Methods for maintaining platelet viability and activation capacity over an extended period of time - Patents.com

Abstract

The compositions and methods described herein provide stabilized platelets through a controlled freezing process coupled with a thawing recovery method, which maintain the ability to be activated after freezing and thawing.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 177203, filed April 20, 2021, which is incorporated by reference in its entirety.

[0002] Federally Funded Research This invention was made with Government support under Grant No. HL147734 awarded by the Small Business Innovation Research (SBIR) program of the National Institutes of Health. The United States Government has certain rights in this invention.

[0003] Reference to sequence listing This application is filed in accordance with 37 C.FR § 1.821(c) with a sequence listing in computer readable form. The text file submitted by EFS, "211509-9003-WO01_sequence_listing_11-MAR-2022_ST25.txt", was created on Mar. 11, 2022, contains two sequences, has a file size of 1.13 Kbytes, and is incorporated herein by reference in its entirety.

[0004] Technical Field The compositions and methods described herein provide stabilized platelets by a controlled freezing process coupled with a thawing recovery method. Stabilized platelets maintain the ability to be activated after freezing and thawing. [Background technology]

[0005] Platelets are anuclear fragments of megakaryocytes that are important for normal hemostasis. In several pathological conditions, thrombosis caused / contributed to by activated platelets can result in high levels of morbidity and mortality. Conversely, low levels of platelets can predispose to severe bleeding. An important feature of platelets is their limited viability, whether used in a diagnostic or therapeutic context. Viable platelets will be able to release granular components that can be conveniently measured by techniques including, but not limited to, flow cytometry and enzyme-linked immunosorbent assay. Typically, platelets are stored at room temperature and become stale within 5 days. Storing platelets at 4°C can extend viability for up to 2 weeks. Other methods for long-term platelet storage, such as freeze-drying, have demonstrated potential utility in treating bleeding patients, but their utility in diagnostic contexts is limited due to the fact that platelets do not have sufficient viability to be activated and release granular components.

[0006] There is a need for methods for stabilizing platelets during cryopreservation that maintain their viability and activation potential in clinical and diagnostic settings. Such stabilized platelets are useful for a variety of research purposes. Summary of the Invention

[0007] One embodiment described herein is a method for cryopreserving platelets that maintains viability and activation capacity, the method comprising: (a) obtaining blood or platelet-rich plasma or apheresis-derived platelets from a subject; (b) isolating platelets from the blood, platelet-rich plasma or apheresis platelets; (c) suspending the platelets in a suspension buffer comprising trehalose; (d) adding a stabilizer to the suspended platelets; (e) cooling the platelets to a temperature of about -80°C; and (f) storing the platelets at about -80°C. In one aspect, the method maintains viability and activation capacity of the platelets for more than two weeks. In another aspect, the suspension buffer comprises about 50 mM trehalose. In another aspect, the stabilizer is about 4% by weight bovine serum albumin (BSA). In another aspect, the platelets of step (c) are cryopreserved at about 1 x 10 6 The platelets are diluted with suspension buffer to a final concentration of about 1×10 platelets / μL. In another aspect, the diluted platelets are incubated at about 37° C. for about 2 hours with periodic agitation. In another aspect, isolating the platelets comprises centrifuging the blood, platelet-rich plasma or apheresis platelets at 100×g for 15 minutes to generate a first pellet and a first supernatant; thereafter, centrifuging the first supernatant at 1000×g for 15 minutes to generate a second pellet and a second supernatant, the first supernatant being platelet-rich and the second supernatant being platelet-poor. In another aspect, the second supernatant is collected, trehalose is added to about 50 mM, and the platelets of step (c) are diluted to about 1×10 6 and using to dilute to a concentration of 100000000 platelets / μL. In another aspect, cooling the platelets comprises decreasing the temperature at a rate of about 2-20° C. per minute. Another embodiment described herein is a cryopreserved platelet prepared by the methods described herein.

[0008] Other embodiments described herein are methods for cryopreserving and recovering platelets from cryopreservation, the methods comprising: (a) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a subject; (b) isolating platelets from the blood, platelet-rich plasma, or apheresis platelets; (c) suspending the platelets in a buffer solution comprising trehalose; (d) adding a stabilizer to the suspended platelets; (e) chilling the platelets to a temperature of about -80°C; (f) storing the platelets at about -80°C for a period of time; and (g) thawing the platelets at about 20-40°C. In one aspect, the suspension buffer comprises about 50 mM trehalose. In another aspect, the stabilizer is about 4% by weight bovine serum albumin (BSA). In another aspect, the platelets of step (c) are thawed to about 1 x 10 6The platelets are diluted with suspension buffer to a final concentration of platelets / μL. In another aspect, the diluted platelets are incubated at about 37° C. for about 2 hours with periodic agitation. In another aspect, the method further comprises the steps of (h) isolating the thawed platelets; and (i) suspending the isolated thawed platelets in an activation buffer comprising about 1% by weight BSA. In another aspect, the isolation of the thawed platelets comprises centrifugation at 1000×g for 15 minutes. In another aspect, the activation buffer further comprises about 50 mM trehalose. In another aspect, the isolation of the thawed platelets comprises centrifugation at 1000×g for 5 minutes. In another aspect, after step (h) and before step (i), the following steps are performed: (h-1) washing the thawed platelets in a wash buffer. In another aspect, isolating the thawed platelets in step (h) comprises centrifuging at 1000×g for 15 minutes and removing the supernatant; and washing the thawed platelets in step (h-1) comprises resuspending the platelets in a wash buffer, centrifuging at 1000×g for 15 minutes and removing the supernatant. In another aspect, the wash buffer further comprises about 50 mM trehalose. In another aspect, isolating the thawed platelets in step (h) comprises centrifuging at 1000×g for 5 minutes and removing the supernatant; and washing the thawed platelets in step (h-1) comprises resuspending the platelets in a wash buffer, centrifuging at 1000×g for 5 minutes and removing the supernatant. In another aspect, the period of time in step (f) is from 1 day to about 5 years. Another embodiment described herein is a collected cryopreserved platelet prepared by the method described herein.

[0009] Another embodiment described herein is a method for activating recovered platelets after cryopreservation, comprising: (a) recovering platelets after cryopreservation according to the methods described herein; (b) treating the recovered platelets with platelet factor 4 (PF4; SEQ ID NO:2); and (c) incubating the PF4-treated platelets with plasma, serum or blood sample from a subject diagnosed with heparin-induced thrombocytopenia (HIT). In one aspect, treating the recovered platelets with PF4 comprises adding PF4 to the platelets at a concentration of about 150 μg / mL and incubating at room temperature for about 20 minutes. In another aspect, platelet activation is assessed by measuring platelet granule contents released after incubation of the PF4-treated platelets with plasma, serum or blood sample from the HIT subject. In another aspect, the platelet granule contents are thrombospondin-1. Another embodiment described herein is activated recovered cryopreserved platelets prepared according to the methods described herein.

[0010] Another embodiment described herein is a method for activating collected platelets after cryopreservation, the method comprising: (a) collecting platelets after cryopreservation according to the methods described herein; and (b) treating the collected platelets with thrombin receptor activating peptide (TRAP; amino acid sequence: SFFLRN; SEQ ID NO:1). In one aspect, treating the collected platelets with TRAP comprises adding TRAP to the platelets at a concentration of about 25 μg / mL and incubating at room temperature for about 30 minutes. In another aspect, platelet activation is assessed by measuring platelet granule contents released after treating the collected platelets with TRAP. In another aspect, the platelet granule contents is thrombospondin-1. Another embodiment described herein is activated collected cryopreserved platelets prepared according to the methods described herein.

[0011] Another embodiment described herein is a method for activating recovered platelets after cryopreservation, the method comprising: (a) recovering platelets after cryopreservation according to the methods described herein; (b) treating the recovered platelets with heparin; and (c) incubating the heparin-treated platelets with a plasma, serum or blood sample from a subject diagnosed with heparin-induced thrombocytopenia (HIT). In one aspect, treating the recovered platelets with heparin comprises adding heparin to the platelets at a concentration of about 0.1-5 U / mL and incubating at room temperature for about 20 minutes. In another aspect, platelet activation is assessed by measuring platelet granule content released after incubation of the heparin-treated platelets with a plasma, serum or blood sample from the HIT subject. In another aspect, the platelet granule content is thrombospondin-1. Another embodiment described herein is activated recovered cryopreserved platelets prepared according to the methods described herein.

[0012] Other embodiments described herein are methods for cryopreserving platelets and recovering platelets from cryopreservation, the methods comprising: (a) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a subject; (b) isolating platelets from the blood, platelet-rich plasma, or apheresis platelets; (c) suspending the platelets in a buffer solution comprising trehalose; (d) adding a stabilizer to the suspended platelets; (e) cooling the platelets to a temperature of about -80°C; (f) storing the platelets at about -80°C for a period of time; (g) thawing the platelets at about 20-40°C; (h) isolating the thawed platelets; and (i) suspending the isolated thawed platelets in an activation buffer comprising about 1% by weight BSA. In one aspect, the suspension buffer comprises about 50 mM trehalose. In another aspect, the stabilizer is about 4% by weight bovine serum albumin (BSA). In another aspect, the platelets of step (c) are suspended in an activation buffer comprising about 1×10 6The platelets are diluted with suspension buffer to a final concentration of platelets / μL. In another aspect, the diluted platelets are incubated at about 37° C. for about 2 hours with periodic agitation. In another aspect, isolating the thawed platelets comprises centrifugation at 1000×g for 15 minutes. In another aspect, the activation buffer further comprises about 50 mM trehalose. In another aspect, isolating the thawed platelets comprises centrifugation at 1000×g for 5 minutes. In another aspect, after step (h) and before step (i), the following steps are performed: (h-1) washing the thawed platelets in a wash buffer. In another aspect, isolating the thawed platelets in step (h) comprises centrifugation at 1000×g for 15 minutes and removing the supernatant; and washing the thawed platelets in step (h-1) comprises resuspending the platelets in a wash buffer, centrifugation at 1000×g for 15 minutes and removing the supernatant. In another aspect, the wash buffer further comprises about 50 mM trehalose. In another aspect, isolating the thawed platelets in step (h) comprises centrifuging at 1000×g for 5 minutes and removing the supernatant; and washing the thawed platelets in step (h-1) comprises resuspending the platelets in a wash buffer, centrifuging at 1000×g for 5 minutes and removing the supernatant. In another aspect, the period of time in step (f) is from 1 day to about 5 years. Another embodiment described herein is a recovered cryopreserved platelet prepared by the methods described herein.

[0013] Another embodiment described herein is a method for activating recovered platelets after cryopreservation, comprising: (a) recovering platelets after cryopreservation according to the methods described herein; (b) treating the recovered platelets with platelet factor 4 (PF4; SEQ ID NO:2); and (c) incubating the PF4-treated platelets with plasma, serum or blood sample from a subject diagnosed with heparin-induced thrombocytopenia (HIT). In one aspect, treating the recovered platelets with PF4 comprises adding PF4 to the platelets at a concentration of about 150 μg / mL and incubating at room temperature for about 20 minutes. In another aspect, platelet activation is assessed by measuring platelet granule contents released after incubation of the PF4-treated platelets with plasma, serum or blood sample from the HIT subject. In another aspect, the platelet granule contents are thrombospondin-1. Another embodiment described herein is activated recovered cryopreserved platelets prepared according to the methods described herein.

