Use of apoptosis inhibitors for cold storage of platelets.

Abstract

The present invention relates to a pharmaceutical composition for the refrigerated storage of platelets, comprising platelets in a suitable storage medium and at least one inhibitor of platelet apoptosis, preferably a selective inhibitor targeting apoptosis induced by refrigerated storage.

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for refrigerated storage of platelets, comprising platelets in a suitable storage medium and at least one inhibitor of platelet apoptosis, preferably a selective inhibitor targeting apoptosis induced by refrigerated storage. [Background technology]

[0002] Platelet transfusion has become an essential therapy in modern medicine. Although the clinical benefits of platelet transfusion are well established, adverse transfusion reactions, especially the transmission of bacterial infections, remain a major challenge. Transfusion of platelet concentrates (PCs) is also essential to reduce blood loss after trauma or to maintain safe platelet (PLT) counts during chemotherapy.

[0003] Currently, PC is stored at room temperature (20° C.-24° C.) with constant agitation to ensure adequate PLT recovery, viability, and sufficient therapeutic efficacy.

[0004] However, storage at room temperature (RT) not only threatens functionality both in vivo and in vitro ("PLT storage injury"), but also increases the potential risk of microbial growth if contaminated. [1~6] For these reasons, the shelf life of PLTs is limited to 4-5 days, depending on national guidelines. [2] However, despite the limited storage time, the incidence of bacterial contamination of PLTs remains high, ranging from 1 to 10 cases per 50,000 units, which is a major disadvantage of PLTs stored at RT for clinical use.

[0005] Refrigerated storage of PC at 4°C may be one option to reduce the risk of bacterial growth. [7、8] A recent study reported that refrigerated stored PLTs were functionally and metabolically superior to PLTs stored at RT. [9~11] .

[0006] Currently, the FDA allows platelets to be stored refrigerated at 1°C to 6°C without stirring for up to 3 days for trauma patients (21 CFR 640.24 and 640.25). Mayo Clinic has recently been promoting the use of such 3-day platelets for trauma patients. The military submitted a variance request to the FDA after considerable research and data collection. The variance was just approved by the FDA in August 2019 with the statement: "Apheresis platelets will be stored at 1°C to 6°C without stirring for up to 14 days. Refrigerated platelet products will be used to treat patients with active bleeding when conventional platelet products are not available or their use is impractical."

[0007] However, a potential limitation of cold storage is the poorer recovery and survival of PLTs after transfusion. However, this issue remains controversial. Indeed, some studies have demonstrated a decreased survival of cold-stored PLTs compared with RT-stored PLTs. [12~14] Other investigators have reported that PLTs stored at 4°C can survive in the circulation for several days. [15、16] Refrigerated platelets have been reported to be rapidly cleared from the circulation, precluding the use of refrigerated storage of platelets for transfusion. Based on these studies, the concept of refrigerated storage has been abandoned in routine clinical practice.

[0008] Recently, the availability of PLT storage in additive solution (PAS) has led to a renaissance in refrigerated storage of PCs. It has been demonstrated that apheresis platelet concentrates (APCs) stored at 4°C are detectable in the mouse circulation for up to 5 hours, implying no enhanced platelet degradation. Platelets stored at 4°C show higher expression of apoptotic markers compared to platelets stored at room temperature, leading to accelerated clearance from the circulation in a humanized animal model compared to human platelets stored at RT (Non-Patent Document 1).

[0009] Leytin (in Non-Patent Document 2) discloses that for many years, programmed cell death known as apoptosis was exclusively that of nucleated cells. Recently, however, apoptosis has been well documented in non-nucleated platelets as well as in the extrinsic and intrinsic pathways of apoptosis in nucleated cells and platelets, platelet apoptosis induced by multiple chemical stimuli and shear stress, markers of platelet apoptosis, mitochondrial control of platelet apoptosis, and apoptosis mediated by the platelet surface receptors PAR-1, GPIIbIIIa, and GPIbα. Furthermore, data have been published on platelet apoptosis caused by in vitro platelet aging during platelet storage, platelet apoptosis in pathological settings in humans and animal models, and inhibition of platelet apoptosis by cyclosporine A, intravenous immunoglobulin, and GPIIbIIIa antagonist drugs.

[0010] Xiang et al. (in Non-Patent Document 3) disclose that cell-impermeable calcium ion chelators such as EGTA inhibited platelet activation and apoptosis in vitro and extended the life span of refrigerated platelets after transfusion. EGTA-treated refrigerated platelets effectively prevented bleeding. They conclude that the addition of EGTA to platelet products may be a simple and effective method to enable refrigerated storage of platelets for transfusion.

