject for surgery, the method comprising administering to a subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, and in an exemplary embodiment, freeze-drying the incubated platelets to form a composition, the composition comprising a platelet derivative, and in a further exemplary embodiment, FDPD, thereby treating a coagulation disorder. In an exemplary embodiment, the composition comprising the platelet derivative is administered to reduce the likelihood of bleeding in the subject, and in an exemplary embodiment, to restore normal hemostasis in the subject. Various characteristics of exemplary embodiments of such FDPD are provided herein. Implementations may include one or more of the following features: The surgery may be an emergency surgery. The surgery may be a scheduled surgery.

[0286] In one aspect, provided herein is a method of improving the effectiveness of an antiplatelet agent in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition comprising platelets, or in an exemplary embodiment, a platelet derivative, or in a further exemplary embodiment, FDPD, thereby treating a coagulation disorder. In an exemplary embodiment, the composition comprising the platelet derivative is administered such that the subject's likelihood of bleeding is reduced, and in an exemplary embodiment, such that normal hemostasis is restored in the subject.

[0287] In one aspect, provided herein is a method of improving the effectiveness of an antiplatelet agent in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form a composition, the composition comprising a platelet derivative, in a further exemplary embodiment, FDPD, thereby treating a coagulation disorder. In exemplary embodiments, the composition comprising the platelet derivative is administered to reduce the likelihood of bleeding in the subject, and in exemplary embodiments, to restore normal hemostasis in the subject.

[0288] In one aspect, provided herein is a method for treating a coagulation disorder, restoring hemostasis in a subject, or reducing the likelihood of bleeding in a subject receiving or having received an antiplatelet agent, comprising administering to a subject in need thereof an effective amount of a composition comprising a platelet derivative, thereby treating the coagulation disorder. In an exemplary embodiment, the platelet derivative is a freeze-dried platelet derivative (FDPD). In a further exemplary embodiment, the composition comprising the platelet derivative is administered to reduce the likelihood of bleeding in the subject, and in an exemplary embodiment, is administered to restore normal hemostasis in the subject.

[0289] In another aspect, provided herein is a method for treating a coagulation disorder, or restoring hemostasis in a subject, or reducing the likelihood of bleeding in a subject who is or has been administered an antiplatelet agent, comprising administering to a subject in need thereof an effective amount of a composition comprising FDPD, wherein the composition comprises a population of FDPDs that has a reduced tendency to aggregate, such that no more than 10% of the FDPDs in the population of FDPDs aggregate under aggregation conditions that include an agonist but do not include platelets, thereby treating the coagulation disorder. In a further exemplary embodiment, the composition comprising FDPD is administered such that the likelihood of bleeding in the subject is reduced, and in an exemplary embodiment, such that normal hemostasis is restored in the subject.

[0290] In another aspect, provided herein are methods for preventing or reducing the likelihood of a coagulation disorder in a subject, the methods comprising: (a) determining that information regarding whether the subject has been administered an antiplatelet agent is unavailable; and (b) administering to the subject a composition comprising an effective amount of a freeze-dried platelet derivative (FDPD). In some embodiments of such methods, the information regarding whether the subject has been administered an antiplatelet agent is unavailable for reasons including an inability to identify the subject. In some embodiments of the methods, the information regarding whether the subject has been administered an antiplatelet agent is unavailable for reasons including an unavailability of the subject's medical history. In further embodiments, the information regarding whether the subject has been administered an antiplatelet agent is unavailable for reasons including the subject's need for emergency treatment.

[0291] In another aspect, provided herein are methods for treating a coagulation disorder in a subject, or for reducing the likelihood of bleeding in a subject, or for restoring hemostasis in a subject, the methods comprising administering to a subject in need thereof an effective amount of a composition comprising a platelet derivative, in an exemplary embodiment comprising FDPD, wherein the subject has been administered an antiplatelet agent and a second agent that reduces platelet function prior to administration of the platelet derivative-containing composition, thereby treating the coagulation disorder. In a further exemplary embodiment, the platelet derivative-containing composition is administered such that the subject's likelihood of bleeding is reduced, and in an exemplary embodiment, such that normal hemostasis is restored in the subject. In an exemplary embodiment, prior to administration of the FDPD-containing composition, the subject was in need thereof due to an increased risk of bleeding caused by or resulting from administration of the antiplatelet agent and the second agent.

[0292]

[0013] In another aspect, provided herein is a composition comprising a freeze-dried platelet derivative (FDPD) for treating a coagulation disorder in a subject, the treatment comprising: administering to a subject in need thereof an effective amount of a composition comprising FDPD such that the subject's likelihood or risk of bleeding is reduced; The subject was administered an antiplatelet agent and a second agent that reduces platelet function, and the subject has a need for this due to the possibility of bleeding or increased risk of bleeding caused by or resulting from the administration of an antiplatelet agent and a second agent; Administering Including, This treats coagulation disorders.

[0293] In another aspect, provided herein is a composition comprising a freeze-dried platelet derivative (FDPD) for treating a coagulation disorder in a subject who is receiving or has been administered an anticoagulant and has an increased likelihood or risk of bleeding as a result, the treatment comprising: administering to a subject having an increased likelihood or risk of bleeding an effective amount of a composition comprising FDPD such that the subject's likelihood or risk of bleeding is reduced; administering a composition comprising FDPD to a population of FDPDs with reduced aggregation propensity, such that 10% or less of the FDPDs in the population aggregate under aggregation conditions that include an agonist but do not include platelets. Including, This treats coagulation disorders.

[0294] In some embodiments, for example, in aspects in which the subject has been administered an antiplatelet agent and a second agent that reduces platelet function, such methods further include determining that the subject has been administered an antiplatelet agent and a second agent that reduces platelet function prior to administration of the composition comprising FDPD. In some embodiments, the antiplatelet agent is a first antiplatelet agent and the second agent is a second antiplatelet agent. In some embodiments, the first antiplatelet agent and the second antiplatelet agent are each different antiplatelet agents selected from aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, cilostazol, prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, and sarpogrelate. In some embodiments, the first antiplatelet agent and the second antiplatelet agent have different mechanisms of action. In some embodiments, the first antiplatelet agent and the second antiplatelet agent are each different antiplatelet agents selected from aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, cilostazol, prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, and sarpogrelate.

[0295] In some embodiments of any of the aspects herein, the subject is or has been administered an antiplatelet agent prior to, immediately prior to, the moment before, the moment before, the moment, and / or at an earlier time point, the administration of a composition comprising a platelet derivative, e.g., FDPD. Furthermore, the subject may be at or have been administered an antiplatelet agent 7, 6, 5, 4, 3, 2, or 1 day, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, or 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute before the administration of the composition comprising a platelet derivative. In some optional embodiments, this is confirmed by laboratory testing. However, in some embodiments, laboratory testing of bleeding risk or any coagulation parameter is not performed 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day or earlier before and / or after the administration of the composition comprising a platelet derivative. Bleeding risk is typically reduced after administration of an effective dose of a composition comprising a platelet derivative, in exemplary embodiments, FDPD. Furthermore, a subject may maintain an increased risk of bleeding after administration of a composition comprising a platelet derivative (e.g., FDPD), depending on the time it takes for the FDPD to reduce the risk in the subject after administration, e.g., 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, or 45 minutes, or 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 8 hours, or longer, after administration. Furthermore, in some embodiments, administration of a composition comprising a platelet derivative (e.g., FDPD) reduces, but does not completely eliminate, the increased risk of bleeding in the subject.

