Thrombosomes as antiplatelet antagonists

Abstract

In some embodiments, provided herein are 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 a platelet derivative and an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, wherein the subject has been or is being treated with an antiplatelet agent.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application 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 which is incorporated by reference in its entirety.

[0002] FIELD OF THE INVENTION This disclosure serves to demonstrate the use of thrombosomes as a treatment for drug-induced coagulation disorders. The use of antiplatelet drugs such as aspirin or clopidogrel can increase the likelihood of bleeding. Here, we demonstrate that thrombosomes can circumvent or overcome this inhibition to restore hemostasis. [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, as stopping therapy may expose the subject to an increased risk of heart attack, stroke, or death, and the drug may need to be reduced before surgery.

[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] 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 agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0007] In some embodiments, provided herein are 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, the composition being prepared by a process comprising incubating platelets with an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0008] 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 agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0009] 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 having been prepared by a process comprising incubating platelets with an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0010] 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 agent 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 scheduled surgery.

[0011] In some embodiments, provided herein are methods for 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 agent 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 scheduled surgery.

[0012] 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.

[0013] In some embodiments, provided herein are methods for improving the effect 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 agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0014] In some embodiments, provided herein are methods of improving the effect 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 agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0015] In some embodiments, the effects of the antiplatelet agent may be the result of an overdose of the antiplatelet agent.

[0016] In some embodiments, the antiplatelet agent may be selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, and supplements.

[0017] 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 lyophilized prior to the administering step. The composition may be rehydrated after the lyophilization step. The incubation agent may include one or more salts selected from phosphate salts, sodium salts, potassium salts, calcium salts, magnesium salts, and combinations of two or more thereof. The incubation agent 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 thrombosomes. [The present invention 1001] 1. A method of restoring normal hemostasis in a subject, comprising administering to a subject in need thereof an effective amount of a composition; the composition comprises platelets or platelet derivatives and an incubation agent comprising one or more salts, buffers, optionally a cryoprotectant, and optionally an organic solvent, and the subject has been or is being treated with an antiplatelet agent; method. [The present invention 1002] 1. A method of restoring normal hemostasis in a subject, comprising administering to a subject in need thereof an effective amount of a composition; the composition has been prepared by a process comprising incubating platelets with an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form the composition, and the subject has been or is being treated with an antiplatelet agent; method. [The present invention 1003] 1. A method of treating a coagulation disorder in a subject, comprising administering to a subject in need thereof an effective amount of a composition; the composition comprises platelets or platelet derivatives and an incubation agent comprising one or more salts, buffers, optionally a cryoprotectant, and optionally an organic solvent, and the subject has been or is being treated with an antiplatelet agent; method. [The present invention 1004] 1. A method of treating a coagulation disorder in a subject, comprising administering to a subject in need thereof an effective amount of a composition; the composition has been prepared by a process comprising incubating platelets with an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form the composition, and the subject has been or is being treated with an antiplatelet agent; method. [The present invention 1005] 1. A method of preparing a subject for surgery, comprising administering to a subject in need thereof an effective amount of a composition; the composition comprises platelets or platelet derivatives and an incubation agent comprising one or more salts, buffers, optionally a cryoprotectant, and optionally an organic solvent, and the subject has been or is being treated with an antiplatelet agent; method. [The present invention 1006] 1. A method of preparing a subject for surgery, comprising administering to a subject in need thereof an effective amount of a composition; the composition has been prepared by a process comprising incubating platelets with an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form the composition, and the subject has been or is being treated with an antiplatelet agent; method. [The present invention 1007] The method of any one of claims 1005 to 1006, wherein the surgery is an emergency surgery. [The present invention 1008] The method of any one of claims 1005 to 1006, wherein said surgery is a planned surgery. [The present invention 1009] The method of any of claims 1001 to 1008, wherein said subject is being treated with an antiplatelet agent. [The present invention 1010] 1009. The method of claim 1009, wherein treatment with the antiplatelet agent is stopped. [The present invention 1011] 1009. The method of claim 1009, wherein treatment with the antiplatelet agent is continued. [The present invention 1012] 1. A method for improving the efficacy of an antiplatelet agent in a subject, comprising administering to a subject in need thereof an effective amount of a composition; the composition comprises platelets or platelet derivatives and an incubation agent comprising one or more salts, buffers, optionally a cryoprotectant, and optionally an organic solvent; method. [The present invention 1013] 1. A method for improving the efficacy of an antiplatelet agent in a subject, comprising administering to a subject in need thereof an effective amount of a composition; the composition is prepared by a process comprising incubating platelets with an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form the composition; method. [The present invention 1014] 1014. The method of claim 1012 or 1013, wherein said effect of said antiplatelet agent is the result of an overdose of said antiplatelet agent. [The present invention 1015] The method of any one of claims 1001 to 1014, wherein the composition further comprises an antifibrinolytic agent. [The present invention 1016] 1015. The method of claim 1015, wherein said antifibrinolytic agent is selected from the group consisting of epsilon-aminocaproic acid (EACA), tranexamic acid, aprotinin, aminomethylbenzoic acid, fibrinogen, and combinations thereof. [The present invention 1017] The method of claim 1015 or claim 1016, wherein said platelets or platelet derivatives are loaded with said antifibrinolytic agent. [The present invention 1018] Any of the methods of claims 1001 to 1017, wherein the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, supplements, and combinations thereof. [The present invention 1019] Any of the methods of claims 1001 to 1017, 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. [The present invention 1020] Any of the methods of claims 1001 to 1017, 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. [The present invention 1021] The method of any one of claims 1001 to 1020, wherein administering comprises topical administration. [The present invention 1022] The method of any of claims 1001 to 1020, wherein the administration comprises parenteral administration. [The present invention 1023] The method of any one of claims 1001 to 1020, wherein the administering comprises intravenous administration. [The present invention 1024] The method of any of claims 1001 to 1020, wherein the administration comprises intramuscular administration. [The present invention 1025] The method of any of claims 1001 to 1020, wherein the administration comprises intrathecal administration. [The present invention 1026] The method of any one of claims 1001 to 1020, wherein the administration comprises subcutaneous administration. [The present invention 1027] The method of any of claims 1001 to 1020, wherein the administration comprises intraperitoneal administration. [The present invention 1028] The method of any one of claims 1001 to 1027, wherein the composition is dried prior to the administering step. [The present invention 1029] The method of claim 1028, wherein the composition is rehydrated after the drying step. [The present invention 1030] The method of any one of claims 1001 to 1028, wherein said composition is lyophilized prior to said administering step. [The present invention 1031] The method of claim 1030, wherein said composition is rehydrated after said freeze-drying step. [The present invention 1032] The method of any of claims 1001 to 1031, wherein said incubation agent comprises one or more salts selected from phosphate salts, sodium salts, potassium salts, calcium salts, magnesium salts, and combinations of two or more thereof. [The present invention 1033] The method of any of claims 1001 to 1032, wherein said incubation agent comprises a carrier protein. [The present invention 1034] The buffer solution may contain HEPES, sodium bicarbonate (NaHCO 3 ), or a combination thereof. [This invention 1035] The method of any one of claims 1001 to 1034, wherein the composition comprises one or more sugars. [The present invention 1036] The method of claim 1035, wherein the one or more sugars include trehalose. [This invention 1037] The method of claim 1035 or claim 1036, wherein the one or more sugars include polysucrose. [The present invention 1038] 1038. The method of any one of claims 1035 to 1037, wherein the one or more sugars include dextrose. [This invention 1039] The method according to any one of claims 1001 to 1038, wherein the composition comprises an organic solvent. [The present invention 1040] The method of any one of claims 1001 to 1039, wherein said platelets or platelet derivatives comprise thrombosomes. [Brief explanation of the drawings]

[0018] [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 datasets from Figure 2. Replicate datasets from Figure 2 are presented as averages. [Figure 5]1 shows the effect of thrombosomes ("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 datasets from Figure 5. Replicate datasets 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 thrombosomal ("Tsome") replacement (approximately 200,000 / uL) 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 thrombosomes (various lots) are clogged 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] FIG. 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 thrombosomes (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 restoration of thrombus formation promoted by thrombosomes 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] Figure 1 shows the restoration of thrombus formation promoted by thrombosomes 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 thrombosomal recruitment 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 thrombosomal recruitment to aspirin (ASA)-inhibited whole blood (500 micromolar) on its interaction with plastic-immobilized porcine collage under high shear, as measured by occlusion (pressure) over time. [Figure 22] 1 shows the effect of thrombosomal recruitment to aspirin (ASA)-inhibited whole blood (100 micromolar) on the interaction with plastic-immobilized porcine collage under high shear as measured by AUC. [Figure 23]Figure 1 shows the effect of thrombosomal recruitment to aspirin (ASA)-inhibited whole blood (100 micromolar) on its interaction with plastic-immobilized porcine collage under high shear, as measured by occlusion (pressure) over time. [Figure 24] 1 shows the effect of thrombosomal recruitment to normal and aspirin-inhibited plasma on peak thrombin. [Figure 25] Figure 25A shows the effect of cangrelor alone or cangrelor plus thrombosomes on platelet occlusion using T-TAS® technology. Figure 25B is a bar graph of occlusion time for the data set from Figure 25A. [Figure 26] Figure 26A shows the effect of tirofiban alone or tirofiban with random donor platelets (RDPs) or thrombosomes on platelet occlusion using T-TAS® technology. Figure 26B is a bar graph of occlusion time for the data set from Figure 26A. [Figure 27] Figure 27A shows the effect of eptifibatide alone or eptifibatide with RDP or thrombosomes on platelet occlusion using T-TAS® technology. Figure 27B is a bar graph of occlusion time for the data set from Figure 27A. [Figure 28] Figure 28A shows the effect of AP2 alone or AP2 with RDP or thrombosomes on platelet occlusion using T-TAS® technology. Figure 28B is a bar graph of occlusion time for the data set from Figure 28A. [Figure 29] Figure 29A shows the effect of thromboses on PRP collected from subjects on aspirin therapy using T-TAS® technology. Figure 29B is a bar graph of occlusion time for the data set from Figure 29A. [Figure 30] Figure 30A shows the effect of thrombosomes on PRP obtained from subjects receiving aspirin therapy on thrombin generation. Figure 30B is a bar graph of thrombin generation parameters for PRP obtained from subjects receiving aspirin therapy with or without added thrombosomes. [Figure 31] Figure 31A shows aggregation measurements of PRP taken from subjects on ibuprofen therapy with the addition of buffer, arachidonic acid, or collagen, and Figure 31B shows the effect of ADP on PRP taken from subjects on ibuprofen therapy with or without thrombosomes. [Figure 32] 1 shows the effect of thrombosomal dosing on bleeding time in mice treated with suprapharmacologic doses of clopidogrel. DETAILED DESCRIPTION OF THE INVENTION