[0014] Another embodiment described herein is a method for activating collected platelets after cryopreservation, the method comprising: (a) collecting platelets after cryopreservation according to the methods described herein; and (b) treating the collected platelets with thrombin receptor activating peptide (TRAP; amino acid sequence: SFFLRN; SEQ ID NO:1). In one aspect, treating the collected platelets with TRAP comprises adding TRAP to the platelets at a concentration of about 25 μg / mL and incubating at room temperature for about 30 minutes. In another aspect, platelet activation is assessed by measuring platelet granule contents released after treating the collected platelets with TRAP. In another aspect, the platelet granule contents are thrombospondin-1. Another embodiment described herein is activated collected cryopreserved platelets prepared according to the methods described herein.

[0015] Another embodiment described herein is a method for activating recovered platelets after cryopreservation, the method comprising: (a) recovering platelets after cryopreservation according to the methods described herein; (b) treating the recovered platelets with heparin; and (c) incubating the heparin-treated platelets with a plasma, serum or blood sample from a subject diagnosed with heparin-induced thrombocytopenia (HIT). In one aspect, treating the recovered platelets with heparin comprises adding heparin to the platelets at a concentration of about 0.1-5 U / mL and incubating at room temperature for about 20 minutes. In another aspect, platelet activation is assessed by measuring platelet granule content released after incubation of the heparin-treated platelets with a plasma, serum or blood sample from the HIT subject. In another aspect, the platelet granule content is thrombospondin-1. Another embodiment described herein is activated recovered cryopreserved platelets prepared according to the methods described herein.

[0016] Another embodiment described herein is collected cryopreserved platelets produced by the following method: (a) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a subject; (b) isolating platelets from the blood, platelet-rich plasma, or apheresis platelets; (c) suspending the platelets in a buffer solution containing trehalose; (d) adding a stabilizer to the suspended platelets; (e) cooling the platelets to a temperature of about -80°C; (f) storing the platelets at about -80°C for a period of time; and (g) thawing the platelets at about 20-40°C.

[0017] Other embodiments described herein are activated, collected, cryopreserved platelets prepared by the following method: (a) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a subject; (b) isolating platelets from the blood, platelet-rich plasma, or apheresis platelets; (c) suspending the platelets in a buffer solution containing trehalose; (d) adding a stabilizer to the suspended platelets; (e) cooling the platelets to a temperature of about -80°C; (f) storing the platelets at about -80°C for a period of time; (g) thawing the platelets at about 20-40°C; (h) isolating the thawed platelets; (i) suspending the isolated, thawed platelets in an activation buffer containing about 1% by weight BSA; (j) treating the collected platelets with heparin or PF4; and (k) incubating the heparin or PF4-treated platelets with a plasma, serum, or blood sample from a subject diagnosed with heparin-induced thrombocytopenia (HIT).

[0018] Another embodiment described herein is activated recovered cryopreserved platelets prepared by the following method: (a) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a subject; (b) isolating platelets from the blood, platelet-rich plasma, or apheresis platelets; (c) suspending the platelets in a buffer solution containing trehalose; (d) adding a stabilizer to the suspended platelets; (e) cooling the platelets to a temperature of about -80°C; (f) storing the platelets at about -80°C for a period of time; (g) thawing the platelets at about 20-40°C; (h) isolating the thawed platelets; (i) suspending the isolated thawed platelets in an activation buffer containing about 1% by weight BSA; and (j) treating the recovered platelets with thrombin receptor activating peptide (TRAP; amino acid sequence: SFFLRN; SEQ ID NO:1).

[0019] Other embodiments described herein are kits for detecting heparin-induced thrombocytopenia (HIT) antibodies in blood, plasma, or serum, the kits comprising two or more of the following: (a) cryopreserved platelets; (b) one or more of platelet factor 4 (PF4; SEQ ID NO:2) or heparin; (c) one or more reagents for detecting platelet activation; (d) optionally, a buffer and container; and (e) optionally, one or more of packaging or instructions for use. In one aspect, the one or more reagents for detecting platelet activation comprise a reagent for detecting thrombospondin-1. In another aspect, the kit further comprises one or more negative control samples negative for pathogenic HIT antibodies that activate platelets; and one or more positive control samples positive for pathogenic HIT antibodies that activate platelets.

[0020] Other embodiments described herein are methods for treating or preventing bleeding in a subject in need thereof, the method comprising: (a) obtaining cryopreserved platelets; (b) thawing the cryopreserved platelets; and (c) contacting the subject with the thawed cryopreserved platelets. In one aspect, steps (a)-(b) comprise: (i) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a second subject; (ii) isolating platelets from the blood, platelet-rich plasma, or apheresis platelets; (iii) suspending the platelets in a buffer solution comprising trehalose; (iv) incubating the trehalose-treated platelets at 37° C. for about 2 hours; (v) adding a stabilizer to the suspended platelets; (vi) cooling the platelets to a temperature of about −80° C.; (vii) storing the platelets at about −80° C. for a period of time; and (viii) thawing the platelets at about 20-40° C. In another aspect, steps (a)-(b) comprise: (i) obtaining platelet-rich plasma or apheresis-derived platelets from the second subject; (ii) adding trehalose to the platelet-rich plasma or apheresis-derived platelets; (iii) incubating the trehalose-treated platelets at 37° C. for about 2 hours; (iv) cooling the platelets to a temperature of about −80° C.; (v) storing the platelets at about −80° C. for a period of time; and (vi) thawing the platelets at about 20-40° C. In another aspect, the contacting in step (c) comprises applying thawed cryopreserved platelets to the site of injury or parenterally administering thawed cryopreserved platelets to the subject.

[0021] Another aspect described herein is the use of the cryopreserved platelets as a reagent, research tool, or medicine to treat a subject in need thereof. Another aspect described herein is the use of thawed cryopreserved platelets as a reagent, research tool, or medicine to treat a subject in need thereof.

[0022] Another aspect described herein is the use of the activated thawed cryopreserved platelets as a reagent, research tool, or medicine to treat a subject in need thereof. [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 illustrates representative process steps for cryopreserving and harvesting platelets as described herein. [Diagram 2] Figure 2 shows 20 lots of platelets from platelet-rich plasma units obtained from 20 different whole blood donors that were cryopreserved and recovered by washing. Platelets were treated with platelet factor 4 (PF4) and tested for reactivity against three HITs (closed circles) and three normal blood samples (open circles). Supernatants from the activation reactions were then assayed for thrombospondin-1 (TSP1) concentrations as a marker of platelet activation. The x-axis shows the 20 different platelet lots and the y-axis shows the amount of TSP1 released from frozen platelets in μg / mL. Data are the average of triplicate determinations. [Diagram 3] FIG. 3 shows seven lots of platelets that were cryopreserved and either after 1 week or less of storage at −80° C. (baseline, B) or after 4 weeks or more of storage at −80° C. (4W) and then recovered by washing. After recovery by washing, platelets were treated with PF4 and tested for reactivity against three HITs (closed circles) and three normal blood samples (open circles). Supernatants from the activation reactions were then assayed for TSP1 concentration as a marker of platelet activation. The x-axis shows the seven different platelet lots separated by vertical lines for both the baseline and 4 week time points, and the y-axis shows the amount of TSP1 released from frozen platelets in μg / mL. Data are the average of triplicate measurements. [Figure 4] Figure 4 shows platelets that were cryopreserved and recovered by washing after 14 weeks of storage at -80°C. After recovery, platelets were treated with PF4 and tested for reactivity against HIT (black) or normal blood samples (white). Supernatants from cryopreserved platelets were then assayed for TSP1 concentration as a marker of platelet activation. The y-axis shows the amount of TSP1 released from frozen platelets in μg / mL. Data are the mean of triplicate determinations + one standard deviation of the mean. [Diagram 5]FIG. 5 shows platelets from a single donor that were cryopreserved and collected by thawing and washing (one wash), thawing and volume reduction (volume reduction), or thawing only (no wash). Platelets were then treated with PF4. HIT (black) or normal blood samples (white) were added to the PF4-treated platelets. Platelet supernatants were assayed for TSP1 concentration as a marker of platelet activation. The y-axis shows the amount of TSP1 released from frozen platelets in μg / mL. Data shown are the mean of triplicate determinations + one standard deviation of the mean. [Figure 6] FIG. 6 shows platelets from a single donor that were cryopreserved and collected by thawing and washing ("wash once"), thawing and volume reduction ("volume reduction"), or thawing only ("no wash"). TRAP (black) or buffer (white) was added to collected platelets. Platelet supernatants were assayed for TSP1 concentration as a marker of platelet activation. The y-axis shows the amount of TSP1 released from frozen platelets in μg / mL. Data are the mean of triplicate determinations + one standard deviation of the mean. [Figure 7] Figure 7 shows platelets that were cryopreserved, recovered by washing, and then treated with unfractionated heparin at concentrations of 0, 0.1, 0.3, 0.5, and 1 U / mL. HIT (black) or normal blood samples (white) were then added to the heparin-treated platelets, and the supernatants were assayed for TSP1 concentration as a measure of platelet activation. The x-axis shows heparin concentration, and the y-axis shows the amount of TSP1 released from frozen platelets in μg / mL. Data are the mean of triplicate determinations + one standard deviation of the mean. [Figure 8] Figure 8 shows platelets cryopreserved with platelet poor plasma, recovered by washing, and treated with PF4. HIT (black) or normal blood samples (white) were then added to the PF4 treated platelets, and the supernatants were assayed for TSP1 concentration as a measure of platelet activation. The y-axis shows the amount of TSP1 released from frozen platelets in μg / mL. Data are the mean of triplicate determinations + one standard deviation of the mean. [Figure 9]Figure 9 shows platelets cryopreserved in the presence of platelet poor plasma and recovered by washing. TRAP (black) or buffer (white) was then added and the supernatant was assayed for TSP1 concentration as a measure of platelet activation. The y-axis shows the amount of TSP1 released from frozen platelets in μg / mL. Data are the mean of triplicate determinations + one standard deviation of the mean. [Figure 10] Figure 10 shows platelets from a single donor that were frozen and recovered by washing (black) or lyophilized and reconstituted (white) and then treated with PF4. PF4-treated platelets were spiked with HIT (left) or normal blood samples (right) and the supernatants were assayed for TSP1 concentration. The y-axis shows the amount of TSP1 released from frozen platelets in μg / mL. Data are the mean of triplicate determinations + one standard deviation of the mean. [Figure 11] FIG. 11 shows platelets from a single donor that were frozen and recovered by washing (black) or freeze-dried and reconstituted (white). TRAP (left) or buffer (right) was added to the platelets and the supernatant was assayed for TSP1 concentration. The y-axis shows the amount of TSP1 released from frozen platelets in μg / mL. Data are the mean of triplicate determinations + one standard deviation of the mean. [Figure 12] FIG. 12 shows platelets that were frozen and recovered by washing using a 15 minute centrifugation without trehalose in the resuspension buffer (left) or a 5 minute centrifugation in the presence of 50 mM trehalose (right). Platelets were spiked with HIT serum (black) or normal serum (white) and the supernatants were assayed for TSP1 concentration. The y-axis shows the amount of TSP1 released from frozen platelets in μg / mL. Data are the mean of triplicate determinations + one standard deviation of the mean. [Figure 13] Figure 13 shows stabilized platelets treated with agonist in the presence and absence of a candidate drug that antagonizes the agonist action. Such stabilized platelets can replace the requirement for fresh platelets in these studies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art.For example, any nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein, and the techniques thereof, are well known and commonly used in the art.In case of conflict, the present disclosure, including definitions, will prevail.Representative methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in carrying out or testing the embodiments and aspects described herein.