[0296] In some embodiments, e.g., in aspects where the subject has received an antiplatelet agent and a second agent that reduces platelet function, administration of the second agent is stopped, e.g., before administering the composition comprising the platelet derivative. In other embodiments of such aspects, administration of the second agent is continued, e.g., after administering the composition comprising the platelet derivative.

[0297] In certain embodiments of any of the aspects provided herein, the method further includes, prior to administering the composition comprising the platelet derivative, determining that the subject has abnormal values ​​for one or more coagulation parameters in a pre-administration assessment. In exemplary embodiments, the pre-administration assessment is an in vitro clinical test.

[0298] In certain embodiments of any of the aspects provided herein, the antiplatelet agent is selected from aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, cilostazol, prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, and sarpogrelate. In other embodiments, the antiplatelet agent is selected from cangrelor, ticagrelor, abciximab, terutroban, picotamide, elinogrel, ibuprofen, vorapaxar, atopaxar, cilostazol, prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, and sarpogrelate.

[0299] In certain embodiments of any of the aspects provided herein, the FDPD comprises a biomolecule (e.g., a detectable amount) targeted by an antiplatelet antagonist administered or being administered to a subject. In some embodiments, the receptor is selected from a P2Y receptor (e.g., a P2Y12 receptor), glycoprotein IIb (i.e., CD41), glycoprotein IIIa (CD61), glycoprotein IIb / IIIa complex, thromboxane synthase or thromboxane receptor, PAR1, PAR4, VPVI, or a collagen receptor (e.g., an α2β1 collagen receptor). Examples of antiplatelet agents targeting these specific biomolecules are provided in other sections herein. In certain embodiments, at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the platelet derivatives, in exemplary embodiments, the FDPD, are positive for (i.e., have detectable levels of) and / or are detectable in the subject's blood for the biomolecule targeted by the antiplatelet agent administered to the subject. As a notable non-limiting example, the antiplatelet agent inhibits the glycoprotein CDIIb / IIIa complex, and at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the platelet derivatives, in exemplary embodiments, FDPDs, are CD41 positive (i.e., contain detectable CD41) and / or are positive for the CDIIb / IIIa complex.

[0300] In certain embodiments of any of the aspects provided herein, the composition comprising FDPD comprises a population of FDPD with a reduced tendency to aggregate, such that under aggregation conditions that include an agonist but no platelets, no more than 2%, 3%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, or 25% of the FDPD in the population of FDPD aggregate. In certain embodiments of any of the aspects provided herein, including embodiments in which the composition comprises a population of FDPD with a reduced tendency to aggregate, such that under aggregation conditions that include an agonist but no platelets, no more than 10% of the FDPD in the population of FDPD aggregate. 6 For example, in some cases, the platelets or platelet derivatives (e.g., FDPD) have a potency of at least 1.2 (e.g., at least 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5) thrombin generating potency units (TGPU) per particle. 6 1.2 to 2.5 TPGU per particle (e.g., 10 6 The composition may have a potency of 1.2 to 2.0, 1.3 to 1.5, 1.5 to 2.25, 1.5 to 2.0, 1.5 to 1.75, 1.75 to 2.5, 2.0 to 2.5, or 2.25 to 2.5 TPGU per particle.

[0301] In certain embodiments of any of the aspects provided herein, including embodiments in which the composition comprising FDPD comprises a population of FDPDs with a reduced tendency to aggregate, such that no more than 10% of the FDPDs in the population aggregate under aggregation conditions that include an agonist but no platelets, the FDPDs have one or more characteristics of hyperactivated platelets, said characteristics being: A) The presence of thrombospondin (TSP) on the surface of FDPD at higher levels than on the surface of resting platelets. B) The presence of von Willebrand factor (vWF) on the surface of FDPDs at levels higher than on the surface of resting platelets, and C) failure to increase the expression of platelet activation markers in the presence of an agonist compared to the expression of platelet activation markers in the absence of an agonist; is selected from.

[0302] In certain embodiments of any of the aspects provided herein, where the composition comprising FDPD comprises a population of FDPDs comprising CD41-positive platelet derivatives, e.g., in non-limiting embodiments, where the population of FDPDs comprises FDPDs with reduced aggregation propensity such that 10% or less of the FDPDs in the population aggregate under aggregation conditions that include an agonist but no platelets, the FDPDs are unable to increase expression of platelet activation markers in the presence of an agonist compared to expression of platelet activation markers in the absence of an agonist. In some embodiments of such methods, the FDPDs are less than 10%. 6 In some embodiments of such methods, less than 5% of the CD41-positive FDPDs are microparticles having a diameter of less than 0.5 μm.

[0303] In certain embodiments of any of the aspects provided herein, the population comprises FDPDs that have a reduced tendency to aggregate, e.g., such that no more than 10% of the FDPDs in the population aggregate under aggregation conditions that include an agonist but no platelets. In non-limiting embodiments, the FDPDs further have one or both of the presence of thrombospondin (TSP) on their surface at a level higher than that on the surface of resting platelets, and the presence of von Willebrand factor (vWF) on their surface at a level higher than that on the surface of resting platelets.

[0304] In certain embodiments of any of the aspects provided herein, the composition comprising FDPD comprises: CD41-positive platelet derivatives, wherein less than 5% of the CD41-positive platelet derivatives are microparticles having a diameter of less than 0.5 μm; The following features: a reduced tendency to aggregate such that no more than 10% of the platelet derivatives in the population aggregate under aggregation conditions that include an agonist but do not include platelets; a failure to increase the expression of the platelet activation marker in the presence of the agonist compared to the expression of the platelet activation marker in the absence of the agonist; the presence of thrombospondin (TSP) on the surface of platelet derivatives at levels higher than on the surface of resting platelets; the presence of von Willebrand factor (vWF) on the surface of platelet derivatives at levels higher than on the surface of resting platelets, and 10 6 A potency of at least 1.5 thrombin generating potency units (TGPU) per platelet derivative and a platelet derivative having one or more, or in an exemplary embodiment, all, of: a population of platelet derivatives, including Including, the FDPD population.

[0305] In some embodiments of any aspect herein, the platelet derivatives, and in exemplary embodiments, FDPD, are surrounded by an impaired plasma membrane. In such embodiments, the platelet derivatives lack an integral membrane around them. In such embodiments, the platelet derivatives are not surrounded by an integral membrane. Instead, the membrane contains pores that are larger than those observed in living cells. Thus, such platelet derivatives have a reduced or incapable of transducing signals from the external environment into intraparticle responses that are typically transduced in living platelets. Furthermore, such platelet derivatives (e.g., FDPD) are not believed to be capable of mitochondrial activation or glycolysis.

[0306] In some embodiments of any aspect herein, the effective amount of the composition comprising FDPD is 1.0×10 7 ~1.0×10 11 particles or FDPD / kg of subject. In some embodiments of any of the aspects herein, the effective amount of a composition comprising FDPD is 1.6×10 7 ~5.1×109 In some embodiments of any of the aspects herein, which may be combined with any of the above embodiments having ranges of particles or FDPD / kg, the effective amount of the composition comprising FDPD is an amount having a potency of 250-5000 TGPU per kg of subject. Further examples of effective amounts are provided in other sections of this specification.

[0307] In one aspect, provided herein is a method for treating a coagulation disorder, or restoring hemostasis in a subject, or reducing the likelihood of bleeding, in a subject who is or has been administered an antiplatelet agent, the method comprising administering to a subject in need thereof an effective amount of a composition comprising a platelet derivative, thereby treating the coagulation disorder, wherein the composition comprising FDPD has properties that can reduce the likelihood of bleeding in the subject, regardless of whether a post-administration assessment of the likelihood of bleeding, if performed, results in a normal result or an abnormal result.