[0019] Detailed Description Before describing the embodiments of the present invention in detail, it should be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled 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 in connection with which the publications are cited. The present disclosure controls for any conflicts with any incorporated publications.

[0020] As used herein and in the appended claims, 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 their equivalents known to those skilled in the art. Furthermore, the use of terms that can be described using equivalent terms includes the use of those equivalent terms. Thus, for example, the use of the term "subject" should be understood to include "patient," "person," "animal," "human," and other terms used in the art to refer to subjects 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.

[0021] 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 these 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."

[0022] As used herein and in the appended claims, the term "platelets" may include whole platelets, fragmented platelets, platelet derivatives, or thrombosomes. "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 be those of a mammal, such as a human, or those of a non-human mammal, etc.

[0023] As used herein, "thrombosomes" (sometimes referred to herein, particularly in the Examples and Figures, as "Tsomes" or "Ts") are platelet derivatives that have been treated with an incubation agent (e.g., any of the incubation agents described herein) and cryopreserved (e.g., lyophilized). In some cases, thrombosomes may be prepared from pooled platelets. Thrombosomes may 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 a thrombosome is THROMBOSOMES®, which is in clinical trials for the treatment of acute bleeding in thrombocytopenic patients. 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 anticoagulants. Other mechanisms of anticoagulants are known, including dabigatran, argatroban, hirudin, rivaroxaban, apixaban, edoxaban, fondaparinux, warfarin, heparin, and low molecular weight heparins.

[0024] As used herein, an "antiplatelet agent" refers to an antithrombotic agent and does not include anticoagulants. 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 refers to an agent that binds to a P2Y receptor (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 alpha2betal collagen receptor antagonists. Non-limiting examples of PAR-1 antagonists include vorapaxar and atopaxar.As used herein, aspirin is considered to be an antiplatelet agent, but not an anticoagulant.Further non-limiting examples of antiplatelet agents include cilostazol, prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, and sarpogrelate.

[0025] In some embodiments, the antiplatelet agent may be selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, 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, 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.

[0026] 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.

[0027] Eptifibatide is a peptide therapeutic agent that blocks the fibrin-binding role of the GPIIb-IIIa receptor on platelets. The drug is typically administered via IV 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.

[0028] 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 following the same oral dose.

[0029] Antiplatelet class drugs are widely used to prevent unwanted clotting episodes that can lead to heart failure, stroke, and the like. In many cases, antiplatelet drugs may need to be reversed or stopped. With advance notice, such as in the context of a preplanned surgery, the dose of the antiplatelet drug may be stopped prior to surgery to prevent unwanted bleeding during surgery. When rapid reversal of an antiplatelet agent is required, reversal agents 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, the compositions described herein (e.g., comprising thrombosomes) are not. In some embodiments, the compositions described herein (e.g., comprising thrombosomes) are active reversal agents. In some embodiments, the hemostatic activity of the compositions described herein (e.g., comprising thrombosomes) is resistant to the antiplatelet drug.

[0030] Some exemplary antiplatelet agents and potential methods of reversal are described below.

[0031] Acetylsalicylic acid (ASA; aspirin) - Aspirin functions as a COX-1 blocker on platelets, which irreversibly inhibits platelet-derived thromboxane formation, rendering them inactive. Clinically, aspirin can be counteracted by platelet transfusion in emergency situations or by halting treatment for upcoming surgery.

[0032] 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 halting treatment when upcoming surgery is planned.

[0033] 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 preferred when reversal is needed after a procedure.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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, thrombosomes are not reactive to these drugs and maintain their ability to support blood clotting. This makes thrombosome-mediated therapy entirely unique and introduces new uses for the product.

[0038] Platelet-derived products are not currently used as anticoagulant / antiplatelet therapy, and there are currently no approved antiplatelet reversal agents. Therefore, emergency treatment (pre-surgery, trauma, etc.) typically involves comprehensive preventative measures to avoid or mitigate bleeding. Non-limiting examples include infusions of plasma, red blood cells, and antifibrinolytic agents. Platelet derivatives (e.g., cryopreserved platelets (e.g., thrombosomes)) may be an effective alternative or complement to these common treatments.

[0039] Without being bound to any particular theory, it is believed that thrombosomes may act, at least in part, by providing a procoagulant negatively charged surface to enhance thrombin generation beyond that inhibited by anticoagulants. Also, without being bound to any particular theory, it is believed that thrombosomes may act, at least in part, by binding to and coaggregating with circulating platelets.

[0040] Described herein are products and methods for controlling bleeding and improving healing. The products and methods described herein can also be used to counteract the activity of antiplatelet agents (e.g., aspirin (also called 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 described herein are directed to embodiments that may aid in wound closure and healing.

[0041] In certain embodiments, compositions containing platelets, such as freeze-dried platelets, or platelet derivatives can be delivered to wounds on or within a patient. In various embodiments, compositions containing platelets, such as freeze-dried platelets, or platelet derivatives can be applied in a selected form, including, but not limited to, adhesive bandages, compression bandages, liquid solutions, aerosols, matrix compositions, and coated sutures or other medical closures. In embodiments, compositions containing platelets, such as freeze-dried platelets, or platelet derivatives can be administered to all or only a portion of the affected area on the patient's surface. In other embodiments, compositions containing platelets, such as freeze-dried platelets, or platelet derivatives can be administered systemically, e.g., via the bloodstream. In embodiments, application of platelet derivatives can provide hemostatic effects for 2 or 3 days, preferably 5 to 10 days, or most preferably up to 14 days.

[0042] Some embodiments provide 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, such as lyophilized platelets, and an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0043] 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 agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0044] In some embodiments of any of the methods described herein, the coagulation disorder is a result of an antiplatelet agent.

[0045] Some embodiments 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 of such treatment an effective amount of a composition comprising platelets or platelet derivatives, such as lyophilized platelets, and an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0046] Some embodiments 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 of such treatment an effective amount of a composition prepared by a process comprising incubating platelets with an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form a composition.

[0047] 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 comprising platelets or platelet derivatives, such as freeze-dried platelets, and an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0048] 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 having been prepared by a process comprising incubating platelets with an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0049] 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 comprising platelets or platelet derivatives, such as lyophilized platelets, and an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0050] 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 having been prepared by a process comprising incubating platelets with an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0051] 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 unwise 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 exposed to the risk of thrombotic events (for example, deep vein thrombosis, pulmonary embolism or stroke)).

[0052] Accordingly, 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 comprising platelets or platelet derivatives, such as freeze-dried platelets, and an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0053] 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 agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0054] Some embodiments provide a method of preparing a subject for surgery who has been or is being treated with an antiplatelet agent, the method comprising administering to the subject in need thereof an effective amount of a composition comprising platelets or platelet derivatives, such as lyophilized platelets, and an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0055] Some embodiments provide a method of preparing a subject for surgery who has been or is being treated with an antiplatelet agent, the method comprising 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 agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition.

[0056] In some embodiments, the surgery may be an emergency surgery (eg, in the case of trauma) or a scheduled surgery.

[0057] In some embodiments, anticoagulant therapy may be stopped (e.g., in preparation for surgery). In some embodiments, anticoagulant therapy may be continued.

[0058] Some embodiments provide a method of improving the effect 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 platelet derivatives, such as lyophilized platelets, and an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0059] Some embodiments provide a method of improving the effect 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 agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form a composition.

[0060] In some cases, it may be necessary to improve the effectiveness of an antiplatelet agent due to inaccurate dosing 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 cases, it may be necessary to improve the effectiveness of an antiplatelet agent due to a possible 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 incorrect dosing of two or more drugs, at least one of which is an antiplatelet agent.

[0061] 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, platelets or platelet derivatives may be loaded with an active agent, such as an antifibrinolytic agent.

[0062] 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 platelet derivatives, such as the freeze-dried platelets described herein, to determine the need for administration of a composition comprising platelets or platelet derivatives described herein, for example. As another example, one or more coagulation parameters of blood can be assessed after administration of a composition comprising platelets or platelet derivatives described herein, for example, to determine the effectiveness of the administered composition, to determine whether additional administration of the composition is warranted, or to determine whether it is safe to perform a surgical procedure.