[0025] As used herein, the terms "amino acid," "nucleotide," "polynucleotide," "vector," "polypeptide," and "protein" have their common meanings as would be understood by one of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0026] As used herein, terms such as "include," "including," "contain," "containing," "having," and the like, mean "comprising." The present disclosure also contemplates other embodiments that "comprise," "consist of," and "consist essentially of" aspects or elements presented herein, whether or not expressly stated.

[0027] As used herein, the terms "a," "an," "the," and similar terms as used in the context of this disclosure (particularly in the context of the claims) are to be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Also, unless otherwise specified, "a," "an," or "the" means "one or more."

[0028] As used herein, the term "or" can be either conjunctive or disjunctive. As used herein, the term "substantially" means to a great extent or to a significant extent, but not completely.

[0029] As used herein, the term "about" or "approximately" as applied to one or more subject values ​​refers to a value that is similar to a recited reference value or within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term "about" refers to any value, including both integer and fractional components, that is within a variation of up to ±10% of the value modified by the term "about". Alternatively, "about" can mean within 3 or more standard deviations, in accordance with the practice in the art. Alternatively, for example, with respect to biological systems or processes, the term "about" can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 1.5-fold of a value. As used herein, the symbol " ~ " means "about" or "approximately."

[0030] All ranges disclosed herein include both endpoints as discrete values, as well as all integers and fractions specified within the range. For example, the range 0.1 to 2.0 includes 0.1, 0.2, 0.3, 0.4...2.0. When an endpoint is modified by the term "about," the specified range is expanded by up to ±10% variation of any value within the range or within 3 or more standard deviations inclusive of the endpoint.

[0031] As used herein, the term "active ingredient" or "active pharmaceutical ingredient" refers to a drug, active ingredient, compound, or substance, composition, or mixture thereof that provides a often beneficial pharmacological effect.

[0032] As used herein, the terms "control" or "reference" are used interchangeably. A "reference" or "control" level can be a predetermined value or range used as a baseline or benchmark for evaluating a measurement result. "Control" also refers to a control experiment or control cells.

[0033] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a substantially non-toxic but sufficient amount of an agent, agent, composition, or cell(s) administered to a subject to prevent, treat, or ameliorate to some extent one or more of the symptoms of a disease or condition experienced or to which the subject is susceptible. The result can be reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired change in a biological system. The effective amount can be based on factors that vary for each subject, including, but not limited to, the subject's age, size, type or extent of disease, stage of disease, route of administration, type or extent of replacement therapy used, ongoing disease course, and type of treatment desired.

[0034] The compositions described herein can be administered parenterally, topically, rectally, nasally, orally, vaginally, or via an implanted reservoir. The term "parenteral" or "parenteral administration" as used herein includes intravenous, intraarterial, subcutaneous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, intracranial injection, or systemic infusion. In some embodiments, the compositions described herein are administered intravenously, intraarterially, or by infusion.

[0035] As used herein, the term "subject" refers to an animal. Typically, the subject is a mammal. Subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), non-human primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate. In one embodiment, the subject is a human. In one aspect, the subject is suspected of having a pathogenic antibody or a platelet-activating antibody. "Subject suspected of having a platelet-activating antibody" or "test subject" as used herein refers to a subject that exhibits clinical signs indicative of a pathogenic antibody, such as, for example, a lower than normal platelet count, a decrease in platelet count, enlargement or elongation of a previously diagnosed clot, or the development of a new clot elsewhere in the body. Symptoms indicative of a pathogenic antibody include shortness of breath, stroke, gangrene of the limbs, skin necrosis, myocardial infarction, or chest pain. In one embodiment, the platelet-activating antibody is an antibody that can cause HIT. In another embodiment, the platelet-activating antibody comprises a platelet-activating heparin-induced thrombocytopenia antibody. "Normal subject" refers to a subject not suffering from any aliment or suspected of having a platelet-activating antibody.

[0036] As used herein, a subject is "in need of treatment" if such a subject would benefit biologically, medically, or in quality of life from said treatment. A subject in need of treatment does not necessarily exhibit symptoms, particularly in the case of preventative or prophylactic treatment.

[0037] As used herein, the terms "inhibit," "inhibition," or "inhibiting" refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant reduction in the baseline activity of a biological activity or process.

[0038] As used herein, "treatment" or "treating" refers to preventing, preventing, inhibiting, arresting, reversing, mitigating, ameliorating or inhibiting the progression of a biological process, including a disorder or disease, or eliminating the disease altogether. Treatment can be performed in either a short-term or long-term manner. The term "treatment" also refers to reducing the severity of a disease or symptoms associated with the disease before the disease is afflicted. "Suppressing" or "ameliorating" a disease, disorder or symptom thereof includes administering a cell, composition or compound described herein to a subject after the clinical appearance of such disease, disorder or symptom. "Preventing" or "preventing" a disease, disorder or symptom thereof includes administering a cell, composition or compound described herein to a subject before the onset of a disease, disorder or symptom thereof. "Suppressing" a disease or disorder includes administering a cell, composition or compound described herein to a subject after the induction of a disease or disorder but before its clinical appearance or symptom.

[0039] As used herein, "identifying" or "diagnosing" refers to classifying a subject as having a pathology or condition, determining the severity (advancement or stage) of a pathology, monitoring the progression of a pathology, or predicting the outcome or likelihood of recovery of a pathology. In one embodiment, a subject is identified or diagnosed as having platelet-activating antibodies in its blood. In one aspect, the platelet-activating antibodies include platelet-activating heparin-induced thrombocytopenia antibodies. Identification or detection of platelet-activating heparin-induced thrombocytopenia antibodies indicates that a subject has or is used to diagnose heparin-induced thrombocytopenia (HIT).

[0040] As used herein, "heparin-induced thrombocytopenia" or "HIT" refers to an adverse reaction to heparin or a thrombotic thrombocytopenic reaction that occurs without exposure to heparin, such as spontaneous HIT and "vaccine-induced immune thrombotic thrombocytopenia" or "VITT," in which affected subjects produce platelet-activating antibodies that bind to molecules such as PF4, IL-8, and NAP-2 in complex with heparin, in complex with platelet membrane components, or alone, resulting in a potentially life-threatening prothrombotic and thrombocytopenic state.

[0041] As used herein, "heparin-induced" refers to antibodies that result from exposure to heparin or that recognize blood factors complexed with heparin or platelet membrane components, such as indicative of HIT, spontaneous HIT or VITT.

[0042] As used herein, "heparin-like compound" refers to a compound having a strong negative charge, such as a polyanion, a heparin derivative, a chemically modified heparin, a heparin-like glycosaminoglycan molecule, a proteoglycan containing multiple heparins or heparin-like glycosaminoglycans, a low molecular weight heparin, a synthetic glycosaminoglycan containing at least 10 sugar units, or a synthetic heparin-like glycosaminoglycan, any of which may be linked to a core molecule directly or via a spacer / linker molecule.

[0043] "Platelets", also known as "thrombocytes", as used herein, refer to anuclear fragments of megakaryocytes involved in blood clotting, hemostasis and thrombus formation. Human platelets are routinely isolated by a variety of methods, including platelet apheresis, platelet pheresis, gel filtration, or differential centrifugation. While isolated platelets are suitable, platelets from other sources, including washed platelets, unwashed platelets, platelet-rich plasma, or purified platelets, can also be used. In one embodiment, the platelets are stabilized for storage by one or more of cooling, freezing, chemical preservation, or lyophilization.

[0044] "Platelet activation", as used herein, refers to the response of platelets when they encounter activation-inducing "platelet activating factor" molecules, such as, inter alia, platelet-activating antibodies, thrombin receptor activating peptide (TRAP, SEQ ID NO: 1), adenosine diphosphate (ADP), arachidonic acid, epinephrine, collagen, thrombin, thromboxane A2 (TxA2), thromboxane A2 (TXA2) mimetic U46619, calcium ionophore A23187, ristocetin, rhodocytin, etc. Platelet activation results in a variety of changes in platelets, such as, inter alia, changes in markers associated with platelet activation, exocytosis of dense granules and alpha granules, activation of the membrane enzyme phospholipase A2, changes in shape, aggregation, agglutination, changes in membrane potential, and changes in integrin conformation. Upon activation, platelets release granule contents including adenosine triphosphate (ATP), adenosine diphosphate (ADP), 5-hydroxytryptamine (serotonin), thrombospondin-1, fibrinogen, CXCL12, thromboxane, platelet factor 4, among other metabolites and proteins, which can be assessed by using a variety of methodologies, such as immunological assays, high performance liquid chromatography (HPLC), fluorescence microscopy, or flow cytometry. Upon activation, platelets have changes in the surface expression of various markers, including P-selectin, CD34, CD41, CD61, phosphatidylserine, among others, which can be assessed by using a variety of methodologies, such as immunological assays, high performance liquid chromatography (HPLC), fluorescence microscopy, or flow cytometry.

[0045] As used herein, "effective amount" refers to an amount of a compound sufficient to affect a desired outcome. In one embodiment, platelets are activated by administering an effective amount of one or more platelet activators described herein.

[0046] Platelet activation level can be measured using any method known in the art, such as measuring the level of markers found on or released from activated platelets in the reaction of platelets from normal donors with blood samples from subjects known to have platelet activation antibodies or HIT (e.g., positive control samples) and blood samples from normal subjects (e.g., negative control samples). Platelet activation can be measured using any marker known to be found on or released from activated platelets, such as one or more CD markers found on activated platelets (e.g., CD9, CD31, CD36, CD41, CD42, CD49b, CD61, CD62P, CD63, CD107, their isoforms, or any marker present on the outside of platelets), such as the marker CD62P, also known as p-selectin. Furthermore, platelet activation can be measured by measuring either increased immunoglobulin binding, phosphatidylserine expression, platelet aggregation, intracellular levels of ionized calcium, changes in integrin conformation, release of platelet granule contents, changes in platelet membrane potential or platelet impedance, levels of Fcγ receptor 2 cleavage fragments, or changes in platelet shape.

[0047] "Increased platelet activation" as used herein refers to an increase in platelet activation that is significantly different from the baseline platelet activation increase in blood samples of normal subjects, or is significantly different compared to known values ​​or past data obtained using the assay described herein.An increase in platelet activation indicates that the patient has platelet activation antibodies, such as antibodies that cause HIT.In one embodiment, the increase in platelet activation is at least two or three times the amount of baseline platelet activation in blood samples of normal subjects or past data obtained for normal subjects using the method described herein.

[0048] As used herein, a "sample" refers to a specimen or culture obtained from a subject. Biological samples can be obtained from a subject and include fluids, solids, tissues, and gases. In one embodiment, the subject's sample is a blood sample. Blood samples include blood products such as whole blood, plasma, serum, platelet-rich plasma, or fractionated blood components such as one of Cohn fractions I-IV, or antibody fractions.