[0308] In some embodiments of any of the aspects herein, a composition comprising FDPD has the property of being able to reduce the bleeding likelihood of a subject, regardless of whether a post-administration assessment of bleeding likelihood, when performed, results in a normal or abnormal result. In some optional embodiments, which are performed in some embodiments and not in some embodiments, such post-administration assessment comprises an in vitro clinical test performed on a sample taken or collected a period of time after administering the composition comprising FDPD to the subject. In other embodiments of any of the aspects herein, a composition comprising FDPD has the property of being able to reduce the bleeding likelihood of a subject who has an increased bleeding likelihood, and in some embodiments, an abnormal value for one or more coagulation parameters, in an in vitro clinical test, such that normal hemostasis is restored in the subject, regardless of whether a post-administration assessment of bleeding likelihood, when performed, results in a normal or abnormal result. In some embodiments, such post-administration assessment comprises an in vitro clinical test performed on a sample taken or collected a period of time after administering the composition comprising FDPD to the subject. The period can be, for example, 0 minutes to 72 hours, or 10 minutes to 72 hours, or 10 minutes to 48 hours, or 10 minutes to 24 hours, or 10 minutes to 4 hours, or 10 minutes to 1 hour, or 10 minutes to 30 minutes, or 30 minutes to 24 hours, or 30 minutes to 4 hours, or 30 minutes to 1 hour after administration of a composition comprising a platelet derivative (e.g., FDPD) to a subject. For example, in certain embodiments, the period is 1 to 4 hours after administration of a composition comprising a platelet derivative (e.g., FDPD). In some embodiments of any of the aspects herein, administration of a composition comprising a platelet derivative is not performed before or after the administration, for example, during the above-mentioned period.

[0309] In some embodiments of any of the aspects herein, a composition comprising a platelet derivative (e.g., FDPD) has the property of being capable of reducing the likelihood of bleeding in a subject with an increased or elevated likelihood of bleeding. In some embodiments, such increased or elevated likelihood of bleeding can be determined by abnormal values ​​of one or more coagulation parameters in an in vitro clinical test performed on a sample drawn within 0 minutes to 72 hours, or 10 minutes to 72 hours, or 10 minutes to 48 hours, or 10 minutes to 24 hours, or 10 minutes to 4 hours, or 10 minutes to 1 hour, or 10 minutes to 30 minutes, or 30 minutes to 24 hours, or 30 minutes to 4 hours, or 30 minutes to 1 hour prior to administration of the composition comprising a platelet derivative (e.g., FDPD). Furthermore, compositions comprising FDPD typically possess the additional surprising property that, after administration to a subject in an effective amount, e.g., to reduce the subject's bleeding potential, the subject has an abnormal value for one or more coagulation parameters in one or more in vitro clinical tests, e.g., a post-administration evaluation performed using, or an in vitro clinical test performed on, a blood sample taken 15 minutes to 4 hours, 30 minutes to 4 hours, 1 hour to 4 hours, or 15 minutes to 2 hours, 30 minutes to 2 hours, or 1 hour to 2 hours, or 15 minutes to 1 hour, or 30 minutes to 1 hour after administration of the FDPD-containing composition. In some subembodiments of this embodiment, the FDPD-containing composition has the property of being able to reduce a subject's bleeding potential to near-normal or normal hemostasis, or to a near-hemostatic level or a hemostatic level in the subject in the absence of an antiplatelet agent. However, in these embodiments, the FDPD-containing composition, after administration to a subject in an effective amount, retains the additional surprising property that such property is independent of post-administration clinical tests for bleeding potential. Thus, in some embodiments, the subject will have abnormal values ​​for one or more coagulation parameters in a post-administration assessment performed using blood samples taken 1 to 4 hours after administration of a composition comprising FDPD, or any of the time ranges listed immediately above, or in an in vitro clinical test performed on those blood samples.It will be understood that for methods involving compositions comprising FDPDs having such properties, or any properties that involve evaluation or testing, unless such testing step is actually recited as a method step, actual testing need not be performed in order to practice such a method.

[0310] In any of the aspects herein, in exemplary embodiments, a composition comprising a platelet derivative or FDPD further comprises additional components, such as components that were present when such platelet derivative was dried or when FDPD was freeze-dried. Such additional components may include one or more salts, buffers, and, in certain embodiments, components of an incubation material, including a cryoprotectant (also called a lyophilizing agent) and / or an organic solvent. For example, such compositions may include one or more sugars, which in exemplary embodiments include trehalose and, in further exemplary embodiments, include polysucrose, as further provided herein.

[0311] In any of the aspects herein, in some embodiments, FDPD is prepared using centrifugation. In some exemplary embodiments, FDPD is prepared using TFF, in further exemplary embodiments, without isolating platelets by centrifugation during the process.

[0312] In some embodiments of any of the aspects herein, the method further includes determining values ​​of the one or more coagulation parameters in a post-administration assessment, where the post-administration assessment is performed after administration. In some embodiments, the post-administration assessment of the one or more coagulation parameters indicates a normal value for at least one of the one or more coagulation parameters. In further embodiments of the method, the result of the post-administration assessment of the one or more coagulation parameters is improved from the result of the assessment of the one or more parameters pre-administration.

[0313] In a further embodiment of the method, administering the antiplatelet agent against medical indication is self-administered by the subject, administered by another, or administered by a medical professional.

[0314] In some embodiments of any of the aspects or embodiments herein including a second agent, typically a second agent that reduces platelet function, the second agent is selected from the group consisting of an antihypertensive agent, a proton pump inhibitor, and combinations thereof. In some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an antibiotic, a cardiovascular agent, an H2 antagonist, a neuropsychiatric agent, and combinations thereof. In some embodiments, the second agent comprises an antidepressant. In further embodiments, the antidepressant is selected from the group consisting of a selective serotonin reuptake inhibitor (SSRI), a serotonin antagonist and reuptake inhibitor (SARI), a serotonin and norepinephrine reuptake inhibitor (SNRI), and combinations thereof. In some embodiments, the second agent is not an anticoagulant.

[0315] In any of the aspects herein, in some embodiments, administration of the antiplatelet agent is stopped before or when a composition comprising a platelet derivative (e.g., FDPD) is administered to the subject. In some aspects of the method, administration of the antiplatelet agent is continued after a composition comprising a platelet derivative (e.g., FDPD) is administered to the subject.

[0316] In any of the aspects herein, in some embodiments, the method further includes determining that the subject has an abnormal value for one or more coagulation parameters in the pre-administration assessment. In some aspects of the method, the method includes determining the value of the one or more coagulation parameters in the post-administration assessment. In some embodiments, the post-administration assessment of the one or more coagulation parameters shows a normal result for at least one of the one or more coagulation parameters. In some embodiments, the result of the post-administration assessment of the one or more coagulation parameters is improved from the result of the assessment of the one or more parameters before administration.

[0317] In any of the aspects herein, in some embodiments, the subject is identified as having abnormal results of one or more pre-administration assessments of intraoperative coagulation parameters. In some embodiments, the surgery is emergency surgery. In some embodiments, the surgery is scheduled surgery.