[0063] Thus, any of the methods described herein can include assessing one or more clotting parameters of the blood before administration of a composition comprising platelets or platelet derivatives described herein, assessing one or more clotting parameters of the blood after administration of a composition comprising platelets or platelet derivatives described herein, or both.

[0064] Any suitable method can be used to evaluate blood coagulation parameters.Non-limiting examples of methods include prothrombin time assay, international normalized ratio (INR), thrombin generation (TGA; for example, can be used to generate parameters such as peak thrombin, endogenous thrombin potential (ETP) and lag time), thromboelastography (TEG), activated clotting time (ACT) and partial thromboplastin time (PTT or aPTT).

[0065] thrombin generation The thrombin generation assay measured thrombin generation after procoagulant-mediated sample activation, resulting 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 until thrombin generation begins, and ETP is the total thrombin potentially generated.

[0066] In some embodiments, a 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.

[0067] 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.

[0068] In hypocoagulable blood, the R time increases and the MA decreases. The R time typically provides a wider range of response than the MA.

[0069] In the Total Thrombus Formation Analysis System (T-TAS®, FUJIMORI KOGYO CO., LTD.), samples are passed through a collagen-coated microchannel using mineral oil. Pressure changes are used to evaluate 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 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.

[0070] 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 about 200-300 seconds. ACT It has.

[0071] 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 comprising platelets or platelet derivatives, such as lyophilized platelets, and an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0072] 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, the composition being prepared by a process comprising incubating platelets with an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0073] 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 comprising platelets or platelet derivatives, such as lyophilized platelets, and an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0074] 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 agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0075] 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 may be measured by any suitable method.

[0076] As used herein, an "effective amount" is the amount of a composition comprising an amount of platelets, such as lyophilized platelets, or platelet derivatives (e.g., thrombosomes) effective to treat a subject. Such an amount of platelets or platelet derivatives (e.g., thrombosomes) comprises any suitable dose of a composition comprising platelets or platelet derivatives described herein that can be administered to a subject. For example, in some embodiments, a dose of a composition comprising platelets or platelet derivatives (e.g., thrombosomes) comprises about 1.0 x 10 7 Particles ~ approx. 1.0 x 10 10 particles, e.g., about 1.6 x 10 7 particles (e.g., thrombosomes) / kg to approximately 1.0 x 10 10 particles / kg (e.g., about 1.6 × 10 7 ~Approx. 5.1×10 9 Particles / kg, approximately 1.6×10 7 ~Approx. 3.0×10 9 Particles / kg, approximately 1.6×10 7 ~Approx. 1.0×10 9 Particles / kg, approximately 1.6×10 7 Particles ~ approx. 5.0 x 10 8 Particles / kg, approximately 1.6×10 7 ~Approx. 1.0×10 8 Particles / kg, approximately 1.6×10 7 ~Approx. 5.0×10 7 Particles / kg, approximately 5.0×10 7 ~Approx. 1.0×10 8 Particles / kg, approximately 1.0×10 8 ~Approx. 5.0×10 8 Particles / kg, approximately 5.0×10 8 ~Approx. 1.0×10 9 Particles / kg, approximately 1.0×10 9 ~Approx. 5.0×10 9 particles / kg, or approximately 5.0 x 10 9 ~Approx. 1.0×10 10 particles / kg).

[0077] In some embodiments of the methods herein, the composition is administered topically. In some embodiments, topical administration may include administration via a solution, cream, gel, suspension, putty, particles, or powder. In some embodiments, topical administration may include administration via a bandage (e.g., an adhesive bandage or compression bandage) or medical closure (e.g., suture, staple), for example, into which the platelet derivative may be embedded, as described in PCT Publication No. WO2017 / 040238 (e.g., paragraphs

[0013] -

[0069] ), which corresponds to U.S. Patent Application No. 15 / 776,255 (incorporated herein by reference in its entirety).

[0078] In some embodiments of the methods herein, the composition is administered parenterally.

[0079] In some embodiments of the methods herein, the composition is administered intravenously.

[0080] In some embodiments of the methods herein, the composition is administered intramuscularly.

[0081] In some embodiments of the methods herein, the composition is administered intrathecally.

[0082] In some embodiments of the methods herein, the composition is administered subcutaneously.

[0083] In some embodiments of the methods herein, the composition is administered intraperitoneally.

[0084] In some embodiments of the methods herein, the composition is dried before the administering step. In some embodiments of the methods, the composition is lyophilized before the administering step. In some embodiments of the methods, the composition is rehydrated after the drying or lyophilization step.

[0085] 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 biloba, 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 biloba.

[0086] In some embodiments, the antiplatelet agent is aspirin.

[0087] In some embodiments, the antiplatelet agent is cangrelor (eg, KENGREAL®).

[0088] In some embodiments, the antiplatelet agent is ticagrelor (e.g., BRILINTA®).

[0089] In some embodiments, the antiplatelet agent is clopidogrel (eg, PLAVIX®).

[0090] In some embodiments, the antiplatelet agent is prasugrel (eg, EFFIENT®).

[0091] In some embodiments, the antiplatelet agent is eptifibatide (eg, INTEGRILIN®).

[0092] In some embodiments, the antiplatelet agent is tirofiban (eg, AGGRASTAT®).

[0093] In some embodiments, the antiplatelet agent is abciximab (eg, REOPRO®).

[0094] In some embodiments, the antiplatelet agent is terutroban.

[0095] In some embodiments, the antiplatelet agent is picotamide.

[0096] In some embodiments, the antiplatelet agent is elinogrel.

[0097] In some embodiments, the antiplatelet agent is ticlopidine.

[0098] In some embodiments, the antiplatelet agent is ibuprofen.

[0099] In some embodiments, the antiplatelet agent is vorapaxar.

[0100] In some embodiments, the antiplatelet agent is atopaxar.

[0101] In some embodiments, the antiplatelet agent is cilostazol.

[0102] In some embodiments, the antiplatelet agent is prostaglandin E1.

[0103] In some embodiments, the antiplatelet agent is epoprostenol.

[0104] In some embodiments, the antiplatelet agent is dipyridamole.

[0105] In some embodiments, the antiplatelet agent is treprostinil sodium.

[0106] In some embodiments, the antiplatelet agent is sarpogrelate.

[0107] In some embodiments, the antiplatelet agent is a supplement.

[0108] In some embodiments, the antiplatelet agent is an herbal supplement.

[0109] In some embodiments, rehydrating a composition comprising platelets or platelet derivatives comprises adding an aqueous liquid to the platelets. 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.

[0110] In some embodiments, the rehydrated platelets or platelet derivatives (e.g., thrombosomes) have coagulation factor levels representing all individual factors associated with clotting (e.g., Factor VII, Factor VIII, and Factor IX) at or above 40 International Units (IU).

[0111] In some embodiments, platelets, such as lyophilized platelets, or platelet derivatives (e.g., thrombosomes) have 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, rehydrated platelets or platelet derivatives (e.g., thrombosomes) have 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.

[0112] In some embodiments, the platelets or platelet derivatives (e.g., thrombosomes), such as freeze-dried platelets, 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 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).

[0113] 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 freeze-dried platelets or platelet derivatives (e.g., thrombosomes) 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 platelet derivatives (e.g., thrombosomes), such as lyophilized platelets, 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 platelet derivatives (e.g., thrombosomes) 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).

[0114] In some embodiments, the platelets are isolated, for example, in a liquid medium, prior to treating the subject.

[0115] 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.

[0116] 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.

[0117] 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).

[0118] 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.

[0119] 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.

[0120] In some embodiments, the platelets are isolated prior to incubation with the incubation agent. In some embodiments, the method further comprises 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.

[0121] The incubation agent may include any suitable components. In some embodiments, the incubation agent may include a liquid medium. In some embodiments, the incubation agent may include one or more salts selected from phosphate salts, sodium salts, potassium salts, calcium salts, magnesium salts, and any other salts that can be found in blood or blood products or that are known to be useful for drying platelets, or any combination of two or more thereof.

[0122] In some embodiments, the incubation agent 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 agent comprises about 0.5 mM to about 100 mM of one or more salts. In some embodiments, the incubation agent 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 agent 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 agent 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.

[0123] 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.

[0124] In some embodiments, the incubation agent 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).

[0125] 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 / carbonic acid, 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 )-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 agent comprises one or more buffers, such as N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), or sodium bicarbonate (NaHCO). In some embodiments, the incubation agent comprises from about 5 to about 100 mM of one or more buffers.In some embodiments, the incubation agent 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 agent comprises about 10 mM, about 20 mM, about 25 mM, or about 30 mM of one or more buffers.

[0126] In some embodiments, the incubation agent 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 agent comprises starch. In some embodiments, the incubation agent comprises polysucrose, which is a polymer of sucrose and epichlorohydrin. In some embodiments, the incubation agent comprises about 10 mM to about 1,000 mM of one or more sugars. In some embodiments, the incubation agent comprises about 50 to about 500 mM of one or more sugars. In 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.

[0127] In some embodiments, compositions comprising platelets or platelet derivatives (e.g., thrombosomes) may include one or more of water or saline. In some embodiments, compositions comprising platelets or platelet derivatives, such as lyophilized platelets, may include DMSO.

[0128] In some embodiments, the incubation agent includes an organic solvent such as alcohol (e.g., ethanol). In such incubation agents, 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).