[0011] Pleines I, et al. (in Non-Patent Document 4) disclose that depletion of BAK / BAX in mouse platelets reveals that intrinsic apoptosis is not required for the development of platelet storage damage. It is described that apoptotic elimination of old platelets from the bloodstream is essential to maintain a functional, hemostatically reactive platelet population. It is described that the restriction of platelet life span by intrinsic apoptosis is crucial to maintain a functional, hemostatically reactive platelet population, and thus inhibition of intrinsic apoptosis in platelets deposited in blood banks is unlikely to provide significant benefit. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Marini I, et al., "Cold storage of platelets in additive solution: the impact of residual plasma inapheresis platelet concentrates." Haematologica. 2019 Jan;104(1):207-214. doi: 10.3324 / haematol.2018.195057.Epub 2018 Aug 16. PMID: 30115655; PMCID: PMC6312032 [Non-Patent Document 2] "Apoptosis in the anucleate platelet." Blood Rev.2012 Mar;26(2):51-63. doi: 10.1016 / j.blre.2011.10.002. Epub 2011 Nov 4. PMID:22055392 [Non-Patent Document 3] Xiang B, et al., "Calcium Ion Chelation Preserves Platelet Function During Cold Storage." Arterioscler Thromb Vasc Biol. 2021Jan;41(1):234-249. doi: 10.1161 / ATVBAHA.120.314879. Epub 2020 Nov 12. PMID :33176450 [Non-Patent Document 4] "Intrinsic apoptosis circumvents the functional decline of circulating platelets but does not cause the storage lesion." Blood.2018 Jul 12;132(2):197-209. doi: 10.1182 / blood-2017-11-816355. Epub 2018 May21. PMID: 29784641 Summary of the Invention [Problem to be solved by the invention]

[0013] As mentioned above, platelet transfusion has become an essential therapy in modern medicine. The above techniques have not provided a conclusive scenario regarding apoptosis during refrigerated storage of platelets. The demand for stable and safe platelet preparations remains high. Therefore, the object of the present invention is to provide a new strategy for preserving platelet preparations. Other objects and advantages of the present invention will become apparent to those skilled in the art upon review of the following more detailed description of the invention, including the figures and examples. [Means for solving the problem]

[0014] In a first aspect of the present invention, the above problem is solved by a pharmaceutical composition for refrigerated storage of platelets, comprising platelets in a suitable storage medium and at least one platelet apoptosis inhibitor, preferably a selective inhibitor targeting refrigerated storage-induced apoptosis, wherein preferably said apoptosis inhibitor is selected from the group of specific RhoA subfamily Rho GTPase inhibitors, RhoA inhibitor G04, specific phosphokinase A (PKA) activators, forskolin, specific caspase-9 inhibitors, z-LEHD-fmk, and combinations thereof, in particular the combination of RhoA inhibitor G04 and forskolin.

[0015] In a second aspect of the present invention, the above problem is solved by a method for making a pharmaceutical composition according to the first aspect of the present invention, comprising providing a sample of platelets stored refrigerated in a suitable storage medium and adding to said platelets at least one platelet apoptosis inhibitor.

[0016] In a third aspect of the invention, the method according to the second aspect of the invention further comprises the step of refrigerating the pharmaceutical composition, wherein the refrigerated storage is at a temperature between about 0° C. and about 10° C., preferably between about 2° C. and about 5° C., more preferably at about 4° C. The refrigerated storage of the pharmaceutical composition is preferably for more than about 4 days, preferably for more than about 7 days, more preferably for more than about 10 days, and most preferably for more than about 14 days.

[0017] In a fourth aspect of the present invention the above problem is solved by a pharmaceutical composition according to the invention for use in the prevention and / or treatment of diseases, preferably for use in platelet transfusion.

[0018] In a fifth aspect of the present invention, the above problem is solved by a method for treating or preventing a disease or condition requiring platelet transfusion, comprising administering to a subject in need of such treatment or prevention an effective amount of a pharmaceutical composition according to the present invention.

[0019] In connection with the experiments carried out, the inventors investigated the effect of various apoptosis inhibitors on platelet viability and function. The invention is based on the surprising finding that in the presence of apoptosis inhibitors, platelets stored in a refrigerator, e.g. at 4°C, have similar viability and functional properties compared to platelets stored at room temperature. Thus, inhibition of apoptosis induced by or associated with cold storage was identified as an approach to prevent the decline in platelet viability and function after cold storage.

[0020] Accordingly, we provide an improved pharmaceutical composition for the refrigerated storage of platelets, comprising platelets in a suitable storage medium and at least one inhibitor of platelet apoptosis.

[0021] In the context of the present invention, refrigerated storage generally refers to a temperature below room temperature (20° C. or 22° C.), generally between room temperature and freezing, for example at about 0° C. to about 10° C., preferably between about 2° C. and about 5° C., more preferably at about 4° C. Refrigerated storage at about 4° C. in a conventional refrigerator is most preferred.

[0022] The platelets to be stored may be provided in any suitable storage medium for refrigerated storage. A preferred pharmaceutical composition according to the invention comprises platelets as platelet concentrates, such as autologous or apheresis platelet concentrates (APC), platelets in additive solution (PAS), or platelet-rich plasma (PRP). Preferably, said PRP does not contain more than about 35% plasma. Autologous platelet concentrates (APC) APC or platelet-rich plasma (PRP) are defined as a substrate that contains a platelet concentration above the basal level, i.e., above the level found in the peripheral blood of a mammal, e.g., a human.