[0318] In any of the aspects herein, in some embodiments, the coagulation parameter includes an assessment of bleeding. In some embodiments, the assessment of bleeding is based on the World Health Organization (WHO) bleeding scale. In some embodiments of the method, prior to administration, the subject has Grade 2, 3, or 4 bleeding based on the WHO bleeding scale. In some embodiments of the method, after administration, the subject has Grade 0 or 1 bleeding based on the WHO bleeding scale. In some embodiments, after administration, the subject has one grade less bleeding based on the WHO bleeding scale than before administration. In some embodiments, after administration, the subject has two grades less bleeding based on the WHO bleeding scale than before administration. In some embodiments, after administration, the subject has three grades less bleeding based on the WHO bleeding scale than before administration.

[0319] In any of the aspects herein, in some embodiments, assessing the coagulation parameter includes assessing the prothrombin time (PT). In some embodiments, an abnormal PT result includes a PT of greater than about 14 seconds. In some embodiments, after administration, the subject has a decrease in PT of at least 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, or more. In some embodiments, after administration, the subject has a normal PT.

[0320] In any of the aspects herein, in some embodiments, the one or more clotting parameters include an assessment of activated partial thromboplastin time (aPTT). In some embodiments, an abnormal aPTT result includes an aPTT of greater than about 40 seconds. In some embodiments, after administration, the subject has a decrease in aPTT of at least 5 seconds, 10 seconds, 15 seconds, 20 seconds, or more. In some embodiments, after administration, the subject has a normal aPTT.

[0321] In any of the aspects herein, in some embodiments, the one or more coagulation parameters include an assessment of thrombin clotting time (TCT). In some embodiments, an abnormal TCT result includes a TCT of greater than about 35 seconds. In some embodiments, after administration, the subject has a decrease in TCT of at least 5 seconds, 10 seconds, 15 seconds, 20 seconds, or more. In some embodiments, after administration, the subject has a normal TCT.

[0322] In any of the aspects herein, in some embodiments, the assessment of one or more coagulation parameters is measured using thromboelastography (TEG). In some embodiments, an abnormal TEG result comprises a maximum amplitude (MA) of less than about 50 mm. In some embodiments, after administration, the subject has an increase in MA of at least 5 mm, 10 mm, 15 mm, 20 mm, or more. In some embodiments, after administration, the subject has normal MA.

[0323] In any of the aspects herein, in some embodiments, the abnormal TEG result comprises a percent aggregation of less than about 85% (in the presence of 1 mmol / L arachidonic acid). In some embodiments, after administration, the subject has an increase in percent aggregation (in the presence of 1 mmol / L arachidonic acid) of at least 2 percentage points, 3 percentage points, 5 percentage points, 8 percentage points, 10 percentage points, 12 percentage points, or more. In some embodiments, after administration, the subject has normal percent aggregation (in the presence of 1 mmol / L arachidonic acid). In some embodiments, TEG is used to assess adenosine diphosphate-induced platelet-fibrin clot strength. In some aspects of the method, TEG is used to assess arachidonic acid-induced platelet-fibrin clot strength.

[0324] In any of the aspects herein, in some embodiments, the assessment of one or more coagulation parameters is measured using a P2Y12 response unit (PRU) or aspirin response unit (ARU) test method.

[0325] In any of the aspects herein, in some embodiments, an abnormal result of a P2Y12 response unit testing method includes a PRU of less than about 195, or less than about 180. In some embodiments, after administration, the subject has an increase in PRU of at least 25, 50, 75, 100, or more. In some embodiments, after administration, the subject has a normal PRU.

[0326] In any of the aspects herein, in some embodiments, an abnormal result of the aspirin response unit test method includes an ARU of less than about 550, or less than about 500. In some embodiments, after administration, the subject has an increase in ARU of at least 25, 50, 75, 100, or more. In some embodiments, after administration, the subject has a normal ARU.

[0327] In any of the aspects herein, in some embodiments, one or more coagulation parameters are measured using multielectrode aggregometry (MEA). In some embodiments, the abnormal result using MEA includes an abnormal result of ADP-induced platelet activity. In some embodiments, the abnormal MEA result includes a result of less than about 50 units (U) of ADP-induced platelet activity. In some embodiments, after administration, the subject has an increase in ADP-induced platelet activity of 5 units, 10 units, 15 units, 20 units, or more. In some embodiments, after administration, the subject has a normal value of ADP-induced platelet activity. In some embodiments, the abnormal MEA result includes an abnormal result of arachidonic acid-induced platelet activity. In some embodiments, the abnormal MEA result includes a result of less than about 70 units (U) of arachidonic acid-induced platelet activity. In some embodiments, after administration, the subject has an increase in arachidonic acid-induced platelet activity of 5 units, 10 units, 15 units, 20 units, or more. In some embodiments, after administration, the subject has a normal value of arachidonic acid-induced platelet activity.

[0328] In any of the aspects herein, in some embodiments, one or more coagulation parameters are measured using light transmittance aggregometry (LTA).

[0329] In any of the aspects herein, in some embodiments, the abnormal LTA results include one or more of: (a) percent aggregation less than about 60% in the presence of 5 μmol / L adenosine diphosphate; (b) percent aggregation less than about 65% in the presence of 2 μg / mL collagen; (c) percent aggregation less than about 65% in the presence of 1 mmol / L arachidonic acid; (d) percent aggregation less than about 69% in the presence of 2 mmol / L arachidonic acid; or (e) percent aggregation less than about 73% in the presence of 5 mmol / L arachidonic acid.

[0330] In any of the aspects herein, in some embodiments, after administration, the subject has an increase in percent aggregation (in the presence of 5 μmol / L adenosine diphosphate) of at least 2 percentage points, 3 percentage points, 5 percentage points, 8 percentage points, 10 percentage points, 12 percentage points, or more, hi some embodiments, after administration, the subject has normal percent aggregation (in the presence of 5 μmol / L adenosine diphosphate).

[0331] In any of the aspects herein, in some embodiments, after administration, the subject has an increase in percent aggregation (in the presence of 2 μg / mL of collagen) of at least 2 percentage points, 3 percentage points, 5 percentage points, 8 percentage points, 10 percentage points, 12 percentage points, or more, hi some embodiments, after administration, the subject has normal percent aggregation (in the presence of 2 μg / mL of collagen).

[0332] In any of the aspects herein, in some embodiments, After administration, the subject has an increase in percent aggregation (in the presence of 1 mmol / L arachidonic acid) of at least 2 percentage points, 3 percentage points, 5 percentage points, 8 percentage points, 10 percentage points, 12 percentage points, or more. In some embodiments, after administration, the subject has normal percent aggregation (in the presence of 1 mmol / L arachidonic acid). In some embodiments, after administration, the subject has an increase in percent aggregation (in the presence of 2 mmol / L arachidonic acid) of at least 2 percentage points, 3 percentage points, 5 percentage points, 8 percentage points, 10 percentage points, 12 percentage points, or more. In some embodiments, after administration, the subject has normal percent aggregation (in the presence of 2 mmol / L arachidonic acid). In some embodiments, after administration, the subject has an increase in percent aggregation (in the presence of 5 mmol / L arachidonic acid) of at least 2 percentage points, 3 percentage points, 5 percentage points, 8 percentage points, 10 percentage points, 12 percentage points, or more. In some embodiments, after administration, the subject has a normal percent aggregation (in the presence of 5 mmol / L arachidonic acid).

[0333] In any of the aspects herein, in some embodiments, the method further comprises administering to the subject an additional antiplatelet agent antagonist. In some embodiments, administration of the composition occurs simultaneously with administration of the additional antiplatelet agent antagonist. In some embodiments, administration of the composition occurs after administration of the additional antiplatelet agent antagonist. In some embodiments, administration of the composition occurs before administration of the additional antiplatelet agent antagonist.