[0129] 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 thrombosomes (e.g., lyophilized platelet derivatives).

[0130] In some embodiments, the incubation agent is incubated with the platelets in the presence of an aqueous medium, hi some embodiments, the incubation agent is incubated in the presence of a medium that includes DMSO.

[0131] In some embodiments, one or more other components may be incubated with the platelets. Exemplary components may include prostaglandin E1 or prostacyclin, and / or EDTA / EGTA to prevent platelet aggregation and activation during the incubation process.

[0132] Non-limiting examples of incubation agent compositions that can be used are shown in Tables 1-5.

[0133] [Table 1]

[0134] [Table 2]

[0135] [Table 3] Table 3. Buffer B may be used, for example, when incubating platelets for flow cytometry. Such incubations may be performed in the dark at room temperature. Albumin is an optional component of Buffer B.

[0136] [Table 4]

[0137] Table 4 is another exemplary incubation agent. The pH can be adjusted to 7.4 with NaOH. Albumin is an optional component of Buffer B.

[0138] [Table 5]

[0139] Table 5 lists other exemplary incubation agents.

[0140] In some embodiments, platelets (e.g., apheresis platelets, platelets isolated from whole blood, pooled platelets, or a combination thereof) are incubated with an incubation agent at different temperatures between 15 and 45°C, or at about 37°C, for different durations.

[0141] 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 agent comprising a liquid medium 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.

[0142] Platelets (e.g., apheresis platelets, platelets isolated from whole blood, pooled platelets, or a combination thereof) can be incubated with the incubation agent for different durations, such as, for example, 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 agent 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 agent 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 thrombosomes 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.

[0143] In some embodiments, platelets (e.g., apheresis platelets, platelets isolated from whole blood, pooled platelets, or a combination thereof) are incubated with an incubation agent 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 as long as 24 to 48 hours. In some embodiments, the platelets are incubated with an incubation agent for different durations as disclosed herein and at temperatures of 15 to 45°C, or about 37°C.

[0144] 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.

[0145] 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, for example, PCT Publication Nos. WO2020 / 113090(A1), WO2020 / 113101(A1), WO2020 / 113035(A1), and WO2020 / 112963(A1). 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 agent. In some embodiments, loading can be carried out in a step separate from the incubation step. In some embodiments, loading can be carried out prior to the incubation step. In some such embodiments, the active agent may be provided to the platelets as a solution or suspension in any of the incubation agents described herein, which may be the same as or different from the incubation agent used for 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 agent (e.g., as a solid or in 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 active agent may be provided to the platelets as a solution or suspension in any of the incubation agents described herein, which may be the same as or different from the incubation agent used for the incubation step.

[0146] 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). The platelets may be applied (as part of an incubation agent or a separate solution or suspension) at a concentration of 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).

[0147] 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.

[0148] In some embodiments, platelets are cryopreserved, frozen (eg, to generate thrombosomes), or lyophilized prior to use in therapy or functional assays.

[0149] Any known technique for drying platelets can be used in accordance with the present disclosure, as 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%, for example, 1%, 0.5%, or 0.1%. Non-limiting examples of suitable techniques are freeze-drying (lyophilization) and spray-drying. Suitable freeze-drying methods are presented in Table A. Additional exemplary freeze-drying methods can be found in U.S. Patent Nos. 7,811,558, 8,486,617, and 8,097,403. An exemplary spray-drying method involves combining nitrogen as the drying gas with an incubation agent 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 ranging from 10 to 35 minutes. The final step in spray-drying is preferentially collecting 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.

[0150] Table A. Exemplary freeze-drying protocol TIFF0007791581000006.tif81128

[0151] In some embodiments, the step of drying platelets obtained as disclosed herein, e.g., freeze-drying platelets obtained as disclosed 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.

[0152] 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. 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%.

[0153] 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 the composition in any suitable amount. For example, the sugar can be present in an amount of 1 mM to 1M. 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 listed above, and one of skill in the art will readily recognize the various concentrations without the need to specifically list 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 listed above.

[0154] In the process provided herein for making the compositions provided herein, the addition of a 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, optionally cryoprotectants, or other components.In some embodiments, the lyophilizing agent is added to an incubation agent and thoroughly mixed to form a dry solution, which is then dispensed into a drying container (e.g., glass or plastic serum vials, lyophilization bags), and subjected to conditions that allow the solution to dry and form a dry composition.

[0155] 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.

[0156] 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.

[0157] 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 some 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.

[0158] In some embodiments, the container of the process herein is a closed or sealed gas-permeable container. In some embodiments, the container is a closed or sealed container, and a portion of it 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 "SA / V ratio") can be adjusted to improve the pH maintenance of the composition 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 cm 2 / 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 9.5cm 2 / mL, maximum approximately 9.4cm 2 / mL, maximum approximately 9.3cm 2 / mL, maximum approximately 9.2cm 2 / mL, maximum approximately 9.1cm 2 / mL, maximum approximately 9.0cm 2 / mL, maximum approximately 8.9cm 2 / mL, maximum approximately 8.8cm 2 / mL, maximum approximately 8.7cm 2 / mL, maximum approximately 8.6cm 2 / mL, maximum approximately 8.5cm 2 / mL, maximum approximately 8.4cm 2 / mL, maximum approximately 8.3cm 2 / mL, maximum approximately 8.2cm2 / mL, maximum approximately 8.1cm 2 / mL, maximum approximately 8.0cm 2 / mL, maximum approximately 7.9cm 2 / mL, maximum approximately 7.8cm 2 / mL, maximum approximately 7.7cm 2 / mL, maximum approximately 7.6cm 2 / mL, maximum approximately 7.5cm 2 / mL, maximum approximately 7.4cm 2 / mL, maximum approximately 7.3cm 2 / mL, maximum approximately 7.2cm 2 / mL, maximum approximately 7.1cm 2 / mL, maximum approximately 6.9cm 2 / mL, maximum approximately 6.8cm 2 / mL, maximum approximately 6.7cm 2 / mL, maximum approximately 6.6cm 2 / mL, maximum approximately 6.5cm 2 / mL, maximum approximately 6.4cm 2 / mL, maximum approximately 6.3cm 2 / mL, maximum approximately 6.2cm 2 / mL, maximum approximately 6.1cm 2 / mL, maximum approximately 6.0cm 2 / mL, maximum approximately 5.9cm 2 / mL, maximum approximately 5.8cm 2 / mL, maximum approximately 5.7cm 2 / mL, maximum approximately 5.6cm 2 / mL, maximum approximately 5.5cm 2 / mL, maximum approximately 5.4cm 2 / 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 approximately 4.2cm 2 / mL, maximum approximately 4.1cm2 / 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. 3.3cm 2 / mL ~ approx. 8.7cm 2 / mL, approx. 3.4cm 2 / mL ~ approx. 8.6cm 2 / mL, approx. 3.5cm 2 / mL ~ approx. 8.5cm 2 / mL, approx. 3.6cm 2 / mL ~ approx. 8.4cm 2 / mL, approx. 3.7cm 2 / mL ~ approx. 8.3cm 2 / mL, approx. 3.8cm 2 / mL ~ approx. 8.2cm 2 / mL, approx. 3.9cm 2 / mL ~ approx. 8.1cm 2 / mL, approx. 4.0cm 2 / mL to approximately 8.0 cm 2 / mL, approximately 4.1 cm 2 / mL to approximately 7.9 cm 2 / mL, approximately 4.2 cm 2 / mL to approximately 7.8 cm 2 / mL, approximately 4.3 cm 2 / mL to approximately 7.7 cm 2 / mL, approximately 4.4 cm 2 / mL to approximately 7.6 cm 2 / mL, approximately 4.5 cm 2 / mL to approximately 7.5 cm 2 / mL, approximately 4.6 cm 2 / mL to approximately 7.4 cm 2 / mL, approximately 4.7 cm 2 / mL to approximately 7.3 cm 2 / mL, approximately 4.8 cm 2 / mL to approximately 7.2 cm 2 / mL, approximately 4.9 cm 2 / mL to approximately 7.1 cm 2 / mL, approximately 5.0 cm 2 / mL to approximately 6.9 cm 2 / mL, approximately 5.1 cm 2 / mL to approximately 6.8 cm 2 / mL, approximately 5.2 cm 2 / mL to approximately 6.7 cm 2 / mL, approximately 5.3 cm 2 / mL to approximately 6.6 cm 2 / mL, approximately 5.4 cm 2 / mL to approximately 6.5 cm 2 / mL, approximately 5.5 cm 2 / mL to approximately 6.4 cm 2 / mL, approximately 5.6 cm 2 / mL to approximately 6.3 cm 2 / mL, approximately 5.7 cm 2 / mL to approximately 6.2 cm 2 / mL, or approximately 5.8 cm 2 / mL to approximately 6.1 cm 2 can be in the range of / mL.

[0159] 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.

[0160] In some embodiments, dried platelets or platelet derivatives (e.g., thrombosomes) may be subjected to heat treatment. Heating may be performed at temperatures above about 25°C (e.g., above about 40°C, 50°C, 60°C, 70°C, 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 may be selected along with the length of time the heating is performed. While any suitable time may be used, typically, freeze-dried platelets are heated for at least 1 hour, but not more than 36 hours. 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, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours. Non-limiting exemplary combinations include heating dried platelets or platelet derivatives (e.g., thrombosomes) at a temperature above 30°C for at least 30 minutes, heating dried platelets or platelet derivatives (e.g., thrombosomes) at a temperature above 50°C for at least 10 hours, heating dried platelets or platelet derivatives (e.g., thrombosomes) at a temperature above 75°C for at least 18 hours, and heating dried platelets or platelet derivatives (e.g., thrombosomes) 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 produce advantageous results compared to lyoprotectants 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., thrombosomes)).