[0049] As used herein, "instructions" refers to publications, figures, or any other medium of expression used to provide directions or steps for practicing the methods described herein. The instructions can be provided in printed form, can be affixed to a container which contains the kit materials, can be shipped with the kit, or can be provided at an internet site.

[0050] One embodiment described herein is a method for cryopreserving platelets that maintains viability and activation capacity, the method comprising: (a) obtaining blood or platelet-rich plasma or apheresis-derived platelets from a subject; (b) isolating platelets from the blood, platelet-rich plasma or apheresis platelets; (c) suspending the platelets in a suspension buffer comprising trehalose; (d) adding a stabilizer to the suspended platelets; (e) cooling the platelets to a temperature of about -80°C; and (f) storing the platelets at about -80°C. In one aspect, the method maintains viability and activation capacity of the platelets for more than two weeks. In another aspect, the suspension buffer comprises about 50 mM trehalose. In another aspect, the stabilizer is about 4% by weight bovine serum albumin (BSA). In another aspect, the platelets of step (c) are cryopreserved at about 1 x 10 6The platelets are diluted with suspension buffer to a final concentration of about 1×10 platelets / μL. In another aspect, the diluted platelets are incubated at about 37° C. for about 2 hours with periodic agitation. In another aspect, isolating the platelets comprises centrifuging the blood, platelet-rich plasma or apheresis platelets at 100×g for 15 minutes to generate a first pellet and a first supernatant; thereafter, centrifuging the first supernatant at 1000×g for 15 minutes to generate a second pellet and a second supernatant, the first supernatant being platelet-rich and the second supernatant being platelet-poor. In another aspect, the second supernatant is collected, trehalose is added to about 50 mM, and the platelets of step (c) are diluted to about 1×10 6 and using to dilute to a concentration of 100000000 platelets / μL. In another aspect, cooling the platelets comprises decreasing the temperature at a rate of about 2-20° C. per minute. Another embodiment described herein is a cryopreserved platelet prepared by the methods described herein.

[0051] Other embodiments described herein are methods for cryopreserving and recovering platelets from cryopreservation, the methods comprising: (a) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a subject; (b) isolating platelets from the blood, platelet-rich plasma, or apheresis platelets; (c) suspending the platelets in a buffer solution comprising trehalose; (d) adding a stabilizer to the suspended platelets; (e) chilling the platelets to a temperature of about -80°C; (f) storing the platelets at about -80°C for a period of time; and (g) thawing the platelets at about 20-40°C. In one aspect, the suspension buffer comprises about 50 mM trehalose. In another aspect, the stabilizer is about 4% by weight bovine serum albumin (BSA). In another aspect, the platelets of step (c) are thawed to about 1 x 10 6The platelets are diluted with suspension buffer to a final concentration of platelets / μL. In another aspect, the diluted platelets are incubated at about 37° C. for about 2 hours with periodic agitation. In another aspect, the method further comprises: (h) isolating the thawed platelets; and (i) suspending the isolated thawed platelets in an activation buffer comprising about 1% by weight BSA. In another aspect, isolating the thawed platelets comprises centrifugation at 1000×g for 15 minutes. In another aspect, the activation buffer further comprises about 50 mM trehalose. In another aspect, isolating the thawed platelets comprises centrifugation at 1000×g for 5 minutes. In another aspect, after step (h) and before step (i), the following steps are performed: (h-1) washing the thawed platelets in a washing buffer. In another aspect, isolating the thawed platelets in step (h) comprises centrifuging at 1000×g for 15 minutes and removing the supernatant; and washing the thawed platelets in step (h-1) comprises resuspending the platelets in a wash buffer, centrifuging at 1000×g for 15 minutes and removing the supernatant. In another aspect, the wash buffer further comprises about 50 mM trehalose. In another aspect, isolating the thawed platelets in step (h) comprises centrifuging at 1000×g for 5 minutes and removing the supernatant; and washing the thawed platelets in step (h-1) comprises resuspending the platelets in a wash buffer, centrifuging at 1000×g for 5 minutes and removing the supernatant. In another aspect, the period of time in step (f) is from 1 day to about 5 years. Another embodiment described herein is a collected cryopreserved platelet prepared by the method described herein.

[0052] Another embodiment described herein is a method for activating recovered platelets after cryopreservation, comprising: (a) recovering platelets after cryopreservation according to the methods described herein; (b) treating the recovered platelets with platelet factor 4 (PF4; SEQ ID NO:2); and (c) incubating the PF4-treated platelets with plasma, serum or blood sample from a subject diagnosed with heparin-induced thrombocytopenia (HIT). In one aspect, treating the recovered platelets with PF4 comprises adding PF4 to the platelets at a concentration of about 150 μg / mL and incubating at room temperature for about 20 minutes. In another aspect, platelet activation is assessed by measuring platelet granule contents released after incubation of the PF4-treated platelets with plasma, serum or blood sample from the HIT subject. In another aspect, the platelet granule contents are thrombospondin-1. Another embodiment described herein is activated recovered cryopreserved platelets prepared according to the methods described herein.

[0053] Another embodiment described herein is a method for activating collected platelets after cryopreservation, the method comprising: (a) collecting platelets after cryopreservation according to the methods described herein; and (b) treating the collected platelets with thrombin receptor activating peptide (TRAP; amino acid sequence: SFFLRN; SEQ ID NO:1). In one aspect, treating the collected platelets with TRAP comprises adding TRAP to the platelets at a concentration of about 25 μg / mL and incubating at room temperature for about 30 minutes. In another aspect, platelet activation is assessed by measuring platelet granule contents released after treating the collected platelets with TRAP. In another aspect, the platelet granule contents is thrombospondin-1. Another embodiment described herein is activated collected cryopreserved platelets prepared according to the methods described herein.

[0054] Another embodiment described herein is a method for activating recovered platelets after cryopreservation, the method comprising: (a) recovering platelets after cryopreservation according to the methods described herein; (b) treating the recovered platelets with heparin; and (c) incubating the heparin-treated platelets with a plasma, serum or blood sample from a subject diagnosed with heparin-induced thrombocytopenia (HIT). In one aspect, treating the recovered platelets with heparin comprises adding heparin to the platelets at a concentration of about 0.1-5 U / mL and incubating at room temperature for about 20 minutes. In another aspect, platelet activation is assessed by measuring platelet granule content released after incubation of the heparin-treated platelets with a plasma, serum or blood sample from the HIT subject. In another aspect, the platelet granule content is thrombospondin-1. Another embodiment described herein is activated recovered cryopreserved platelets prepared according to the methods described herein.

[0055] Other embodiments described herein are methods for cryopreserving platelets and recovering platelets from cryopreservation, the methods comprising: (a) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a subject; (b) isolating platelets from the blood, platelet-rich plasma, or apheresis platelets; (c) suspending the platelets in a buffer solution comprising trehalose; (d) adding a stabilizer to the suspended platelets; (e) cooling the platelets to a temperature of about -80°C; (f) storing the platelets at about -80°C for a period of time; (g) thawing the platelets at about 20-40°C; (h) isolating the thawed platelets; and (i) suspending the isolated thawed platelets in an activation buffer comprising about 1% by weight BSA. In one aspect, the suspension buffer comprises about 50 mM trehalose. In another aspect, the stabilizer is about 4% by weight bovine serum albumin (BSA). In another aspect, the platelets of step (c) are suspended in an activation buffer comprising about 1×10 6The platelets are diluted with suspension buffer to a final concentration of platelets / μL. In another aspect, the diluted platelets are incubated at about 37° C. for about 2 hours with periodic agitation. In another aspect, isolating the thawed platelets comprises centrifugation at 1000×g for 15 minutes. In another aspect, the activation buffer further comprises about 50 mM trehalose. In another aspect, isolating the thawed platelets comprises centrifugation at 1000×g for 5 minutes. In another aspect, after step (h) and before step (i), the following steps are performed: (h-1) washing the thawed platelets in a wash buffer. In another aspect, isolating the thawed platelets in step (h) comprises centrifugation at 1000×g for 15 minutes and removing the supernatant; and washing the thawed platelets in step (h-1) comprises resuspending the platelets in a wash buffer, centrifugation at 1000×g for 15 minutes and removing the supernatant. In another aspect, the wash buffer further comprises about 50 mM trehalose. In another aspect, isolating the thawed platelets in step (h) comprises centrifuging at 1000×g for 5 minutes and removing the supernatant; and washing the thawed platelets in step (h-1) comprises resuspending the platelets in a wash buffer, centrifuging at 1000×g for 5 minutes and removing the supernatant. In another aspect, the period of time in step (f) is from 1 day to about 5 years. Another embodiment described herein is a recovered cryopreserved platelet prepared by the methods described herein.

[0056] Another embodiment described herein is a method for activating recovered platelets after cryopreservation, comprising: (a) recovering platelets after cryopreservation according to the methods described herein; (b) treating the recovered platelets with platelet factor 4 (PF4; SEQ ID NO:2); and (c) incubating the PF4-treated platelets with plasma, serum or blood sample from a subject diagnosed with heparin-induced thrombocytopenia (HIT). In one aspect, treating the recovered platelets with PF4 comprises adding PF4 to the platelets at a concentration of about 150 μg / mL and incubating at room temperature for about 20 minutes. In another aspect, platelet activation is assessed by measuring platelet granule contents released after incubation of the PF4-treated platelets with plasma, serum or blood sample from the HIT subject. In another aspect, the platelet granule contents are thrombospondin-1. Another embodiment described herein is activated recovered cryopreserved platelets prepared according to the methods described herein.

[0057] Another embodiment described herein is a method for activating collected platelets after cryopreservation, the method comprising: (a) collecting platelets after cryopreservation according to the methods described herein; and (b) treating the collected platelets with thrombin receptor activating peptide (TRAP; amino acid sequence: SFFLRN; SEQ ID NO:1). In one aspect, treating the collected platelets with TRAP comprises adding TRAP to the platelets at a concentration of about 25 μg / mL and incubating at room temperature for about 30 minutes. In another aspect, platelet activation is assessed by measuring platelet granule contents released after treating the collected platelets with TRAP. In another aspect, the platelet granule contents are thrombospondin-1. Another embodiment described herein is activated collected cryopreserved platelets prepared according to the methods described herein.

[0058] Another embodiment described herein is a method for activating recovered platelets after cryopreservation, the method comprising: (a) recovering platelets after cryopreservation according to the methods described herein; (b) treating the recovered platelets with heparin; and (c) incubating the heparin-treated platelets with a plasma, serum or blood sample from a subject diagnosed with heparin-induced thrombocytopenia (HIT). In one aspect, treating the recovered platelets with heparin comprises adding heparin to the platelets at a concentration of about 0.1-5 U / mL and incubating at room temperature for about 20 minutes. In another aspect, platelet activation is assessed by measuring platelet granule content released after incubation of the heparin-treated platelets with a plasma, serum or blood sample from the HIT subject. In another aspect, the platelet granule content is thrombospondin-1. Another embodiment described herein is activated recovered cryopreserved platelets prepared according to the methods described herein.

[0059] Another embodiment described herein is collected cryopreserved platelets produced by the following method: (a) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a subject; (b) isolating platelets from the blood, platelet-rich plasma, or apheresis platelets; (c) suspending the platelets in a buffer solution containing trehalose; (d) adding a stabilizer to the suspended platelets; (e) cooling the platelets to a temperature of about -80°C; (f) storing the platelets at about -80°C for a period of time; and (g) thawing the platelets at about 20-40°C.