[0334] In any of the aspects herein, in some embodiments, the composition further comprises an antifibrinolytic agent. In some embodiments, the antifibrinolytic agent is selected from the group consisting of epsilon-aminocaproic acid (EACA), tranexamic acid, aprotinin, aminomethylbenzoic acid, fibrinogen, and combinations thereof. In some embodiments, the platelets or platelet derivatives are loaded with the antifibrinolytic agent.

[0335] In any of the aspects herein, in some embodiments, administration comprises topical administration, parenteral administration, intravenous administration, intramuscular administration, intrathecal administration, subcutaneous administration, intraperitoneal administration, or a combination thereof.

[0336] In any of the aspects herein, in some embodiments, the composition is dried before the administering step, hi some embodiments, the composition is rehydrated after the drying step.

[0337] In any of the aspects herein, in some embodiments, the composition is freeze-dried prior to the administering step, hi some embodiments, the composition is rehydrated after the freeze-drying step.

[0338] In any of the aspects herein, in some embodiments, the incubation substance comprises one or more salts selected from phosphate salts, sodium salts, potassium salts, calcium salts, magnesium salts, and combinations of two or more thereof. In some embodiments, the incubation substance comprises a carrier protein. In some embodiments, the incubation substance comprises a buffer comprising HEPES, sodium bicarbonate (NaHCO), or a combination thereof.

[0339] In any of the aspects herein, in some embodiments, the composition comprises one or more sugars. In some embodiments, the one or more sugars comprise trehalose. In some embodiments, the one or more sugars comprise polysucrose. In some embodiments, the one or more sugars comprise dextrose.

[0340] In some aspects of the method, the composition comprises an organic solvent.

[0341] In some embodiments of any of the aspects herein, the antiplatelet agent is present in the subject at the time the composition comprising FDPD is administered at a level that increases the subject's likelihood of bleeding, hi some embodiments, the antiplatelet agent is present at Cmax within 15, 30, or 45 minutes, or within 1, 2, 3, 4, 6, or 8 hours after the composition comprising FDPD is administered, or after the first or last dose of the composition comprising FDPD is administered.

[0342] In any of the aspects herein, in some embodiments, the antiplatelet agent comprises aspirin, which has been or is being administered at a dose of about 80 mg to about 700 mg once, twice, three times, or four times daily. In some embodiments, the antiplatelet agent is administered to a subject with a C of about 3 to about 25 mg / L. max aspirin that has been or is being administered to a subject to achieve

[0343] In any of the aspects herein, in some embodiments, the antiplatelet agent comprises cangrelor, which has been or is being administered to the subject at an initial dosage of about 25 to about 30 μg / kg body weight of the subject, or at subsequent dosages of about 3 to about 5 μg / kg body weight of the subject / min. In some embodiments, the antiplatelet agent is administered to the subject at a C of about 400 to about 500 ng / mL. max The present invention includes cangrelor that has been or is being administered to a subject to achieve the above.

[0344] In any of the aspects herein, in some embodiments, the antiplatelet agent comprises ticagrelor, which has been or is being administered to the subject at an initial dosage of about 170 to about 190 mg, or at a subsequent dosage of about 80 to about 100 mg twice daily in the first year of treatment, or at a subsequent dosage of about 50 to about 70 mg twice daily in the second year of treatment, optionally in combination with aspirin. In some embodiments, the antiplatelet agent is administered to the subject at a C of about 550 to about 650 ng / mL. max The present invention includes ticagrelor that has been or is being administered to a subject to achieve the above.

[0345] In any of the aspects herein, in some embodiments, the antiplatelet agent comprises clopidogrel, which has been or is being administered to the subject at an initial dosage of about 275 to about 325 mg, or at subsequent dosages of about 70 to about 80 mg once daily, optionally in combination with aspirin. In some embodiments, the antiplatelet agent is administered to the subject at a C of about 6 to about 20 ng / mL. max The present invention includes clopidogrel that has been or is being administered to a subject to achieve the above.

[0346] In any of the aspects herein, in some embodiments, the antiplatelet agent comprises prasugrel, which has been or is being administered to the subject at an initial dosage of about 50 to about 70 mg, or at subsequent dosages of about 3 to about 12 mg once daily, optionally in combination with aspirin. In some embodiments, the antiplatelet agent is administered to the subject at a C of about 200 to about 525 ng / mL. max Prasugrel that has been administered or is being administered to a subject to achieve the above.

[0347] In any of the aspects herein, in some embodiments, the antiplatelet agent comprises eptifibatide that has been or is being administered to the subject at an initial dosage of about 170 to about 190 mcg / kg body weight of the subject, optionally at a second initial dosage of about 170 to about 190 mcg / kg body weight of the subject, or at subsequent dosages of about 1 to about 2 mcg / kg body weight of the subject / minute.

[0348] In any of the aspects herein, in some embodiments, the antiplatelet agent comprises tirofiban, which has been or is being administered to the subject at an initial dosage of about 0.3 to about 0.5 μg / kg body weight of the subject / minute for about 30 minutes, or at subsequent dosages of about 0.1 μg / kg body weight of the subject / minute.

[0349] In any of the aspects herein, in some embodiments, the antiplatelet agent comprises abciximab, which has been or is being administered to the subject at an initial dosage of about 0.2 to about 0.3 mg / kg body weight of the subject, or at subsequent dosages of about 0.10 to about 0.15 μg / kg body weight of the subject. In some embodiments, the antiplatelet agent comprises abciximab, which has been or is being administered to the subject at an initial dosage of about 0.2 to about 0.3 mg / kg body weight of the subject, or at subsequent dosages of about 8 to about 10 μg / min.

[0350] In any of the aspects herein, in some embodiments, the antiplatelet agent comprises ticlopidine that has been or is being administered to the subject at a dosage of about 240 to about 260 mg twice daily.

[0351] In any of the aspects herein, in some embodiments, the antiplatelet agent comprises ibuprofen, which has been or is being administered to the subject in a dosage of about 100 to about 600 mg once, twice, three times, or four times daily.

[0352] In any of the aspects herein, in some embodiments, the antiplatelet agent comprises vorapaxar, which has been or is being administered to the subject in a dosage of about 2 to about 3 mg once daily, optionally with aspirin or clopidogrel.

[0353] In any of the aspects herein, in some embodiments, the antiplatelet agent comprises cilostazol, which has been or is being administered to the subject at a dosage of about 40 to about 110 mg twice daily.

[0354] In any of the aspects herein, in some embodiments, the antiplatelet agent comprises epoprostenol, which has been or is being administered to the subject at an initial dosage of about 2 ng / kg body weight of the subject / min, or subsequent dosages of about 4, 6, 8, 10, 12, 14, 16, 18, or 20 ng / kg body weight of the subject / min.

[0355] In any of the aspects herein, in some embodiments, the antiplatelet agent comprises dipyridamole that has been or is being administered to the subject at a dosage of about 60 to about 110 mg four times daily.

[0356] In any of the aspects herein, in some embodiments, the antiplatelet agent comprises treprostinil sodium that has been administered or is being administered to the subject at a dosage of about 0.5 to about 1.3 ng / kg body weight of the subject / min.

[0357] In some embodiments of the methods of any of the aspects herein, in some aspects, the subject does not have cancer.