[0161] In some embodiments, platelets or platelet derivatives (e.g., thrombosomes) 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.

[0162] In some embodiments, the method further comprises cryopreserving the platelets or platelet derivatives (e.g., in an incubation agent, e.g., an incubation agent described herein) prior to administering the platelets or platelet derivatives.

[0163] In some embodiments, the method further comprises drying the composition comprising platelets or platelet derivatives (e.g., with an incubation agent, e.g., an incubation agent described herein) prior to administering the platelets or platelet derivatives (e.g., thrombosomes). In some embodiments, the method may further comprise heating the composition after the drying step. In some embodiments, the method may further comprise rehydrating the composition after the lyophilization or heating step.

[0164] In some embodiments, the method further comprises lyophilizing the composition comprising the platelets or platelet derivatives (e.g., with an incubation agent, e.g., an incubation agent described herein) prior to administering the platelets or platelet derivatives (e.g., thrombosomes). In some embodiments, the method may further comprise heating the composition after the lyophilization step. In some embodiments, the method may further comprise rehydrating the composition after the lyophilization or heating step.

[0165] In some embodiments, the method further comprises cold storing the platelets, platelet derivatives, or thrombosomes (e.g., in an incubation agent, e.g., an incubation agent described herein) prior to administering the platelets, platelet derivatives, or thrombosomes.

[0166] Storage conditions include, for example, standard room temperature storage (e.g., storage at a temperature ranging from about 20 to about 30°C) or cold 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 agent, e.g., an incubation agent described herein), lyophilizing, thawing, rehydrating, and combinations thereof, a composition comprising platelets or platelet derivatives (e.g., thrombosomes) prior to administering the platelets or platelet derivatives (e.g., thrombosomes). For example, in some embodiments, the method further includes drying (e.g., lyophilizing) (e.g., with an incubation agent, e.g., an incubation agent described herein) a composition comprising platelets or platelet derivatives (e.g., thrombosomes) (e.g., to form thrombosomes) prior to administering the platelets or platelet derivatives (e.g., thrombosomes). In some embodiments, the method can further include rehydrating the composition resulting from the drying step.

[0167] In some embodiments, provided herein is a composition comprising platelets or platelet derivatives (e.g., thrombosomes), polysucrose, and trehalose, the composition having been 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 agent 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 lyophilized composition comprising platelets or platelet derivatives (e.g., thrombosomes), polysucrose, and trehalose.

[0168] In some embodiments, provided herein is a method of making a freeze-dried platelet composition comprising platelets or platelet derivatives (e.g., thrombosomes), 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 agent 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 freeze-drying the second mixture to form a freeze-dried composition comprising platelets or platelet derivatives (e.g., thrombosomes), polysucrose, and trehalose.

[0169] 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.

[0170] 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; freeze-drying the second composition to form freeze-dried platelets; and heating the freeze-dried platelets at 80°C for 24 hours.

[0171] Certain embodiments disclosed herein may be further limited in the claims using the language "consisting of" or "consisting essentially of."

[0172] Illustrative Embodiments 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 a platelet derivative and an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0173] 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 agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form a composition.

[0174] 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 a platelet derivative and an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0175] 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 agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form a composition.

[0176] 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 agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0177] 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 agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form a composition.

[0178] Embodiment 7 is the method of embodiment 5 or 6, wherein the surgery is emergency surgery.

[0179] Embodiment 8 is the method of embodiment 5 or 6, wherein the surgery is a planned surgery.

[0180] 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.

[0181] Embodiment 10 is the method of embodiment 9, wherein treatment with the antiplatelet agent is stopped.

[0182] Embodiment 11 is the method of embodiment 9, wherein treatment with the antiplatelet agent is continued.

[0183] Embodiment 12 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 comprising platelets or a platelet derivative and an incubation agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

[0184] 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 agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent to form a composition.

[0185] Embodiment 14 is the method of embodiment 12 or embodiment 13, wherein the antiplatelet agent effect is the result of an overdose of the antiplatelet agent.

[0186] Embodiment 15 is the method of any one of embodiments 1 to 14, wherein the composition further comprises an antifibrinolytic agent.

[0187] 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.

[0188] Embodiment 17 is the method of embodiment 15 or embodiment 16, wherein the platelets or platelet derivatives are loaded with an antifibrinolytic agent.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] Embodiment 21 is the method of any one of embodiments 1 to 20, wherein administering comprises topical administration.

[0193] Embodiment 22 is the method of any one of embodiments 1 to 20, wherein administering comprises parenteral administration.

[0194] Embodiment 23 is the method of any one of embodiments 1 to 20, wherein administering comprises intravenous administration.

[0195] Embodiment 24 is the method of any one of embodiments 1 to 20, wherein administering comprises intramuscular administration.

[0196] Embodiment 25 is the method of any one of embodiments 1 to 20, wherein administering comprises intrathecal administration.

[0197] Embodiment 26 is the method of any one of embodiments 1 to 20, wherein administering comprises subcutaneous administration.

[0198] Embodiment 27 is the method of any one of embodiments 1 to 20, wherein administering comprises intraperitoneal administration.

[0199] Embodiment 28 is the method of any one of embodiments 1 to 27, wherein the composition is dried prior to the administering step.

[0200] Embodiment 29 is the method of embodiment 28, wherein the composition is rehydrated after the drying step.

[0201] Embodiment 30 is the method of any one of embodiments 1 to 28, wherein the composition is lyophilized prior to the administering step.

[0202] Embodiment 31 is the method of embodiment 30, wherein the composition is rehydrated after the lyophilization step.

[0203] Embodiment 32 is the method of any one of embodiments 1 to 31, wherein the incubation agent comprises one or more salts selected from phosphate salts, sodium salts, potassium salts, calcium salts, magnesium salts, and combinations of two or more thereof.

[0204] Embodiment 33 is the method of any one of embodiments 1 to 32, wherein the incubation agent comprises a carrier protein.

[0205] 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.

[0206] Embodiment 35 is the method of any one of embodiments 1 to 34, wherein the composition comprises one or more sugars.

[0207] Embodiment 36 is the method of embodiment 35, wherein the one or more sugars comprise trehalose.

[0208] Embodiment 37 is the method of embodiment 35 or embodiment 36, wherein the one or more sugars comprise polysucrose.

[0209] Embodiment 38 is the method of any one of embodiments 35-37, wherein the one or more sugars comprise dextrose.

[0210] Embodiment 39 is the method of any one of embodiments 1 to 38, wherein the composition comprises an organic solvent.

[0211] Embodiment 40 is the method of any one of embodiments 1 to 39, wherein the platelets or platelet derivatives comprise thrombosomes. [Example]

[0212] The following results demonstrate the effects of thrombosomal products in an in vitro model of patients taking antiplatelet medications. Thrombosomal and other freeze-dried platelet products are designed to be infused into a patient's bloodstream after trauma or a diagnosis of hemostatic failure. These 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.

[0213] Example 1 - P2Y 12 inhibitors Cangrelor, like clopidogrel, ticagrelor, and prasugrel, inhibits P2Y agonists on platelets. 12 (ADP) receptors. Cangrelor is used in this example as a representative of this class of drugs.

[0214] Thrombosomes were prepared in accordance with the procedure in Example 4. Light transmission aggregometry and T-TAS® experiments were performed according to Example 4.

[0215] The effect of cangrelor on platelet aggregation in platelet-rich plasma (PRP; collected from humans as whole blood and processed to isolate platelets in plasma without white blood cells (WBCs) or red blood cells (RBCs)) was assessed by transmitted light aggregometry. Platelet aggregation in response to agonist-induced activation (platelet-rich plasma) demonstrated complete inhibition of 10 μM adenosine diphosphate (ADP)-induced aggregation by cangrelor at therapeutic concentrations of 0.5 μM to 3.5 μM (Figure 1). All doses of cangrelor investigated completely eliminated ADP-induced platelet aggregation in PRP.

[0216] The effect of cangrelor on platelet occlusion under shear stress was assessed by T-TAS®. Fresh platelet-rich plasma (platelet concentration 278,000 / μL; PRP generally has a platelet concentration of approximately 200,000 / μL to approximately 300,000 / μL) stimulated in vitro with 10 μM ADP occluded faster than unstimulated platelets (PRP) under high shear stress, as determined by AR chip (collagen and tissue thromboplastin) using T-TAS® technology (Figure 2). Cangrelor alone (1 μM) showed no inhibition of occlusion, but when combined with ADP (10 μM), it essentially eliminated platelet adhesion and occlusion. These results are further illustrated in Figures 3 and 4. Without being bound by any particular theory, it is believed that this pattern is observed because platelets have other ADP receptors that respond to ADP and are not blocked by cangrelor, where blocking the ADP receptor P2Y12 causes shape changes and aggregation that inhibit collagen binding; therefore, platelets may bind to each other due to ADP stimulation but prevent collagen binding on the coated chip.

[0217] In Figure 3, area under the curve (AUC) values ​​(derived from data in Figure 2; replicates are averaged and plotted once) represent a combined measure of how quickly thrombus develops and, if it does develop, how substantial it is. PRP AUC increased with ADP stimulation. Cangrelor had little effect on AUC values, but when combined with ADP stimulation, AUC decreased to nearly zero.