[0060] Other embodiments described herein are activated, collected, cryopreserved platelets prepared by the following method: (a) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a subject; (b) isolating platelets from the blood, platelet-rich plasma, or apheresis platelets; (c) suspending the platelets in a buffer solution containing trehalose; (d) adding a stabilizer to the suspended platelets; (e) cooling the platelets to a temperature of about -80°C; (f) storing the platelets at about -80°C for a period of time; (g) thawing the platelets at about 20-40°C; (h) isolating the thawed platelets; (i) suspending the isolated, thawed platelets in an activation buffer containing about 1% by weight BSA; (j) treating the collected platelets with heparin or PF4; and (k) incubating the heparin or PF4-treated platelets with a plasma, serum, or blood sample from a subject diagnosed with heparin-induced thrombocytopenia (HIT).

[0061] Another embodiment described herein is activated recovered cryopreserved platelets prepared by the following method: (a) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a subject; (b) isolating platelets from the blood, platelet-rich plasma, or apheresis platelets; (c) suspending the platelets in a buffer solution containing trehalose; (d) adding a stabilizer to the suspended platelets; (e) cooling the platelets to a temperature of about -80°C; (f) storing the platelets at about -80°C for a period of time; (g) thawing the platelets at about 20-40°C; (h) isolating the thawed platelets; (i) suspending the isolated thawed platelets in an activation buffer containing about 1% by weight BSA; and (j) treating the recovered platelets with thrombin receptor activating peptide (TRAP; amino acid sequence: SFFLRN; SEQ ID NO:1).

[0062] Other embodiments described herein are kits for detecting heparin-induced thrombocytopenia (HIT) antibodies in blood, plasma, or serum, the kits comprising two or more of the following: (a) cryopreserved platelets; (b) one or more of platelet factor 4 (PF4; SEQ ID NO:2) or heparin; (c) one or more reagents for detecting platelet activation; (d) optionally, a buffer and container; and (e) optionally, one or more of packaging or instructions for use. In one aspect, the one or more reagents for detecting platelet activation comprise a reagent for detecting thrombospondin-1. In another aspect, the kit further comprises one or more negative control samples negative for pathogenic HIT antibodies that activate platelets; and one or more positive control samples positive for pathogenic HIT antibodies that activate platelets.

[0063] Other embodiments described herein are methods for treating or preventing bleeding in a subject in need thereof, the method comprising: (a) obtaining cryopreserved platelets; (b) thawing the cryopreserved platelets; and (c) contacting the subject with the thawed cryopreserved platelets. In one aspect, steps (a)-(b) comprise: (i) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a second subject; (ii) isolating platelets from the blood, platelet-rich plasma, or apheresis platelets; (iii) suspending the platelets in a buffer solution comprising trehalose; (iv) incubating the trehalose-treated platelets at 37° C. for about 2 hours; (v) adding a stabilizer to the suspended platelets; (vi) cooling the platelets to a temperature of about −80° C.; (vii) storing the platelets at about −80° C. for a period of time; and (viii) thawing the platelets at about 20-40° C. In another aspect, steps (a)-(b) comprise: (i) obtaining platelet-rich plasma or apheresis-derived platelets from the second subject; (ii) adding trehalose to the platelet-rich plasma or apheresis-derived platelets; (iii) incubating the trehalose-treated platelets at 37° C. for about 2 hours; (iv) cooling the platelets to a temperature of about −80° C.; (v) storing the platelets at about −80° C. for a period of time; and (vi) thawing the platelets at about 20-40° C. In another aspect, the contacting in step (c) comprises applying thawed cryopreserved platelets to the site of injury or parenterally administering thawed cryopreserved platelets to the subject.

[0064] Another aspect described herein is the use of the cryopreserved platelets as a reagent, research tool, or medicine to treat a subject in need thereof. Another aspect described herein is the use of thawed cryopreserved platelets as a reagent, research tool, or medicine to treat a subject in need thereof.

[0065] Another aspect described herein is the use of the activated thawed cryopreserved platelets as a reagent, research tool, or medicine to treat a subject in need thereof. It will be apparent to those skilled in the art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and uses described herein can be made without departing from the scope of any embodiment or aspect thereof. The compositions and methods provided are representative and are not intended to limit the scope of any of the particular embodiments. All of the various aspects, aspects, and options disclosed herein can be combined in any variation or iteration. The scope of the compositions, formulations, methods, and processes described herein encompasses all actual or potential combinations of the aspects, aspects, options, examples, and preferences described herein. The representative compositions and formulations described herein can exclude any component, can be substituted with any component disclosed herein, or can include any component disclosed elsewhere herein. The ratio of the mass of any component of any composition or formulation disclosed herein to the mass of any other component in the formulation or the total mass of other components in the formulation is disclosed herein as if they were expressly disclosed. If the meaning of any term in any of the patents or publications incorporated by reference conflicts with the meaning of the term used in this disclosure, the meaning of the term or phrase in this disclosure shall control. Moreover, the foregoing discussion discloses and describes merely exemplary embodiments, and all patents and publications cited herein are hereby incorporated by reference for their specific teachings.