[0358] Any of the method aspects herein may be uses for a composition comprising a platelet derivative (e.g., FDPD) provided herein, or for a kit comprising such a composition, as described in the following aspects including such "use" language. It will be understood that when such aspects refer to FDPD, they may instead refer to a platelet derivative. It will be further understood that such aspects may include any of the elements provided herein with respect to the method aspects, and any of the embodiments provided herein. For example, administering a composition comprising an effective amount of a platelet derivative (e.g., FDPD) may be to reduce the subject's likelihood of bleeding, in an exemplary embodiment, to restore normal hemostasis. When such aspects refer to a disorder, the disorder in an exemplary embodiment is a bleeding disorder. Such a disorder may be identified, for example, because a sample from a subject with such a disorder produces abnormal values ​​for one or more coagulation parameters.

[0359] In one aspect, provided herein is the use of a composition comprising a platelet derivative, in exemplary embodiments, a freeze-dried platelet derivative (FDPD), in the manufacture of a kit for treating a coagulation disorder in a subject receiving or who has received an antiplatelet agent, the use of the kit comprising: (a) determining, in a pre-administration assessment, that the subject has abnormal values ​​for one or more coagulation parameters; and (b) subsequent to (a), administering to the subject in need thereof an effective amount of a composition comprising a freeze-dried platelet derivative (FDPD). In some embodiments, the use further comprises, prior to administration, determining that the subject has been administered an antiplatelet agent. In some embodiments, the use further comprises, prior to administration, determining that information regarding whether the subject has been administered an antiplatelet agent is unavailable. In some embodiments, the use further comprises, prior to administration, determining that the subject has abnormal values ​​for one or more coagulation parameters in a pre-administration assessment prior to administering the composition comprising the freeze-dried platelet derivative. In some embodiments, the antiplatelet agent is a first antiplatelet agent and the second agent is a second antiplatelet agent.

[0360] In one aspect, provided herein is the use of a composition comprising a platelet derivative, in an exemplary embodiment, a freeze-dried platelet derivative (FDPD), in the manufacture of a kit for treating a coagulation disorder in a subject who is or has been administered an antiplatelet agent, wherein the use of the kit comprises administering an effective amount of FDPD to a subject in need thereof, and the composition comprising FDPD comprises a population of FDPD with a reduced tendency to aggregate, such that no more than 10% of the FDPD in the population aggregate under aggregation conditions that include an agonist but no platelets.

[0361] In one aspect, provided herein is the use of a composition comprising a platelet derivative, in an exemplary embodiment, a freeze-dried platelet derivative (FDPD), in the manufacture of a kit for treating a coagulation disorder in a subject, the use of the kit comprising administering to a subject in need thereof an effective amount of the composition comprising the freeze-dried platelet derivative (FDPD), wherein the subject has been administered an antiplatelet agent and a second agent that reduces platelet function.

[0362] In one aspect, provided herein is the use of a composition comprising a platelet derivative of any aspect provided herein, in an exemplary embodiment, a freeze-dried platelet derivative (FDPD), in the manufacture of a kit for treating a coagulation disorder in a subject receiving or who has received an antiplatelet agent.

[0363] In one aspect, provided herein is the use of a composition comprising a platelet derivative of any aspect provided herein, in an exemplary embodiment, a freeze-dried platelet derivative (FDPD), in the preparation of a medicament for use in treating a coagulation disorder in a subject receiving or who has received an antiplatelet agent.

[0364] In one aspect, provided herein is a composition comprising a platelet derivative of any aspect provided herein, in an exemplary embodiment, a freeze-dried platelet derivative (FDPD), for use in the manufacture of a kit for treating a coagulation disorder in a subject receiving or who has received an antiplatelet agent.

[0365] In one aspect, provided herein is a composition comprising a platelet derivative of any aspect provided herein, in an exemplary embodiment a freeze-dried platelet derivative (FDPD), for use as a medicament for treating a coagulation disorder in a subject receiving or who has received an antiplatelet agent.

[0366] In one aspect, provided herein is a composition comprising a platelet derivative of any aspect provided herein, in an exemplary embodiment, a freeze-dried platelet derivative (FDPD), for use in treating a coagulation disorder in a subject receiving or who has received an antiplatelet agent.

[0367] In one aspect, provided herein is a composition comprising a platelet derivative of any aspect provided herein, in an exemplary embodiment, a freeze-dried platelet derivative (FDPD), for use in the manufacture of a kit for treating a disorder in a subject who is or has been administered an antiplatelet agent.

[0368] In one aspect, provided herein is a composition comprising a platelet derivative of any aspect provided herein, in an exemplary embodiment a freeze-dried platelet derivative (FDPD), for use as a medicament for treating a disorder in a subject who is receiving or has been administered an antiplatelet agent.

[0369] In one aspect, provided herein is a composition comprising a platelet derivative of any aspect provided herein, in an exemplary embodiment, a freeze-dried platelet derivative (FDPD), for use in treating a disorder in a subject receiving or who has received an antiplatelet agent.

[0370] In any of the aspects provided herein involving a composition comprising a platelet derivative, in an exemplary embodiment, a freeze-dried platelet derivative (FDPD), for use in the treatment of a disorder, or in the manufacture of a kit, or as a medicament, the disorder is selected from the group consisting of alopecia areata, von Willebrand's disease, hemophilia, thrombasthenia, thrombocytopenia, thrombocytopenic purpura, trauma, or a combination thereof.

[0371] In any of the aspects provided herein involving a composition comprising a platelet derivative, in exemplary embodiments, a freeze-dried platelet derivative (FDPD), for treating a coagulation disorder or disorder, or for manufacturing a kit, or for use as a medicament, the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, cilostazol, prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, sarpogrelate, and combinations thereof.

[0372] In any of the aspects provided herein, including a composition comprising a platelet derivative, in exemplary embodiments, a freeze-dried platelet derivative (FDPD), for use in treating a coagulation disorder or disorder, or in the manufacture of a kit, or as a medicament, the treatment, or use of the kit, or medicament comprises administering to a subject in need thereof an effective amount of FDPD, wherein the composition comprising FDPD comprises a population of FDPD with a reduced tendency to aggregate, such that no more than 10% of the FDPD in the population aggregate under aggregation conditions that include an agonist but no platelets. 6 The platelet derivatives have a potency of at least 1.5 thrombin generating potency units (TGPU) per platelet derivative.

[0373] In any of the aspects provided herein, including a composition comprising a platelet derivative, in exemplary embodiments, freeze-dried platelet derivatives (FDPD), for treating a coagulation disorder or disorder, or for manufacturing a kit, or for use as a medicament, at least 50% of the FDPD are CD41-positive platelet derivatives, less than 5% of the CD41-positive FDPD are microparticles having a diameter of less than 0.5 μm, and the FDPD is less than 10 6 The platelet derivatives have a potency of at least 1.5 thrombin generating potency units (TGPU) per platelet derivative.

[0374] In any of the aspects provided herein, including a composition comprising a platelet derivative, in exemplary embodiments, a freeze-dried platelet derivative (FDPD), for use in treating a coagulation disorder or disorder, or in the manufacture of a kit, or as a medicament, the FDPD has one or more characteristics of a superactivated platelet selected from A) the presence of thrombospondin (TSP) on the surface of the FDPD at a level higher than on the surface of resting platelets, B) the presence of von Willebrand factor (vWF) on the surface of the FDPD at a level higher than on the surface of resting platelets, and C) a failure to increase expression of a platelet activation marker in the presence of an agonist compared to expression of the platelet activation marker in the absence of the agonist.