[0218] In Figure 4, the time to occlusion of the AR T-TAS® chip with drug treatment was evaluated. PRP occluded the chip channel in approximately 20 minutes, during which time platelet stimulation by ADP decreased. Cangrelor had little effect on occlusion time, but the addition of ADP stimulation to the PRP sample essentially completely inhibited occlusion.

[0219] In the presence of cangrelor, with ADP stimulation at concentrations shown to be inhibitory for platelets, thrombosomes ("thromb" in Figures 5-7) were not inhibited, indicating that thrombosomes can aid clot formation even in the presence of cangrelor at therapeutic levels.

[0220] The effect of cangrelor on thrombosomes under shear stress was assessed by T-TAS®. Figure 5 shows that thrombosomes (after 60, 90, or 115 minutes of rehydration, as indicated) retain hemostatic function in the absence or presence of cangrelor (1 μM) in the presence of ADP (10 μM). Unlike platelets, thrombosome blockage in the T-TAS® AR chip is not affected by the antiplatelet effect of cangrelor plus ADP. This suggests that thrombosomes will maintain expected functionality when infused into patients receiving cangrelor and similar agents. These results are further illustrated in Figures 6 and 7.

[0221] In Figure 6, the AUC values ​​(derived from the data in Figure 5) indicate thrombus formation. Cangrelor and ADP had no effect on thrombosome adhesion and blockage of the T-TAS® AR chip in plasma; thrombosomes caused thrombus formation regardless of cangrelor and ADP. The same doses of cangrelor and ADP completely inhibited freshly harvested platelets.

[0222] In Figure 7, the time to thrombosomal occlusion for the AR T-TAS® chip with drug treatment (derived from the data in Figure 5) was evaluated. There was no effect of cangrelor plus ADP on the time to thrombosomal occlusion using the T-TAS® AR chip in plasma. The same dose of cangrelor and ADP completely inhibited freshly harvested platelets.

[0223] Example 2. GPIIb-IIIa inhibitors. The following results demonstrate the effects of thrombosis in an in vitro model of patients taking GPIIb-IIIa inhibitors. Eptifibatide, a common antiplatelet drug, competitively inhibits the GPIIb-IIIa receptor on platelets, which interacts with fibrinogen and von Willebrand factor.

[0224] 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.

[0225] Thrombosomes were prepared in accordance with the procedure in Example 4. Light transmission aggregometry and T-TAS® experiments were performed according to Example 4.

[0226] Platelet aggregation (in platelet-rich plasma) was assessed using transmitted light aggregometry. Eptifibatide completely inhibited collagen-induced (10 μg / mL) platelet aggregation in PRP at all concentrations tested, as detected by transmitted light aggregometry in PRP (FIG. 8).

[0227] The effect of thrombosomes on shortening clotting time in the presence of eptifibatide was also studied. The ability of thrombosomes to restore occlusion time was studied on the T-TAS® system, which measures occlusion time under collagen- and thromboplastin-stimulated shear forces. The occlusion and AUC whole blood profiles on the AR T-TAS® chip were prolonged and decreased, respectively, by eptifibatide. Eptifibatide prolonged the occlusion time of whole blood on the T-TAS® AR chip in a dose-dependent manner. In this experiment, whole blood occluded in 8 minutes, and 6 μM eptifibatide prolonged the occlusion time to 16 minutes (FIG. 9). Thrombosomes antagonized the inhibitory effect of eptitibatide on clot formation. Eptifibatide inhibition of whole blood occlusion on the T-TAS® AR chip was antagonized by the addition of thrombosomes at approximately 200,000 / μL (N=3). When thrombosomes (approximately 200k / μL) were added to eptifibatide-inhibited whole blood samples, the time to occlusion decreased to "normal" at 9 minutes (FIG. 10).

[0228] The area under the curve values ​​for thrombosomal treatment were also increased by thrombosomal compared with those for normal whole blood samples. Figure 11 demonstrates the time to thrombosomal occlusion on the AR T-TAS® chip with drug treatment, and eptifibatide inhibition of T-TAS® AR chip occlusion was almost completely antagonized by the addition of thrombosomal (200,000 / μL; N=3). In Figure 12, the area under the curve values ​​indicate thrombus formation in which thrombosomal inhibition by eptifibatide was restored to normal levels, and eptifibatide inhibition of platelet adhesion and occlusion to the T-TAS® AR chip was overcome by the addition of thrombosomal (200,000 / μL; N=3).

[0229] Unlike platelets, thrombosomes are not inhibited in their ability to clog under shear in the presence of eptifibatide (Figure 13). Figure 13 shows that the thrombus formation profiles of various lots of thrombosomes on the AR T-TAS® system were not altered by eptifibatide treatment. Thrombososomes in platelet-poor plasma (PPP) were flowed through the T-TAS® AR chip with and without 6 μM eptifibatide. There was no effect of eptifibatide on thrombosome adhesion and clog. All thrombosome concentrations were approximately 300,000 / μL.

[0230] The AUC and occlusion values ​​by T-TAS for thrombosomes in plasma (approximately 300,000 / μL) were similar regardless of whether eptifibatide was used or not (Figures 14-15). Figure 14 shows that the area under the curve values, indicating clot formation, were not altered by eptifibatide in platelet-poor plasma. There was no effect of 6 μM eptifibatide on the AUC of thrombosome occlusion in the T-TAS® AR chip. Figure 15 shows that the time to occlusion of thrombosomes on the AR T-TAS® chip was not altered by eptifibatide. There was no significant effect of 6 μM eptifibatide on the thrombosome occlusion time in the T-TAS® AR chip in platelet-poor plasma.

[0231] Example 3. COX inhibitors. The following results demonstrate the effects of thrombosis in an in vitro model of patients taking COX inhibitors. Aspirin, a common antiplatelet drug, blocks the COX1 enzyme in platelets. COX1 is involved in the conversion of arachidonic acid to prostaglandins.

[0232] 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 following the same oral dose.

[0233] Thrombosomes were prepared in accordance with the procedure in Example 4. Light transmission aggregometry and T-TAS® experiments were performed according to Example 4.

[0234] Platelets aggregate in response to collagen and arachidonic acid stimulation. Arachidonic acid-stimulated platelets could be completely inhibited, whereas collagen-stimulated platelet aggregation could only be partially inhibited at concentrations of 100–400 μM aspirin (Figure 16). Figure 16 shows light transmission aggregometry in PRP containing collagen (10 μg / mL) and arachidonic acid (AA; 500 μg / mL), which induced platelet aggregation. This aggregation was inhibited by all doses of aspirin (ASA) tested. Aspirin completely eliminated arachidonic acid-induced platelet aggregation. Using the PL-chip system on the T-TAS®, we mimicked in vitro platelet binding and aggregation by exposing collagen to the vasculature under shear conditions. This effect on platelets was largely limited in the presence of 100 and 500 μM aspirin, but could be at least partially reversed in the presence of thrombosomes (approximately 200,000–400,000 / μL; Figure 17). FIG. 17 shows area under the curve measurements of whole blood, demonstrating that clot formation on the PL T-TAS® chip was inhibited by aspirin and partially reversed by thrombosomes.

[0235] Example 4. Protocol Thrombosome Production. Thrombosomes were prepared in accordance with the procedures described in U.S. Patent Nos. 8,486,617 (e.g., Examples 1-5, etc.) and 8,097,403 (e.g., Examples 1-3, etc.), which are incorporated by reference in their entireties.

[0236] Light transmission aggregation measurement A plasma sample containing platelets, thrombosomes, or a combination of both is loaded into a cuvette and placed in the aggregometry chamber. The chamber warms the sample and provides constant agitation. Aggregation can be initiated by several types of inhibitors, including but not limited to thrombin, ADP, collagen, and any agent known to stimulate platelet aggregation. Samples may also be collected ex vivo or supplemented with inhibitors in vitro. The instrument begins the assay by first recording light transmission prior to a two-minute stimulation. The technician then introduces the stimulus of interest, and the change in light transmission is recorded over time. An increase in light transmission corresponds to an increase in platelet aggregation.

[0237] T-TAS® Assessment Using the AR Chip The AR chip features single channels containing collagen and tissue factor, which can be used to analyze coagulation and platelet function.

[0238] The T-TAS® instrument was prepared for use according to the manufacturer's instructions. The AR tip (Diapharma catalog no. TC0101) and AR tip calcium corn trypsin inhibitor (CaCTI; Diapharma catalog no. TR0101) were warmed to room temperature. 300 μL of rehydrated thrombosomes were transferred to a 1.7 mL microcentrifuge tube and centrifuged at 3900 g for 10 minutes to pellet the thrombosomes. The thrombosome pellet was resuspended in George King (GK) pooled normal human plasma or autologous plasma with or without autologous platelets to a concentration of approximately 100,000–450,000 / μL, as determined by AcT count (Beckman Coulter AcT Diff 2 Cell Counter). 20 μL of CaCTI was gently mixed with 480 μL of the thrombosome sample in GK plasma using a pipette. Samples were loaded and run on the T-TAS® according to the manufacturer's instructions.

[0239] Evaluation by T-TAS (registered trademark) using PL chip The PL chip is performed similarly to the AR chip, but this chip is coated with collagen alone.