[0066] Various aspects and aspects of the invention described herein are summarized by the following clauses: Clause 1. A method for cryopreserving platelets that maintains viability and activatability, comprising: (a) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a subject; (b) isolating platelets from blood, platelet-rich plasma, or apheresis platelets; (c) suspending the platelets in a suspension buffer containing trehalose; (d) adding a stabilizer to the suspended platelets; (e) cooling the platelets to a temperature of about −80° C.; and (f) storing platelets at approximately −80°C; The method comprising: Clause 2. The method of clause 1, wherein said method maintains platelet viability and activatability for more than two weeks. Clause 3. The method of clause 1 or 2, wherein the suspension buffer comprises about 50 mM trehalose. Clause 4. The method of any one of clauses 1 to 3, wherein the stabilizer is about 4% by weight bovine serum albumin (BSA). The platelets of step (c) of clause 5 are about 1 x 10 6 5. The method of any one of clauses 1-4, wherein the suspension buffer is diluted to a final concentration of platelets / µL. Clause 6. The method of any one of clauses 1-5, wherein the diluted platelets are incubated at about 37° C. for about 2 hours with regular agitation. Clause 7. The method of any one of clauses 1-6, wherein isolating platelets comprises centrifuging blood, platelet-rich plasma or apheresis platelets at 100×g for 15 minutes to produce a first pellet and a first supernatant; and then centrifuging the first supernatant at 1000×g for 15 minutes to produce a second pellet and a second supernatant, wherein the first supernatant is platelet-rich and the second supernatant is platelet-poor. Clause 8. Collect the second supernatant, add trehalose to about 50 mM, and resuspend the platelets of step (c) in a solution of about 1×10 6 The method according to any one of clauses 1 to 7, wherein the platelet concentration is diluted to a concentration of 10 ... Clause 9. The method of any one of clauses 1 to 8, wherein cooling the platelets comprises decreasing the temperature at a rate of about 2 to 20° C. per minute. Clause 10. Cryopreserved platelets prepared by the method of any one of clauses 1 to 9. Clause 11. A method for cryopreserving and recovering platelets from cryopreservation, comprising: (a) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a subject; (b) isolating platelets from blood, platelet-rich plasma, or apheresis platelets; (c) suspending the platelets in a buffer solution containing trehalose; (d) adding a stabilizer to the suspended platelets; (e) cooling the platelets to a temperature of about −80° C.; (f) storing the platelets at about −80° C. for a period of time; and (g) thawing the platelets at approximately 20-40°C; The method comprising: Clause 12. The method of clause 11, wherein the suspension buffer comprises about 50 mM trehalose. Clause 13. The method of clause 11 or 12, wherein the stabilizer is about 4% by weight bovine serum albumin (BSA). Clause 14. The platelets of step (c) are about 1×10 6 14. The method of any one of clauses 11-13, wherein the suspension is diluted with suspension buffer to a final concentration of platelets / µL. Clause 15. The method of any one of clauses 11-14, wherein the diluted platelets are incubated at about 37° C. for about 2 hours with regular agitation. Article 16. Furthermore: (h) isolating the thawed platelets; and (i) suspending isolated, thawed platelets in an activation buffer containing about 1% by weight BSA; 16. The method according to any one of clauses 11 to 15, comprising: Clause 17. Any one of clauses 11 to 16, wherein isolation of the thawed platelets comprises centrifugation at 1000 x g for 15 minutes. Clause 18. The method of any one of clauses 11-17, wherein the activation buffer further comprises about 50 mM trehalose. Clause 19. The method of any one of clauses 11 to 18, wherein isolation of the thawed platelets comprises centrifugation at 1000 x g for 5 minutes. Clause 20. The method according to any one of clauses 11 to 19, wherein after step (h) and before step (i), the following step is carried out: (h-1) A step of washing the thawed platelets in a washing buffer. Clause 21. The method of clause 20, wherein isolating the thawed platelets in step (h) comprises centrifuging at 1000 x g for 15 minutes and removing the supernatant; and washing the thawed platelets in step (h-1) comprises resuspending the platelets in a wash buffer, centrifuging at 1000 x g for 15 minutes and removing the supernatant. Clause 22. The method of any one of clauses 11-21, wherein the wash buffer further comprises about 50 mM trehalose. Clause 23. The method of any one of clauses 11 to 22, wherein isolating the thawed platelets in step (h) comprises centrifuging at 1000×g for 5 minutes and removing the supernatant; and washing the thawed platelets in step (h-1) comprises resuspending the platelets in a washing buffer, centrifuging at 1000×g for 5 minutes and removing the supernatant. Clause 24. The method according to any one of clauses 11 to 24, wherein the fixed period of time in step (f) is from 1 day to about 5 years. Clause 25. A collected cryopreserved platelet prepared by the method of any one of clauses 11 to 24. Article 26. A method for activating platelets recovered after cryopreservation, comprising: (a) recovering platelets after cryopreservation by the method of any one of clauses 11 to 24; (b) treating the collected platelets with platelet factor 4 (PF4; SEQ ID NO:2); and (c) incubating the PF4-treated platelets with a plasma, serum or blood sample from a subject diagnosed with heparin-induced thrombocytopenia (HIT); The method comprising: Clause 27. The method of clause 26, wherein treating the collected platelets with PF4 comprises adding PF4 to the platelets at a concentration of about 150 μg / mL and incubating at room temperature for about 20 minutes. Clause 28. The method according to clause 26 or 27, wherein platelet activation is assessed by measuring platelet granule contents released after incubation of PF4-treated platelets with plasma, serum or blood samples from HIT subjects. Clause 29. The method of any one of clauses 26 to 28, wherein the platelet granule content is thrombospondin-1. Clause 30. Activated harvested cryopreserved platelets prepared by the method of any one of clauses 26 to 29. Article 31. A method for activating platelets recovered after cryopreservation, comprising: (a) recovering platelets after cryopreservation by the method of any one of clauses 11 to 24; and (b) treating the collected platelets with thrombin receptor-activating peptide (TRAP; amino acid sequence: SFFLRN; SEQ ID NO: 1); The method comprising: Clause 32. The method of clause 31, wherein treating the collected platelets with TRAP comprises adding TRAP to the platelets at a concentration of about 25 μg / mL and incubating at room temperature for about 30 minutes. Clause 33. The method of clause 31 or 32, wherein platelet activation is assessed by measuring platelet granule contents released after treatment of harvested platelets with TRAP. Clause 34. The method of any one of clauses 31 to 33, wherein the platelet granule content is thrombospondin-1. Clause 35. Activated harvested cryopreserved platelets prepared by the method of any one of clauses 31 to 34. Article 36. A method for activating platelets recovered after cryopreservation, comprising: (a) recovering platelets after cryopreservation by the method of any one of clauses 11 to 24; (b) treating the collected platelets with heparin; and (c) incubating the heparin-treated platelets with a plasma, serum or blood sample from a subject diagnosed with heparin-induced thrombocytopenia (HIT); The method comprising: Clause 37. The method of clause 36, wherein treating the collected platelets with heparin comprises adding heparin to the platelets at a concentration of about 0.1 to 5 U / mL and incubating at room temperature for about 20 minutes. Clause 38. The method of clause 36 or 37, wherein platelet activation is assessed by measuring platelet granule contents released after incubation of heparin-treated platelets with plasma, serum or blood samples from HIT subjects. Clause 39. The method of any one of clauses 36 to 38, wherein the platelet granule content is thrombospondin-1. Clause 40. Activated harvested cryopreserved platelets prepared by the method according to any one of clauses 36 to 39. Clause 41. A method for cryopreserving platelets and recovering platelets from cryopreservation, comprising: (a) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a subject; (b) isolating platelets from blood, platelet-rich plasma, or apheresis platelets; (c) suspending the platelets in a buffer solution containing trehalose; (d) adding a stabilizer to the suspended platelets; (e) cooling the platelets to a temperature of about −80° C.; (f) storing the platelets at about −80° C. for a period of time; (g) thawing the platelets at approximately 20-40°C; (h) isolating the thawed platelets; and (i) suspending isolated, thawed platelets in an activation buffer containing about 1% by weight BSA; The method comprising: Clause 42. The method of clause 41, wherein the suspension buffer comprises about 50 mM trehalose. Clause 43. The method of clause 41 or 42, wherein the stabilizer is about 4% by weight bovine serum albumin (BSA). Clause 44. The platelets of step (c) are about 1 × 10 6 44. The method of any one of clauses 41 to 43, wherein the suspension is diluted with suspension buffer to a final concentration of platelets / µL. Clause 45. The method of any one of clauses 41-44, wherein the diluted platelets are incubated at about 37° C. for about 2 hours with periodic agitation. Clause 46. The method of any one of clauses 41 to 45, wherein isolation of the thawed platelets comprises centrifugation at 1000 x g for 15 minutes. Clause 47. The method of any one of clauses 41-46, wherein the activation buffer further comprises about 50 mM trehalose. Clause 48. The method of any one of clauses 41 to 47, wherein isolation of the thawed platelets comprises centrifugation at 1000 x g for 5 minutes. Clause 49. The method according to any one of clauses 41 to 48, wherein after step (h) and before step (i), the following step is carried out: (h-1) A step of washing the thawed platelets in a washing buffer. Clause 50. The method of any one of clauses 41 to 49, wherein isolating the thawed platelets in step (h) comprises centrifuging at 1000×g for 15 minutes and removing the supernatant; and washing the thawed platelets in step (h-1) comprises resuspending the platelets in a washing buffer, centrifuging at 1000×g for 15 minutes and removing the supernatant. Clause 51. The method of any one of clauses 41 to 50, wherein the wash buffer further comprises about 50 mM trehalose. Clause 52. The method of any one of clauses 41 to 51, wherein isolating the thawed platelets in step (h) comprises centrifuging at 1000×g for 5 minutes and removing the supernatant; and washing the thawed platelets in step (h-1) comprises resuspending the platelets in a washing buffer, centrifuging at 1000×g for 5 minutes and removing the supernatant. Clause 53. The method according to any one of clauses 41 to 52, wherein the fixed period of time in step (f) is from 1 day to about 5 years. Clause 54. Recovered cryopreserved platelets prepared by the method of any one of clauses 41 to 53. Article 55. A method for activating platelets recovered after cryopreservation, comprising: (a) recovering platelets after cryopreservation by the method of any one of clauses 41 to 53; (b) treating the collected platelets with platelet factor 4 (PF4; SEQ ID NO:2); and (c) incubating the PF4-treated platelets with a plasma, serum or blood sample from a subject diagnosed with heparin-induced thrombocytopenia (HIT); The method comprising: Clause 56. The method of clause 55, wherein treating the collected platelets with PF4 comprises adding PF4 to the platelets at a concentration of about 150 μg / mL and incubating at room temperature for about 20 minutes. Clause 57. The method of clause 55 or 56, wherein platelet activation is assessed by measuring platelet granule contents released following incubation of PF4-treated platelets with plasma, serum or blood samples from HIT subjects. Clause 58. The method of any one of clauses 55 to 57, wherein the platelet granule content is thrombospondin-1. Clause 59. Activated harvested cryopreserved platelets prepared by the method of any one of clauses 55 to 58. Article 60. A method for activating platelets recovered after cryopreservation, comprising: (a) recovering platelets after cryopreservation by the method of any one of clauses 41 to 53; and (b) treating the collected platelets with thrombin receptor-activating peptide (TRAP; amino acid sequence: SFFLRN; SEQ ID NO: 1); The method comprising: Clause 61. The method of clause 60, wherein treating the collected platelets with TRAP comprises adding TRAP to the platelets at a concentration of about 25 μg / mL and incubating at room temperature for about 30 minutes. Clause 62. The method of clause 60 or 61, wherein platelet activation is assessed by measuring platelet granule contents released after treatment of harvested platelets with TRAP. Clause 63. The method of any one of clauses 60 to 62, wherein the platelet granule content is thrombospondin-1. Clause 64. Activated harvested cryopreserved platelets prepared by the method of any one of clauses 60 to 62. Article 65. A method for activating platelets recovered after cryopreservation, comprising: (a) recovering platelets after cryopreservation by the method of any one of clauses 41 to 53; (b) treating the collected platelets with heparin; and (c) incubating the heparin-treated platelets with a plasma, serum or blood sample from a subject diagnosed with heparin-induced thrombocytopenia (HIT); The method comprising: Clause 66. The method of clause 65, wherein treating the collected platelets with heparin comprises adding heparin to the platelets at a concentration of about 0.1 to 5 U / mL and incubating at room temperature for about 20 minutes. Clause 67. The method of clause 65 or 66, wherein platelet activation is assessed by measuring platelet granule contents released after incubation of heparin-treated platelets with plasma, serum or blood samples from HIT subjects. Clause 68. The method of any one of clauses 65 to 67, wherein the platelet granule content is thrombospondin-1. Clause 69. Activated harvested cryopreserved platelets prepared by the method of any one of clauses 65 to 68. Article 70. The following methods: (a) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a subject; (b) isolating platelets from blood, platelet-rich plasma, or apheresis platelets; (c) suspending the platelets in a buffer solution containing trehalose; (d) adding a stabilizer to the suspended platelets; (e) cooling the platelets to a temperature of about −80° C.; (f) storing the platelets at about −80° C. for a period of time; and (g) thawing the platelets at approximately 20-40°C; Recovered cryopreserved platelets produced by. Article 71. The following methods: (a) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a subject; (b) isolating platelets from blood, platelet-rich plasma, or apheresis platelets; (c) suspending the platelets in a buffer solution containing trehalose; (d) adding a stabilizer to the suspended platelets; (e) cooling the platelets to a temperature of about −80° C.; (f) storing the platelets at about −80° C. for a period of time; (g) thawing the platelets at approximately 20-40°C; (h) isolating the thawed platelets; (i) suspending isolated, thawed platelets in an activation buffer containing about 1% by weight BSA; (j) treating the collected platelets with heparin or PF4; and (k) incubating the heparin or PF4 treated platelets with a plasma, serum or blood sample from a subject diagnosed with heparin-induced thrombocytopenia (HIT); Activated harvested cryopreserved platelets prepared by. Article 72. The following methods: (a) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a subject; (b) isolating platelets from blood, platelet-rich plasma, or apheresis platelets; (c) suspending the platelets in a buffer solution containing trehalose; (d) adding a stabilizer to the suspended platelets; (e) cooling the platelets to a temperature of about −80° C.; (f) storing the platelets at about −80° C. for a period of time; (g) thawing the platelets at approximately 20-40°C; (h) isolating the thawed platelets; (i) suspending the isolated, thawed platelets in an activation buffer containing about 1% by weight BSA; and (j) treating the collected platelets with thrombin receptor activating peptide (TRAP; amino acid sequence: SFFLRN; SEQ ID NO: 1); Activated harvested cryopreserved platelets prepared by. Clause 73. A kit for detecting heparin-induced thrombocytopenia (HIT) antibodies in blood, plasma or serum, comprising: (a) Cryopreserved platelets; (b) one or more of platelet factor 4 (PF4; SEQ ID NO:2) or heparin; (c) one or more reagents for detecting platelet activation; (d) optionally, buffers and containers; and (e) Optionally, one or more of packaging or instructions for use; The kit further comprises two or more of the following: Clause 74. The kit of clause 73, wherein the one or more reagents for detecting platelet activation include a reagent for detecting thrombospondin-1. Clause 75. The kit of clause 73 or 74, wherein the kit further comprises one or more negative control samples that are negative for pathogenic HIT antibodies that activate platelets; and one or more positive control samples that are positive for pathogenic HIT antibodies that activate platelets. Article 76. A method for treating or preventing bleeding in a subject in need thereof, comprising: (a) Obtaining frozen platelets; (b) thawing frozen platelets; (c) contacting the subject with thawed cryopreserved platelets; The method comprising: Clause 77. Steps (a) and (b) (i) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a second subject; (ii) isolating platelets from blood, platelet-rich plasma, or apheresis platelets; (iii) suspending the platelets in a buffer solution containing trehalose; (iv) incubating the trehalose-treated platelets at 37° C. for approximately 2 hours; (v) adding a stabilizer to the suspended platelets; (vi) cooling the platelets to a temperature of about −80° C.; (vii) storing the platelets at about −80° C. for a period of time; and (viii) thawing the platelets at approximately 20-40°C; 77. The method of claim 76, comprising: Clause 78. Steps (a) and (b) (i) obtaining platelet-rich plasma or apheresis-derived platelets from a second subject; (ii) adding trehalose to platelet-rich plasma or apheresis-derived platelets; (iii) incubating the trehalose-treated platelets at 37° C. for approximately 2 hours; (iv) cooling the platelets to a temperature of about −80° C.; (v) storing the platelets at about −80° C. for a period of time; and (vi) thawing the platelets at approximately 20-40°C; 77. The method of claim 76, comprising: Clause 79. The method of any one of clauses 76 to 78, wherein the contacting in step (c) comprises applying thawed cryopreserved platelets to the site of injury or parenterally administering thawed cryopreserved platelets to the subject. Article 80. Use of cryopreserved platelets as a reagent, research tool, or medicine to treat a subject in need thereof. Article 81. Use of thawed cryopreserved platelets as a reagent, research tool, or medicine to treat a subject in need thereof. Article 82. Use of activated thawed cryopreserved platelets as a reagent, research tool, or medicine to treat a subject in need thereof. EXAMPLES

[0067] Example 1 Cryopreservation of platelets in buffer solution Platelet-rich plasma was anticoagulated with citrate phosphate dextrose anticoagulant (CPD, 15.6 mM citric acid, 89 mM sodium citrate dihydrate, 16 mM NaH 2 PO 4 H 2Platelets were collected (45 mL) in cryopreservation buffer (117 mM sodium citrate, 136 mM dextrose, pH 5.9; final dilution 1:10) and 5 mL of acidic citrate dextrose buffer (117 mM sodium citrate, 136 mM dextrose, pH 5.9; final dilution 1:10) was added along with prostaglandin-E1 (final concentration 50 ng / mL). The combination was centrifuged at 100 × g for 15 min. The supernatant (40 mL) was collected and centrifuged at 1000 × g for 15 min, and the supernatant was removed and discarded. Platelets were stored in cryopreservation buffer (9.5 mM HEPES; 100 mM NaCl; 4.8 mM KCl; 5.0 mM glucose; 12 mM NaHCO 3 Platelets were counted and resuspended at 1 × 10 in cryopreservation buffer (50 mM trehalose, pH 6.8). 6 Platelets were incubated at 37° C. for 2 hours with inversion every 30 minutes. After the 2 hour incubation, bovine serum albumin (BSA) was added dropwise to a final concentration of 4% by weight. 1 mL aliquots were dispensed into cryovials and cooled at a rate of 4° C. / min to a final temperature of −80° C. Vials were transferred to a −80° C. freezer for long term storage. See FIG. 1.