[0375] In any of the aspects provided herein that include a composition comprising a platelet derivative, in exemplary embodiments, freeze-dried platelet derivative (FDPD), for use in treating a coagulation disorder or disorder, or in the manufacture of a kit, or as a medicament, an effective amount of the composition comprising FDPD is 1.0 x 10 7 ~1.0×10 11 In some embodiments, the effective amount of a composition comprising FDPD is 1.6×10 7 ~5.1×10 9 In some embodiments, an effective amount of a composition comprising FDPD is an amount having a potency of 250-5000 TGPU / kg of subject. In some embodiments, an effective amount of a composition comprising FDPD is an amount having a potency of 300-3800 TGPU / kg of subject.

[0376] Further exemplary aspects and embodiments are provided below.

[0377] In some embodiments, provided herein is a method of treating a coagulation disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition comprising platelets or a platelet derivative and an incubation material comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0378] In some embodiments, provided herein is a method of treating a coagulation disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0379] In some embodiments, provided herein are methods of restoring normal hemostasis in a subject, the methods comprising administering to a subject in need thereof an effective amount of a composition comprising platelets or platelet derivatives and an incubation material comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0380] In some embodiments, provided herein are methods of restoring normal hemostasis in a subject, the methods comprising administering to a subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0381] In some embodiments, provided herein are methods of preparing a subject for surgery, the methods comprising administering to a subject in need thereof an effective amount of a composition comprising platelets or platelet derivatives and an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent. Implementations may include one or more of the following features: The surgery may be an emergency surgery. The surgery may be a planned surgery.

[0382] In some embodiments, provided herein are methods of preparing a subject for surgery, the methods comprising administering to a subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form a composition. Implementations may include one or more of the following features: The surgery may be an emergency surgery. The surgery may be a planned surgery.

[0383] In some embodiments of the above methods, the subject has been or is being treated with an antiplatelet agent. In some embodiments, the antiplatelet agent treatment can be stopped. In some embodiments, the antiplatelet agent treatment can be continued.

[0384] In some embodiments, provided herein are methods of improving the effectiveness of an antiplatelet agent in a subject, the methods comprising administering to a subject in need thereof an effective amount of a composition comprising platelets or a platelet derivative and an incubation material comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0385] In some embodiments, provided herein are methods of improving the effectiveness of an antiplatelet agent in a subject, the methods comprising administering to a subject in need thereof an effective amount of a composition, the composition being prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0386] In some embodiments, the effects of the antiplatelet agent may be the result of an overdose of the antiplatelet agent.

[0387] In some embodiments, the antiplatelet agent may be selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, and supplements.

[0388] Some embodiments of any of the methods herein may include one or more of the following features: The administering may include topical administration. The administering may include parenteral administration. The administering may include intravenous administration. The administering may include intramuscular administration. The administering may include intrathecal administration. The administering may include subcutaneous administration. The administering may include intraperitoneal administration. The composition may be dried prior to the administering step. The composition may be rehydrated after the drying step. The composition may be freeze-dried prior to the administering step. The composition may be rehydrated after the freeze-drying step. The incubation substance may include one or more salts selected from sodium salts, potassium salts, calcium salts, magnesium salts, and combinations of two or more thereof. The incubation substance may include a carrier protein. The buffer may include HEPES, sodium bicarbonate (NaHCO3), or a combination thereof. The composition may include one or more sugars. The one or more sugars may include trehalose. The one or more sugars may include polysucrose. The one or more sugars may include dextrose. The composition may include an organic solvent. The platelets or platelet derivatives may include FDPD.

[0389] Further non-limiting example embodiments are provided in numbered form as follows:

[0390] Embodiment 1 is a method of treating a coagulation disorder in a subject, comprising administering to a subject in need thereof an effective amount of a composition comprising platelets or platelet derivatives and an incubation material comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0391] Embodiment 2 is a method of treating a coagulation disorder in a subject, comprising administering to a subject in need thereof an effective amount of a composition, wherein the composition has been prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form a composition.

[0392] Embodiment 3 is a method of restoring normal hemostasis in a subject, comprising administering to a subject in need thereof an effective amount of a composition comprising platelets or platelet derivatives and an incubation material comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0393] Embodiment 4 is a method of restoring normal hemostasis in a subject, comprising administering to a subject in need thereof an effective amount of a composition, wherein the composition has been prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form a composition.

[0394] Embodiment 5 is a method of preparing a subject for surgery, comprising administering to a subject in need thereof an effective amount of a composition comprising platelets or platelet derivatives and an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0395] Embodiment 6 is a method of preparing a subject for surgery, comprising administering to a subject in need thereof an effective amount of a composition, wherein the composition has been prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form a composition.

[0396] Embodiment 7 is the method of embodiment 5 or 6, wherein the surgery is emergency surgery.

[0397] Embodiment 8 is the method of embodiment 5 or 6, wherein the surgery is a planned surgery.

[0398] Embodiment 9 is the method of any one of embodiments 1 to 8, wherein the subject has been or is being treated with an antiplatelet agent.

[0399] Embodiment 10 is the method of embodiment 9, wherein the antiplatelet agent treatment is stopped.

[0400] Embodiment 11 is the method of embodiment 9, wherein treatment with the antiplatelet agent is continued.

[0401] Embodiment 12 is a method of improving the effectiveness of an antiplatelet agent in a subject, comprising administering to a subject in need thereof an effective amount of a composition comprising platelets or a platelet derivative and an incubation material comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0402] Embodiment 13 is a method of improving the effect of an antiplatelet agent in a subject, comprising administering to a subject in need thereof an effective amount of a composition, wherein the composition is prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form a composition.

[0403] Embodiment 14 is the method of embodiment 12 or embodiment 13, wherein the antiplatelet agent effect is the result of an antiplatelet agent overdose.

[0404] Embodiment 15 is the method of any one of embodiments 1 to 14, wherein the composition further comprises an antifibrinolytic agent.

[0405] Embodiment 16 is the method of embodiment 15, wherein the antifibrinolytic agent is selected from the group consisting of epsilon-aminocaproic acid (EACA), tranexamic acid, aprotinin, aminomethylbenzoic acid, fibrinogen, and combinations thereof.

[0406] Embodiment 17 is the method of embodiment 15 or embodiment 16, wherein the platelets or platelet derivatives are loaded with an antifibrinolytic agent.

[0407] Embodiment 18 is the method of any one of embodiments 9 to 16, wherein the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, supplements, and combinations thereof.

[0408] Embodiment 19 is the method of any one of embodiments 9 to 16, wherein the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, and combinations thereof.

[0409] Embodiment 20 is the method of any one of embodiments 9 to 16, wherein the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, cilostazol, prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, sarpogrelate, and combinations thereof.

[0410] Embodiment 21 is the method of any one of embodiments 1 to 20, wherein administering comprises topical administration.

[0411] Embodiment 22 is the method of any one of embodiments 1 to 20, wherein administering comprises parenteral administration.

[0412] Embodiment 23 is the method of any one of embodiments 1 to 20, wherein administering comprises intravenous administration.

[0413] Embodiment 24 is the method of any one of embodiments 1 to 20, wherein administering comprises intramuscular administration.

[0414] Embodiment 25 is the method of any one of embodiments 1 to 20, wherein administering comprises intrathecal administration.

[0415] Embodiment 26 is the method of any one of embodiments 1 to 20, wherein administering comprises subcutaneous administration.

[0416] Embodiment 27 is the method of any one of embodiments 1 to 20, wherein administering comprises intraperitoneal administration.

[0417] Embodiment 28 is the method of any one of embodiments 1 to 27, wherein the composition is dried prior to the administering step.

[0418] Embodiment 29 is the method of embodiment 28, wherein the composition is rehydrated after the drying step.