[0240] thrombin generation Reagent Preparation. For thrombin generation, the following materials were used from the manufacturers as follows: FluCa kit (Diagnostica Stago, catalog number 86197), thrombin calibrator (Diagnostica Stago, catalog number 86197), PRP reagent (Diagnostica Stago, catalog number 86196), OCTOPLAS®, solvent detergent-treated human pooled plasma (Octapharma, catalog number 8-68209-952-04). All frozen reagents were thawed in a 37°C water bath before use. All reagents were rehydrated with sterile water using the volumes printed on the reagent label. After approximately 2 minutes of rehydration, the reagents were mixed by inverting the vial five times so no clumps or powder remained; no vortexing was used. This procedure was repeated approximately 10 minutes after rehydration. All reagents were incubated at room temperature for approximately another 10 minutes (approximately 20 minutes total after rehydration). A 30% OCTOPLAS® solution was prepared by mixing 4.66 ml of thrombosomal control buffer (Table B) with 2 ml of OCTOPLAS®.

[0241] Table B: Thrombosomal Control Buffer TIFF0007791581000007.tif61128

[0242] Sample Analysis—Plate Preparation and Testing. For experiments containing thrombosomes, a thrombosome dilution series was generated for each experimental and reference thrombosome (typically, 194.4K, 64.8K, 21.6K, and 7.2K dilutions per μL were used; cell counts were determined by flow cytometry). Unless otherwise indicated, thrombosomes were rehydrated. The highest concentration dilution (e.g., 194.4K thrombosomes) was prepared by combining thrombosomes, OCTAPLAS®, and thrombosome control buffer. The remaining dilution series was prepared by serial 1:3 dilutions of OCTAPLAS®. For each test sample, 20 μL of PRP reagent was added to each sample well (Immulon 2HB Clear, round-bottom 96-well plate (VWR, catalog number 62402-954)), and 20 μL of thrombin calibrator was added to each calibrator well. To each sample well and calibrator well, 80 μL of each of the thrombosomal dilutions was added, continuing until the final dilution. The plate was then incubated for 10 minutes in a Fluoroskan Ascent 96-well fluorescence plate reader (Thrombinoscope) (ThermoFisher Scientific). During this incubation step, FluCa solution was prepared by adding 40 μL of FluCa substrate to 1.6 ml of thawed Fluo-Buffer, vortexing, and returning the solution to the water bath. Once incubation was complete, the FluCa solution was added to the Fluroskan instrument according to the manufacturer's instructions. Plate fluorescence was monitored at 20-second intervals and at a temperature of 40–41°C for 75 minutes.

[0243] Example 5. Additional experiments were performed with cangrelor and aspirin. Thrombosomes were prepared in accordance with the procedures in Example 4. Light transmittance aggregometry, T-TAS®, and thrombin generation experiments were performed according to Example 4.

[0244] The effect of thrombosomes on clot recovery was evaluated using T-TAS® technology and AR chips. Figure 18 shows the occlusion time of whole blood treated with various combinations of thrombosomes (at a concentration of 250,000 thrombosomes per μL), aspirin (200 μM), cangrelor (1 μM), anti-integrin alpha-2 (CD49B) antibody 6F1 (40 μg; for product / manufacturer information, see dshb.biology.uiowa.edu / integrin-alpha-2-alpha2betal?sc=7&category=-107), and anti-GPIIb / IIIa receptor antibody AP2 (20 μg / mL; for product / manufacturer information, see kerafast.com / product / 2010 / anti-glycoprotein-gpiiiagpiib-complex-ap-2-antibody). Figure 19 shows the occlusion over time of untreated whole blood and whole blood treated with a mixture containing thrombosomes (at a concentration of 250,000 thrombosomes per μL), 6F1 (40 μg / mL; anti-CD49b), ASA (aspirin; 200 μM), and cangrelor (1 μM), or a combination thereof.

[0245] The effect of thrombosomes on clot recovery was also evaluated using T-TAS® technology and PL chips. Figure 20 shows the occlusion time of whole blood treated with buffer, aspirin (500 μM), or aspirin (500 μM) and thrombosomes (at a concentration of 250,000 thrombosomes per μL). Figure 21 shows the occlusion time of whole blood, whole blood treated with aspirin (500 μM), or aspirin (500 μM) and thrombosomes (250,000 / μL). Figures 22 and 23 show data from a similar experiment using 100 μM aspirin instead of 500 μM aspirin.

[0246] The effect of aspirin treatment (concentration) on thrombin generation was measured. Thrombosomes were assessed at concentrations of 1450, 1150, 850, 650, 450, 150, 50, and 0 kJ / µL in PPP and normal plasma (INR = 1) from patients taking baby aspirin daily. Figure 24 shows that peak thrombin values ​​in aspirin plasma in the absence of thrombosomes were below the normal range (approximately 45 nM, with the normal range being approximately 66-166 nM), but addition of thrombosomes brought the values ​​back within the normal range, even at the lowest thrombosome concentration used (50 kJ / µL). Resaturation with approximately 800 kJ thrombosomes raised the values ​​to 220 nM (increased from 45 to 220 nM), five times higher than the values ​​in this plasma in the absence of thrombosomes.

[0247] Example 6. Thrombosomes antagonized cangrelor-induced prolonged PRP occlusion time Additional experiments were performed with cangrelor. Thrombosomes were prepared in accordance with the procedure in Example 4. T-TAS® was performed according to Example 4.

[0248] Figures 25A and 25B show that platelet-rich plasma treated with 100 ng / mL cangrelor and ADP extended the occlusion time from 19 to 26 minutes on the T-TAS® flow system (collagen and tissue factor coated channel). Addition of 150 kJ / μL thrombosomes reduced the time back to 15.3 minutes.

[0249] Example 7. Thrombosomes, but not random donor platelets (RDP), antagonized the prolonged occlusion time induced by tirofiban in PRP Additional experiments were performed with tirofiban. Thrombosomes were prepared in accordance with the procedure in Example 4. T-TAS® was performed according to Example 4. Random donor platelets were prepared from whole blood.

[0250] Figures 26A and 26B show that platelet-rich plasma treated with 100 ng / mL tirofiban extended the occlusion time from 18.43 minutes to no occlusion on the T-TAS® flow system (collagen and tissue factor coated channel). Addition of 150 kJ / μL thrombosomes reduced the time back to 12.94 minutes, but RDP was only partially restored at the same number.

[0251] Example 8. Random donor thrombosomes, but not platelets, antagonized the prolonged occlusion time induced by eptifibatide in PRP Additional experiments were performed with eptifibatide. Thrombosomes were prepared consistent with the procedure in Example 4. T-TAS® was performed according to Example 4. Random donor platelets were prepared from whole blood.

[0252] 27A and 27B show that platelet-rich plasma treated with 9 μM eptifibatide extended the occlusion time from 18.43 min to over 30 min on the T-TAS® flow system (collagen and tissue factor coated channel). Addition of 150 kJ / μL thrombosomes reduced the time back to 11.56 min, but occlusion was not seen with the same number of RDPs.

[0253] Example 9. Thrombosomes antagonized the prolonged occlusion time induced by AP2 (anti-GpIIb / IIIa) in PRP Additional experiments were performed with AP2. Thrombosomes were prepared in accordance with the procedure in Example 4. T-TAS® was performed according to Example 4. Random donor platelets were prepared from whole blood.

[0254] Figures 28A and 28B show that platelet-rich plasma treated with 10 μg / mL AP-2 extended the occlusion time from 18.43 minutes to over 30 minutes on the T-TAS® flow system (collagen and tissue factor coated channel). Addition of 150 kJ / μL thrombosomes reduced the time back to 13.14 minutes, and occlusion was seen at 17.43 minutes with the same number of RDPs.

[0255] Example 10. Thrombosomes antagonized long-term obstruction in PRP from subjects on aspirin therapy Additional experiments were performed using aspirin. Thrombosomes were prepared in accordance with the procedure of Example 4. T-TAS® was performed according to Example 4. Random donor platelets were prepared from whole blood. Subjects were taking a standard dose of 81 mg / day of aspirin.

[0256] Figures 29A and 29B show that platelet-rich plasma from an aspirin-treated patient failed to occlude on the T-TAS® flow system (channels coated with collagen and tissue factor). Addition of 200 kJ / μL of thrombosomes restored the normal occlusion time to ~16 min.

[0257] Example 11. Thrombosomes restore thrombin generation in ex vivo aspirin-rich platelet plasma Additional experiments were performed with aspirin. Thrombosomes were prepared in accordance with the procedure in Example 4. Thrombin generation was performed according to Example 4.

[0258] Figure 30A shows that thrombin generation in platelet-rich plasma from aspirin patients versus normals stimulated with PRP reagent was antagonized with 50 kJ / μL thrombosomes. Figure 30B shows the shift and return from normal thrombin generation, time to peak production, and lag time in three replicate aspirin ex vivo samplings containing thrombosomes (50 kJ / μL). (n=3 thrombosome lots, n=2 individuals).

[0259] Example 12. Thrombosomes restore hemostasis in PRP from subjects on NSAID ibuprofen therapy. Additional experiments were performed using ibuprofen, an NSAID. Thrombosomes were prepared in accordance with the procedure in Example 4. Aggregometry and T-TAS® were performed according to Example 4.

[0260] Platelet-rich plasma was collected from a subject taking 800 mg of ibuprofen. Figure 31A shows that the lack of aggregation in response to arachidonic acid confirms the presence of NSAIDs in PRP. Figure 31B shows blockage on the T-TAS® flow system (collagen and tissue factor coated channel), where PRP from an ibuprofen patient demonstrated blockage, while the addition of ADP eliminated the blockage. The addition of 150 kJ / μL of thrombosomes restored the blockage.