[0068] Platelets were obtained from 20 different whole blood donors and cryopreserved in buffer as described. Cryopreserved platelets were stored at -80°C until use and harvested by washing. After harvesting, platelets were treated with platelet factor 4 (PF4) and added to one of three blood samples from patients with confirmed heparin-induced thrombocytopenia (HIT) or one of three normal control sera. After treatment of the PF4-treated platelets with the patient / normal samples, the supernatants were collected and assayed for thrombospondin-1 concentrations using a thrombospondin-1 (TSP1) ELISA kit. As shown in Figure 2, the three HIT serum samples and the three normal serum test samples were clearly distinguishable for each of the 20 different platelet lots tested.

[0069] Of the 20 different cryopreserved platelet lots shown in Figure 2, seven were tested after storage at -80°C for 4 weeks or more. Cryopreserved platelets were harvested by washing, treated with PF4, and activated with HIT or normal blood samples. As shown in Figure 3, all platelet lots tested after 4 weeks of storage clearly differentiated between HIT and normal test samples.

[0070] Platelets from one of the cryopreserved lots shown in Figure 2 were tested after 14 weeks of storage at -80°C. The cryopreserved platelets were harvested by washing, treated with PF4, and activated in HIT or normal serum samples. As shown in Figure 4, after 14 weeks of storage, the cryopreserved platelets retain the ability to be activated.

[0071] Example 2 Recovery of cryopreserved platelets by washing A 1 mL aliquot of cryopreserved platelets was thawed in a 37° C. water bath for 3 min. The platelets were then centrifuged at 1000×g for 15 min and the supernatant was removed and discarded. The platelets were washed with 1 mL of platelet wash buffer (108 mM NaCl, 3.8 mM KCl, 1.7 mM NaHCO 3 Platelets were resuspended in 175 μL of phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM NaCl, 22.9 mM sodium citrate, 27.8 mM glucose, pH 6.5) containing 1% BSA. The platelets were centrifuged at 1000×g for 15 min and the supernatant was removed and discarded. The platelets were resuspended in 175 μL of phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM NaCl, 22.9 mM sodium citrate, 27.8 mM glucose, pH 6.5) containing 1% BSA. 2 HPO 4 , 1.47 mM KH 2 PO 4 The cells were then resuspended in 100 mM NaCl (pH 7.4) (see Figure 1).

[0072] Platelet factor 4 (PF4) treatment and activation of collected cryopreserved platelets using blood samples from patients with heparin-induced thrombocytopenia (HIT) PF4 (SEQ ID NO:2) was added to collected, washed, cryopreserved platelets at a concentration of 150 μg / mL and incubated for 20 min at room temperature. An 80 μL aliquot of PF4-treated platelets was added to 20 μL of HIT patient serum sample or normal serum sample and incubated for 30 min at room temperature. The platelet reaction mixture was centrifuged at 1000×g for 15 min and 40 μL of supernatant was collected for evaluation of thrombospondin-1 (a platelet α-granule component) concentration as a marker of platelet activation.

[0073] Measurement of thrombospondin-1 A commercially available thrombospondin-1 (TSP1) ELISA kit (R&D Systems) was used to quantitate TSP1 in platelet supernatants as a measure of platelet activation. Briefly, ELISA plates were coated with 1 μg / mL of anti-TSP1 capture antibody for at least 8 hours at 4°C. ELISA plates were washed three times with 400 μL of ELISA plate wash buffer and blocked with ELISA reagent diluent. Supernatants from platelet reactions were diluted 1:1000 or 1:2000. Standards were prepared according to the manufacturer's instructions and incubated on the ELISA plates. ELISA plates were washed three times with 400 μL of ELISA plate wash buffer and incubated with 100 ng / mL of biotin-labeled detection antibody. ELISA plates were washed again three times with 400 μL of ELISA plate wash buffer and incubated with 1:200 horseradish peroxidase-labeled streptavidin. The ELISA plate was washed again three times with 400 μL of ELISA plate wash buffer and incubated with tetramethylbenzidine for 10 min. 2 SO 4 The reaction was terminated at 4 °C and the optical density at 450 nm was measured. TSP1 concentrations were determined using a four-parameter logistic regression on the standard curve.

[0074] Recovery of cryopreserved platelets by volume reduction A 1 mL aliquot of cryopreserved platelets was thawed in a 37° C. water bath for 3 minutes. The platelets were centrifuged at 1000×g for 15 minutes and the supernatant was removed and discarded. The platelets were resuspended in 175 μL of PBS containing 1% BSA by weight, pH 7.4.

[0075] Recovery of frozen platelets by thawing A 1 mL aliquot of cryopreserved platelets was thawed for 3 minutes in a water bath at 37° C. The platelets were used without further processing.

[0076] Activation of harvested cryopreserved platelets with thrombin receptor-activating peptide(TRAP). An 80 μL aliquot of collected cryopreserved platelets was added to 20 μL of 125 μg / mL TRAP peptide (TRAP final concentration: 25 μg / mL; amino acid sequence: SFFLRN; SEQ ID NO:1) or 20 μL of PBS pH 7.4 containing 1% BSA (control) and incubated at room temperature for 30 min. The platelet reaction mixture was centrifuged at 1000×g for 15 min and 40 μL of supernatant was collected for evaluation of thrombospondin-1 concentration as a marker of platelet activation.

[0077] The recovery method of cryopreserved platelets was also investigated. Platelets from a single donor were cryopreserved in buffer. Cryopreserved platelets were washed, reduced in volume, or used directly upon thawing. After recovery, platelets were treated with PF4 and activated by HIT or normal serum samples. Additionally, recovered platelets were treated with thrombin receptor activating peptide (TRAP), a potent platelet agonist, or buffer as a control. Both washed or volume-reduced human sample (Figure 5) and TRAP (Figure 6) cryopreserved platelets have a higher degree of activation potential than only thawed cryopreserved platelets.

[0078] Example 3 Heparinization and activation of cryopreserved platelets using blood samples from patients with heparin-induced thrombocytopenia (HIT) Unfractionated heparin was added to the collected washed cryopreserved platelets at concentrations of 0.1, 0.3, 0.5, 1 U / mL or 0 U / mL as a control, and the mixture was incubated at room temperature for 20 min. An 80 μL aliquot of heparin-treated collected cryopreserved platelets was added to 20 μL of HIT or normal patient serum samples and incubated at room temperature for 30 min. The platelet reaction mixture was centrifuged at 1000 × g for 15 min, and 40 μL of the supernatant was collected for evaluation of thrombospondin-1 concentration as a marker of platelet activation.

[0079] FIG. 7 shows that heparin-treated cryopreserved platelets can be used to distinguish between HIT and normal blood samples, whereby HIT samples activate platelets but normal samples do not.

[0080] Example 4 Cryopreservation of platelets in the presence of platelet-poor plasma Platelet-rich plasma (45 mL) was collected in citrate phosphate dextrose anticoagulant (CPD) and 5 mL of acidic citrate dextrose buffer was added along with prostaglandin-E1 (final concentration 50 ng / mL). The platelet-rich plasma mixture was centrifuged at 100 × g for 15 min. The supernatant (40 mL) was collected and centrifuged at 1000 × g for 15 min. The supernatant (platelet-poor plasma, PPP) was removed and used as described below. Platelets were frozen in 1 mL of cryopreservation buffer (9.5 mM HEPES; 100 mM NaCl; 4.8 mM KCl; 5.0 mM glucose; 12 mM NaHCO 3 50 mM trehalose, pH 6.8). Trehalose (200 μL of 1 M trehalose) was added to 4 mL of PPP to make PPP containing 50 mM trehalose. Platelets were counted and resuspended at 1 × 10 in PPP containing 50 mM trehalose. 6 Platelets were diluted to a final concentration of 1000 platelets / μL. Platelets were incubated at 37° C. for 2 hours with inversion every 30 minutes. After the 2 hour incubation, 1 mL aliquots of platelets were dispensed into cryovials and cooled at a rate of 4° C. / min to a final temperature of −80° C. Vials were transferred to a −80° C. freezer for long-term storage.

[0081] The platelets cryopreserved in PPP were then collected by washing. After collection, the platelets were treated with PF4 and activated by HIT or normal serum. The collected platelets were further treated with TRAP or buffer as a control. Similar to platelets cryopreserved in buffer, platelets cryopreserved in PPP have the ability to be activated by HIT antibody (Figure 8) or TRAP (Figure 9).

[0082] Example 5 Freeze-drying of cryopreserved platelets Platelets were frozen and stored in buffer as described above in Example 1, and the vials were transferred to a freeze dryer equilibrated to -40°C at atmospheric pressure for 4 hours. Vacuum (100 mTorr) was applied at -30°C for 13 hours. The temperature was then gradually increased from -30°C to +30°C over 12 hours while a vacuum of 100 mTorr was applied. The vials were then transferred to a -80°C freezer.

[0083] Reconstitution, PF4 treatment and processing of freeze-dried platelets in HIT and normal blood samples 1 mL aliquot of H 2 O was added to each freeze-dried platelet aliquot. PF4 was added to the reconstituted freeze-dried platelets at a concentration of 150 μg / mL and incubated at room temperature for 20 min. An 80 μL aliquot of PF4-treated cryopreserved platelets was added to 20 μL of HIT or normal serum and incubated at room temperature for 30 min. The platelet reaction mixture was centrifuged at 1000 × g for 15 min and 40 μL of supernatant was collected for evaluation of thrombospondin-1 concentration as a marker of platelet activation.