[0419] Embodiment 30 is the method of any one of embodiments 1 to 28, wherein the composition is freeze-dried prior to the administering step.

[0420] Embodiment 31 is the method of embodiment 30, wherein the composition is rehydrated after the freeze-drying step.

[0421] Embodiment 32 is the method of any one of embodiments 1 to 31, wherein the incubation substance comprises one or more salts selected from phosphate salts, sodium salts, potassium salts, calcium salts, magnesium salts, and combinations of two or more thereof.

[0422] Embodiment 33 is the method of any one of embodiments 1 to 32, wherein the incubation substance comprises a carrier protein.

[0423] Embodiment 34 is the method of any one of embodiments 1 to 33, wherein the buffer comprises HEPES, sodium bicarbonate (NaHCO3), or a combination thereof.

[0424] Embodiment 35 is the method of any one of embodiments 1 to 34, wherein the composition comprises one or more sugars.

[0425] Embodiment 36 is the method of embodiment 35, wherein the one or more sugars comprise trehalose.

[0426] Embodiment 37 is the method of embodiment 35 or embodiment 36, wherein the one or more sugars comprise polysucrose.

[0427] Embodiment 38 is the method of any one of embodiments 35-37, wherein the one or more sugars comprises dextrose.

[0428] Embodiment 39 is the method of any one of embodiments 1 to 38, ...

Claims

1. A composition for use in a method of controlling bleeding in a subject, the composition comprising an effective amount of freeze-dried platelet derivative particles in a dry form and an incubation material comprising one or more salts, buffers, and sugars, the composition comprising: The method further comprising: determining that the subject has abnormal values ​​for one or more coagulation parameters in a pre-administration assessment; rehydrating the composition to form a rehydrated composition; administering the rehydrated composition to the subject; Including, the subject is being treated with an antiplatelet agent; said administering including parenteral administration; the one or more coagulation parameters are selected from the group consisting of thrombin generation, World Health Organization (WHO) bleeding scale, prothrombin time (PT) assay, thromboelastography (TEG), multielectrode aggregometry, light transmittance aggregometry (LTA), activated clotting time (ACT), aspirin response unit (ARU) test, and partial thromboplastin time (PTT or aPTT); after said administration, said subject has an improved coagulation parameter compared to before said administration; and the effective amount of freeze-dried platelet derivative particles is between 1.0×10 9 and 5.0×10 9 particles per kg of the subject, or between 5.0×10 9 and 1.0×10 10 particles per kg of the subject; The composition.

2. The composition of claim 1, wherein the one or more coagulation parameters is thrombin generation, and thrombin generation is assessed by peak thrombin, thrombin generation assay (TGA), endogenous thrombin potential (ETP), and lag time.

3. The composition of claim 2, wherein the one or more coagulation parameters is peak thrombin, and prior to administration, the subject's peak thrombin is less than 66 nM, and after administration, the subject's peak thrombin is greater than 66 nM.

4. A composition for use in a method of controlling bleeding in a subject, the composition comprising an effective amount of freeze-dried platelet derivative particles in a dry form and an incubation material comprising one or more salts, buffers, and sugars, the composition comprising: The method further comprising: rehydrating the composition to form a rehydrated composition; administering the rehydrated composition to the subject; Including, said administering including parenteral administration; the subject has been treated with an antiplatelet agent such that the most recent dose of the antiplatelet agent was within 3 days of administration of the rehydrated composition to the subject; and the effective amount of freeze-dried platelet derivative particles is between 1.0×10 9 and 5.0×10 9 particles per kg of the subject, or between 5.0×10 9 and 1.0×10 10 particles per kg of the subject; The composition.

5. The composition of claim 4, wherein the method includes determining, prior to said administering, in a pre-administration assessment, that the subject has abnormal values ​​for one or more coagulation parameters, the one or more coagulation parameters being selected from the group consisting of thrombin generation, World Health Organization (WHO) bleeding scale, prothrombin time (PT) assay, thromboelastography (TEG), multiple electrode aggregometry, light transmittance aggregometry (LTA), activated clotting time (ACT), aspirin response unit (ARU) test, and partial thromboplastin time (PTT or aPTT), and wherein after said administering, the subject has improved coagulation parameters compared to before said administering.

6. The composition described in claim 4, wherein the most recent dose of antiplatelet agent is within 36 hours of administration of the rehydrated composition to the subject.

7. The composition of claim 1, wherein the effective amount of freeze-dried platelet derivative particles is between 1.0×10 9 and 5.0×10 9 particles per kg of the subject.

8. The composition of claim 1, wherein the effective amount of freeze-dried platelet derivative particles is between 5.0×10 9 and 1.0×10 10 particles per kg of the subject.

9. The composition of any one of claims 1 to 6, wherein the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, cilostazol, prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, and sarpogrelate.

10. The composition of any one of claims 1 to 6, wherein the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, ibuprofen, and combinations thereof.

11. The composition of any one of claims 1 to 6, wherein the subject is being treated with two antiplatelet agents, and the two antiplatelet agents are selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, cilostazol, prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, and sarpogrelate.

12. The composition of claim 11, wherein the subject is being treated with aspirin and an antiplatelet agent selected from cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, cilostazol, prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, and sarpogrelate.

13. The composition of claim 12, wherein the effective amount of freeze-dried platelet derivative particles is between 5.0×10 9 and 1.0×10 10 particles per kg of the subject.

14. The composition of any one of claims 1 to 6, wherein the subject is being treated with an antiplatelet agent such that treatment with the antiplatelet agent is not stopped and is continued before and after administration.

15. The composition of any one of claims 1 to 6, wherein the method restores hemostasis in the subject.

16. The composition of claim 1 or 5, wherein the one or more coagulation parameters comprises an assessment of bleeding.

17. said bleeding assessment being based on the World Health Organization (WHO) bleeding scale; prior to said administration, said subject has bleeding grade 2, 3, or 4 based on the WHO bleeding scale, and after said administration, said subject has bleeding grade 0 or 1 based on the WHO bleeding scale; said effective amount of freeze-dried platelet derivative particles is between 1.0×10 7 and 1.0×10 11 particles per kg of said subject; and The antiplatelet agent is selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, cilostazol, prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, and sarpogrelate; 17. The composition of claim 16.

18. The composition of any one of claims 1 to 6, wherein administration comprises intravenous administration.

19. The composition of any one of claims 1 to 6, wherein the rehydrated composition comprises trehalose in an amount ranging from 10 mM to 500 mM and sugars comprising polysucrose in an amount ranging from 3% to 10% w / v, and at least 50% of the freeze-dried platelet derivative particles in the rehydrated composition have a particle size of 0.3 μm to 5.0 μm.

20. Within 1 hour after the rehydrated composition is administered to the subject, the antiplatelet agent is ｍａｘ The composition of any one of claims 1 to 6, wherein 21. The composition of any one of claims 1 to 6, wherein the dried platelet derivative composition is prepared by a process comprising incubating platelets with an incubation substance comprising one or more salts, a buffer, and a sugar to form incubated platelets, and freeze-drying the incubated platelets to form the dried platelet derivative composition comprising freeze-dried platelet derivative particles, at least 50% of the freeze-dried platelet derivative particles having a particle size of about 0.3 μm to 5.0 μm, and the sugar comprises trehalose in an amount in the range of 10 mM to 500 mM.

22. The composition of claim 21, wherein the process further comprises, after lyophilization, heat-treating the freeze-dried platelet derivative composition by heating the freeze-dried platelet derivative composition at a temperature in the range of 70°C to 85°C for at least 1 hour, but not more than 36 hours.

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