[0261] Example 13. Thrombosome® restores bleeding times in NOD-SCID mice treated with suprapharmacological clopidogrel Additional experiments were performed with clopidogrel. Thrombosomes were prepared in accordance with the procedure in Example 4.

[0262] Mice were treated with clopidogrel for 5 days. Mice were anesthetized, and the tip of the tail was amputated, followed by immediate administration of thrombosomes. The time from the dock until bleeding ceased was recorded by visual inspection.

[0263] NOD / SCID mice were treated with approximately three times the clinical dose of clopidogrel for 5 days and then evaluated in a tail dock bleeding model. Bleeding time (minutes) was prolonged to 17.8 minutes with clopidogrel treatment compared to 9 minutes with no treatment (data not shown). Treatment with 8 μL / gram of thrombosomes (1.8 × 10^9 particles / mL in 200 μL) reduced bleeding to 12.31 minutes (Figure 32).

[0264] It should be noted that while the foregoing description is directed to preferred embodiments of the present invention, other variations and modifications will be apparent to those skilled in the art and can be made without departing from the spirit or scope of the invention. Furthermore, features described in connection with one embodiment of the invention may be used in conjunction with other embodiments, even if not explicitly described above. Moreover, those skilled in the art will readily appreciate that the invention as discussed above may be practiced with a different sequence of steps and / or with hardware elements in a different configuration than that disclosed. Thus, while the invention has been described based on these preferred embodiments, it will be apparent to those skilled in the art that certain modifications, variations, and alternative configurations will be apparent while remaining within the spirit and scope of the invention. The embodiments of the invention so claimed are operative as essentially or explicitly described herein. Accordingly, reference should be made to the appended claims to determine the scope and metes and bounds of the invention.

Claims

1. 1. A composition for use in a method of controlling bleeding in a subject, the composition comprising an effective amount of freeze-dried platelet particles and an incubation agent comprising one or more salts, buffers, and sugars, and in a dry form, the method comprising: rehydrating the composition to form a rehydrated composition; and administering the rehydrated composition to the subject in need thereof, wherein the subject is being treated with an antiplatelet agent. Including, the administering step comprises parenteral administration; the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, ticagrelor, prasugrel, abciximab, elinogrel, clopidogrel, eptifibatide, tirofiban, ticlopidine, and ibuprofen; The effective amount is about 5.0 x 10 per kg of the subject's body weight. ７ ~Approx. 1.0×10 ８ particles, approximately 1.0 x 10 ８ ~Approx. 5.0×10 ８ particles, approximately 5.0 x 10 ８ ~Approx. 1.0×10 ９ particles, approximately 1.0 x 10 ９ ~Approx. 5.0×10 ９ particles, or about 5.0 x 10 ９ ~Approx. 1.0×10 １０ particle, 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; composition.

2. The effective amount is about 1.0 x 10 per kg of the subject's body weight. ９ ~Approx. 5.0×10 ９ particles, or about 5.0 x 10 ９ ~Approx. 1.0×10 １０ The composition of claim 1 which is a particle.

3. The effective amount is about 5.0 x 10 per kg of the subject's body weight. ９ ~Approx. 1.0×10 １０ The composition of claim 1 or 2, which is a particle.

4. The composition of any one of claims 1 to 3, wherein at least 50% of the freeze-dried platelet particles have a particle size between 0.3 μm and 5.0 μm.

5. The composition of any one of claims 1 to 4, wherein the incubation agent further comprises 0.1% (v / v) to 5.0% (v / v) of an organic solvent.

6. The composition of any one of claims 1 to 5, wherein the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, clopidogrel, eptifibatide, tirofiban, ibuprofen, and combinations thereof.

7. 7. The composition of claim 6, wherein the subject is being treated with an antiplatelet agent such that the treatment with the antiplatelet agent is continued before and after administration without cessation of the treatment.

8. 6. The composition of any one of claims 1 to 5, wherein the subject is being treated with an antiplatelet agent selected from the group consisting of cangrelor, ticagrelor, prasugrel, elinogrel, clopidogrel, ticlopidine, ibuprofen, and combinations thereof, such that treatment with the antiplatelet agent is continued before and after administration without cessation of the treatment.

9. 9. The composition of any one of claims 1 to 8, wherein the sugar is selected from sucrose, maltose, trehalose, glucose, mannose, and xylose in an amount ranging from 10 mM to 500 mM.

10. 10. The composition of claim 9, wherein the sugar is trehalose.

11. The composition according to claim 9 or 10, wherein the composition in dry form further comprises polysucrose in an amount ranging from 3% to 7%.

12. 12. The composition of any one of claims 1 to 11, wherein the method further comprises performing surgery on the subject after administering the composition to the subject, and wherein the dose of the antiplatelet agent is not reduced prior to the surgery.

13. The composition of any one of claims 1 to 12, wherein the subject is being treated with two antiplatelet agents.

14. the subject is being treated with aspirin and an antiplatelet agent selected from the group consisting of cangrelor, ticagrelor, prasugrel, abciximab, elinogrel, clopidogrel, eptifibatide, tirofiban, ticlopidine, and ibuprofen, and the effective amount is about 5.0 x 10 per kg of the subject's body weight ９ ~Approx. 1.0×10 １０ The composition according to any one of claims 1 to 5 and 9 to 13, which is in the form of particles.

15. 15. The composition of any one of claims 1 to 14, wherein the administering step comprises administering the rehydrated composition intravenously.

16. 16. The composition of any one of claims 1 to 15, wherein the subject has a peak thrombin of greater than 70 nM after administration.

17. 17. The composition of any one of claims 1-5, 9-13, 15, and 16, wherein the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, elinogrel, ticlopidine, ibuprofen, and combinations thereof.

18. 17. The composition of any one of claims 1-5, 9-13, 15, and 16, wherein the antiplatelet agent is selected from the group consisting of cangrelor, ticagrelor, prasugrel, abciximab, ibuprofen, elinogrel, and combinations thereof.

19. 17. The composition of any one of claims 1-5, 9-13, 15, and 16, wherein the antiplatelet agent is selected from the group consisting of cangrelor, ticagrelor, prasugrel, ibuprofen, elinogrel, and combinations thereof.

20. 1. Use of an effective amount of freeze-dried platelet particles for the preparation of a rehydrated platelet composition for use in a method for controlling bleeding in a subject, the method comprising: rehydrating a composition in dry form comprising an effective amount of freeze-dried platelet particles and an incubation agent comprising one or more salts, buffers, and sugars to form the rehydrated platelet composition; and administering the rehydrated platelet composition parenterally to the subject in need thereof, wherein the subject has been or is being treated with an antiplatelet agent. Including, the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, ticagrelor, prasugrel, abciximab, elinogrel, clopidogrel, eptifibatide, tirofiban, ticlopidine, ibuprofen, and combinations thereof; The effective amount is about 5.0 x 10 per kg of the subject's body weight. ７ ~Approx. 1.0×10 ８ particles, approximately 1.0 x 10 ８ ~Approx. 5.0×10 ８ particles, approximately 5.0 x 10 ８ ~Approx. 1.0×10 ９ particles, approximately 1.0 x 10 ９ ~Approx. 5.0×10 ９ particles, or about 5.0 x 10 ９ ~Approx. 1.0×10 １０ particle, 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; use.

21. a composition in a dry form comprising an effective amount of freeze-dried platelet particles prepared by a method comprising: incubating platelets with an incubation agent comprising one or more salts, a buffer, and a sugar to form incubated platelets; and freeze-drying the incubated platelets to form the composition in a dry form comprising an effective amount of freeze-dried platelet particles, wherein at least 50% of the freeze-dried platelet particles have a particle size of about 0.3 μm to 5.0 μm, and the sugar comprises trehalose in an amount ranging from 10 mM to 500 mM; 21. The use according to claim 20.

22. 22. The use of claim 20 or 21, wherein the method further comprises, after freeze-drying, heating the composition, in dry form, comprising an effective amount of freeze-dried platelet particles, at a temperature in the range of 70°C to 85°C for at least 1 hour but not more than 36 hours to heat-treat the freeze-dried platelet particles.

23. The effective amount is about 1.0 x 10 per kg of the subject's body weight. ９ ~Approx. 5.0×10 ９ particles, or about 5.0 x 10 ９ ~Approx. 1.0×10 １０ The use according to any one of claims 20 to 22, which is a particle.

24. The use according to any one of claims 20 to 23, wherein the antiplatelet agent is selected from the group consisting of cangrelor, ticagrelor, prasugrel, ibuprofen, elinogrel, and combinations thereof.

25. 25. The use according to any one of claims 20 to 24, wherein the subject is being treated with an antiplatelet agent such that the treatment with the antiplatelet agent is continued before and after administration without stopping the treatment.

26. 26. The use of any one of claims 20 to 25, wherein the subject is being treated with an antiplatelet agent, such as being treated with an antiplatelet agent selected from the group consisting of aspirin and cangrelor, ticagrelor, prasugrel, abciximab, elinogrel, clopidogrel, eptifibatide, tirofiban, ticlopidine, and ibuprofen.

27. The effective amount is about 5.0 x 10 per kg of the subject's body weight. ９ ~Approx. 1.0×10 １０ The use according to any one of claims 20 to 22 and 24 to 26, which is a particle.

28. 28. The use of any one of claims 20 to 27, wherein after administration the subject has a peak thrombin of greater than 70 nM.

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