[0084] Reconstitution and activation of freeze-dried platelets with TRAP Freeze-dried platelets were placed in 1 mL of HO. 2Platelets were reconstituted in 0. An 80 μL aliquot of reconstituted platelets was added to 20 μL of 125 μg / mL TRAP peptide (TRAP final concentration: 25 μg / mL; amino acid sequence: SFFLRN; SEQ ID NO:1) or 20 μL of PBS, pH 7.4, containing 1% BSA by weight (control) and incubated at room temperature for 30 min. The platelet reaction mixture was centrifuged at 1000 × g for 15 min and 40 μL of supernatant was collected for evaluation of thrombospondin-1 concentration as a marker of platelet activation.

[0085] Freeze-drying is another technique commonly used to cryopreserve cells. Platelets from a single donor were cryopreserved in buffer and a subset of aliquots were subjected to freeze-drying. Frozen platelets were harvested by washing and freeze-dried platelets were subjected to HO. 2 After collection, freeze-dried or frozen platelets were treated with PF4 and activated by HIT or normal serum samples. Additionally, platelets were treated with TRAP or buffer as a control. Unlike frozen platelets, freeze-dried platelets do not have the ability to be activated by HIT samples (Figure 10) or TRAP (Figure 11).

[0086] Example 6 Modified recovery, platelet factor 4 (PF4) treatment and activation of cryopreserved platelets in blood samples from patients with heparin-induced thrombocytopenia (HIT) The modified recovery refers to (1) different centrifugation conditions (5 min vs. 15 min as above) and (2) the use of trehalose in the resuspension buffers (wash buffer and activation buffer) versus the use of trehalose only in the cryopreservation buffer used to freeze the platelets.

[0087] Platelets were diluted to 1 x 10 in a volume of 500 μL. 6Platelets were stored frozen at a concentration of 1000 x g / μL and thawed in a 37°C water bath for 3 min. Platelets were centrifuged at 1000 x g for 5 min, and the supernatant was removed and discarded. Platelets were resuspended with 500 μL of platelet wash buffer containing 50 mM trehalose. Platelets were centrifuged at 1000 x g for 5 min, and the supernatant was removed and discarded. Platelets were resuspended with 80 μL of PBS pH 7.4 containing 1 wt% BSA, 50 mM trehalose, and 150 μg / mL PF4 (SEQ ID NO: 2), and the mixture was incubated at room temperature for 20 min. A 20 μL aliquot of HIT patient serum sample or normal patient serum sample was added, and the mixture was incubated at room temperature for an additional 30 min. Platelet reaction mixture was centrifuged at 1000 x g for 5 min, and 40 μL of supernatant was collected for evaluation of thrombospondin-1 (a platelet alpha granule component) concentration as a marker of platelet activation.

[0088] The modified activation procedure was also used to enhance the signal (HIT) to background (normal) ratio obtained with frozen platelets. Briefly, platelets were cryopreserved in the presence of 50 mM trehalose, then harvested by washing with RCD containing 50 mM trehalose, and resuspended in PBS containing 1% by weight BSA, 50 mM trehalose, and 150 μg / mL PF4 (SEQ ID NO: 2). In addition, the centrifugation time of all centrifugation steps after thawing was changed from 15 minutes to 5 minutes. Figure 12 compares the standard activation procedure (left) with this modified procedure (right). The signal to background ratio is 4.4 times higher for the standard procedure and 7.2 times higher for the modified procedure.

[0089] In addition to demonstrating the utility of frozen platelets in detecting samples with platelet-activating HIT antibodies, FIG. 13 presents a model in which these platelets can be used for antiplatelet drug discovery.

[0090] Example 7 Therapeutic Uses of Cryopreserved Platelets Platelet transfusions are life-saving in the context of prevention and treatment of bleeding in a variety of conditions, including but not limited to trauma, cancer, and autoimmune disorders. Donor platelets intended for transfusion are typically stored at room temperature and have a shelf life of 5-7 days. At 4°C, they can be stored for slightly longer periods (2 weeks). This limitation to platelet shelf life based on current technology poses logistical challenges in treating bleeding patients, or thrombocytopenic patients at risk of bleeding, especially in resource-poor environments such as rural areas, conflict zones, and developing countries, as well as during periods of blood shortages such as those frequently found after natural disasters and severe weather. Freeze-dried platelets, which have a long shelf life (months to years), have been proposed as an alternative, but these platelet preparations are suboptimal because they do not degranulate upon stimulation while already in an activated state, exposing surface phospholipids on top of which thromboses can form. Platelet granules contain important molecules that can further activate platelets, such as ADP, as well as other components that regulate the coagulation cascade. Therefore, there is a critical need to develop a platelet product that not only has a long shelf life but also has the ability to degranulate upon activation, similar to fresh platelets.

[0091] Platelets having a long shelf life and capable of degranulation and intended for transfusion are prepared similarly to the diagnostic platelet preparations described in this application, with some modifications as described below.

[0092] Source of platelets and isolation of platelets There are several methods for collecting and isolating platelets for cryopreservation and therapeutic use: platelets are collected by apheresis or a platelet-rich plasma component is produced by centrifugation techniques from a standard whole blood collection.

[0093] Preparation of platelets for cryopreservation Trehalose is added to the platelet product to achieve a concentration of about 50 mM. This can be accomplished in several ways. For example, platelets can be collected directly into a blood bag containing trehalose; this can be done with apheresis platelets or with platelet-rich plasma isolated by centrifugation from a whole blood unit. Another way to introduce trehalose to platelets is by adding a concentrated trehalose solution to isolated platelets obtained by apheresis or by generating platelet-rich plasma from whole blood. Alternatively, collected platelets can be pelleted by centrifugation of apheresis platelets or platelets of platelet-rich plasma, and the pellet resuspended in a trehalose-containing buffer, similar to the procedure described herein for diagnostic cryopreserved platelet products. After addition of trehalose, the platelets are incubated at about 37° C. for about 2 hours.

[0094] If the trehalose-treated platelets are in platelet-rich plasma or apheresis-derived plasma (which already contains human albumin), no further addition of human albumin is necessary. If the platelets are pelleted and resuspended in platelet-poor plasma (which already contains human albumin), no further addition of human albumin is necessary. If the platelet pellet is resuspended in a buffer containing trehalose and no albumin, add a final concentration of approximately 4-5% human albumin to the trehalose-treated platelet preparation.

[0095] Cryopreservation Platelets prepared by any of the methods described herein are cooled slowly or in an uncontrolled manner at a rate of 2-20°C / min to about -80°C. Prior to freezing, it may be desirable, but not required, to concentrate the platelets by centrifugation and discard a portion of the platelet-free supernatant to reduce the volume of platelets to be frozen and therefore the volume of platelets to be transfused to the patient. The platelets may be frozen in bags or tubes.

[0096] Thawing and therapeutic use Frozen stored platelets are thawed at 20-40°C. This may take 5 minutes to 1 hour depending on the volume of platelets being frozen. The thawed platelets can be infused parenterally directly into the patient either prophylactically, such as in thrombocytopenic patients at high risk of bleeding, or therapeutically, in actively bleeding patients.

Claims

1. 1. A method for cryopreserving platelets that maintain viability and activatability, comprising: (a) obtaining blood or platelet-rich plasma or apheresis-derived platelets from a subject; (b) isolating platelets from blood, platelet-rich plasma, or apheresis platelets; (c) suspending the platelets in a suspension buffer containing trehalose; (d) adding a stabilizer to the suspended platelets; (e) cooling the platelets to a temperature of about −80° C. at a rate of about 2-20° C. per minute; and (f) storing the platelets at about −80° C.; The method comprising:

2. 2. The method of claim 1, wherein isolating platelets comprises centrifuging blood, platelet-rich plasma or apheresis platelets at 100×g for 15 minutes to produce a first pellet and a first supernatant; and then centrifuging the first supernatant at 1000×g for 15 minutes to produce a second pellet and a second supernatant, wherein the first supernatant is platelet-rich and the second supernatant is platelet-poor, and the second supernatant is collected and trehalose is added to about 50 mM and may be used to dilute the platelets of step (c) to a concentration of about 1×106 platelets / μL.

3. 2. Cryopreserved platelets prepared by the method of claim 1.

4. 1. A method for cryopreserving and recovering platelets from cryopreservation, comprising: (a) obtaining blood, platelet-rich plasma, or apheresis-derived platelets from a subject; (b) isolating platelets from blood, platelet-rich plasma, or apheresis platelets; (c) suspending the platelets in a buffer solution containing trehalose; (d) adding a stabilizer to the suspended platelets; (e) cooling the platelets to a temperature of about −80° C. at a rate of about 2-20° C. per minute; (f) storing the platelets at about −80° C. for a period of time; and (g) thawing the platelets at about 20-40°C; The method comprising:

5. 5. The method of claim 1 or 4, wherein the stabilizer is about 4% by weight of bovine serum albumin (BSA). 。

6. The platelets of step (c) are about 1×10 ６ Dilute with suspension buffer to a final concentration of platelets / μL; and The diluted platelets are incubated at about 37° C. for about 2 hours with regular agitation, and may further include the steps of: (h) isolating the thawed platelets, comprising centrifugation of the thawed platelets at 1000×g for 15 minutes; and (i) suspending the isolated, thawed platelets in an activation buffer containing about 1% by weight BSA; The method according to claim 4.

7. The method of claim 6, wherein after step (h) and before step (i), the following step is carried out: (h-1) washing the thawed platelets in a wash buffer, comprising resuspending the thawed platelets in a wash buffer, centrifuging the resuspended platelets at 1000×g for 15 minutes, and removing the supernatant.

8. The method of claim 6 or 7, wherein the suspension buffer, activation buffer, or wash buffer further contains about 50 mM trehalose.

9. 9. The method of claim 8, wherein the period of time in step (f) is from 1 day to about 5 years.

10. 1. A method for activating platelets recovered after cryopreservation, comprising: (a) recovering platelets after cryopreservation according to the method of claim 4; (b) treating the collected platelets with platelet factor 4 (PF4; SEQ ID NO:2) or heparin; and (c) incubating the PF4-treated platelets or the heparin-treated platelets with a plasma, serum or blood sample from a subject diagnosed with heparin-induced thrombocytopenia (HIT); The method comprising:

11. Treating the collected platelets with PF4 comprises adding PF4 to the platelets at a concentration of about 150 μg / mL and incubating at room temperature for about 20 minutes; or 11. The method of claim 10, wherein treating the collected platelets with heparin comprises adding heparin to the platelets at a concentration of about 0.1-5 U / mL and incubating at room temperature for about 20 minutes.

12. 11. The method of claim 10, wherein platelet activation is assessed by measuring platelet granule content released following incubation of PF4-treated or heparin-treated platelets with plasma, serum or blood samples from HIT subjects, and the platelet granule content can be thrombospondin-1.

13. 1. A kit for detecting heparin-induced thrombocytopenia (HIT) antibodies in blood, plasma or serum, comprising: (a) Cryopreserved platelets; (b) one or more of platelet factor 4 (PF4; SEQ ID NO:2) or heparin; (c) one or more reagents for detecting platelet activation; (d) optionally, buffers and containers; and (e) optionally, one or more of packaging or instructions; The kit further comprises two or more of the following:

14. The kit of claim 13, wherein the one or more reagents for detecting platelet activation include a reagent for detecting thrombospondin-1.

15. The kit of claim 13, wherein the kit further comprises one or more negative control samples that are negative for pathogenic HIT antibodies that activate platelets; and one or more positive control samples that are positive for pathogenic HIT antibodies that activate platelets.

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