[0023] Platelet additive solutions (PAS) are becoming increasingly popular for platelet storage, and PAS are steadily replacing plasma as the storage medium for platelets. PAS are electrolyte solutions intended for platelet storage and are used to regulate platelet quality by the addition of certain components. All currently available PAS contain acetate. Acetate reduces the amount of glucose that oxidizes to lactate, thereby preventing a decrease in pH that would reduce platelet quality. In addition, the oxidation of acetate leads to the production of bicarbonate, which acts as a buffering agent. The presence of potassium and magnesium in PAS prevents a decrease in pH and reduces the degree of spontaneous activation of platelets during storage. In hospitals, platelets stored in PAS cause approximately half as many allergic transfusion reactions compared to platelets in plasma. Post-transfusion recovery and survival, as well as corrected thrombocytopenia, are at least as good for platelets in PAS as for plasma, and recent data suggest that they may be even better. Therefore, PAS (e.g. SSP+) or plasma are preferred for storage of platelet concentrates (van der Meer PF. PAS or plasma for storage of platelets? A concise review. Transfus Med. 2016 Oct;26(5):339-342. doi: 10.1111 / tme.12325. Epub 2016 Jun 7. PMID: 27273164).

[0024] Preferably, the apoptosis inhibitor added is a selective inhibitor that targets apoptosis in platelets induced by cold storage. Various references describe factors involved in apoptosis in platelets induced by cold storage (see above), such as 14-3-3ζ (Yan Y, Xie R, Ning Z., "14-3-3ζ Regulates the Platelet Apoptosis During Storage." Indian J Hematol Blood Transfus. 2020 Apr;36(2):324-329.doi: 10.1007 / s12288-019-01229-z. Epub 2019 Nov 14. PMID: 32425384; PMCID:PMC7229142).

[0025] Preferably, the apoptosis inhibitor used in the context of the present invention is selected from the group of specific RhoA subfamily Rho GTPase inhibitors, specific phosphokinase A (PKA) activators, specific caspase-9 inhibitors, specific 14-3-3ζ activators, and combinations thereof (Shang X, et al., "Small-molecule inhibitors targeting G-protein-coupled Rho guaninenucleotide exchange factors." Proc NatlAcad Sci US A. 2013 Feb 19;110(8):3155-60. doi: 10.1073 / pnas.1212324110. Epub2013 Feb 4. PMID: 23382194; PMCID: PMC3581902; Evelyn CR, et al., "Small-molecule inhibitors targeting G-protein-coupled Rho guaninenucleotide exchange factors." Proc NatlAcad Sci US A. 2013 Feb 19;110(8):3155-60. doi: 10.1073 / pnas.1212324110. Epub2013 Feb 4. PMID: 23382194; PMCID: PMC3581902; al., "CCG-1423: a small-molecule inhibitor of RhoA transcriptional signaling." Mol Cancer Ther. 2007 Aug;6(8):2249-60.doi: 10.1158 / 1535-7163.MCT-06-0782. PMID: 17699722). Further preferred examples are the RhoA inhibitors G04, forskolin, and z-LEHD-fmk. Particularly preferred are combinations, such as the RhoA inhibitors G04 and forskolin, and the RhoA inhibitors G04 and z-LEHD-fmk.

[0026] The amount and concentration of the apoptosis inhibitor used can be easily determined by the skilled artisan and is usually found between 0.1 μM and 500 μM. In case of synergistic effects, such as those expected for example for the combination of the RhoA inhibitor G04 with forskolin or the RhoA inhibitor G04 with z-LEHD-fmk, the individual concentrations can be adjusted accordingly.

[0027] The platelets used, stored and / or transfused in the context of the present invention may be of autologous or allogeneic origin, preferred is a pharmaceutical composition according to the invention, wherein said platelets are derived from a mammalian donor, in particular a rodent donor or a human donor.

[0028] Refrigerated stored pharmaceutical compositions according to the present invention exhibit several advantages, for example, platelets stored refrigerated in a pharmaceutical composition according to the present invention exhibit a percent platelet (PLT) viability of greater than about 50%, preferably greater than about 55%, and more preferably greater than about 60% at day 10.

[0029] More preferably, platelets stored refrigerated in a pharmaceutical composition according to the invention exhibit less than about 40% apoptosis (%), preferably less than about 35% at day 10. Even more preferably, platelets stored refrigerated in a pharmaceutical composition according to the invention exhibit more than about 60% PLT aggregation (%), preferably more than about 65% at day 7. Methods for testing each of these parameters are disclosed herein and in the respective literature.

[0030] Platelet transfusion has become an essential therapy in modern medicine. Although the clinical benefits of platelet transfusion are well established, adverse reactions during transfusion, especially the transmission of bacterial infections, remain a major challenge. Bacterial contamination is more likely with storage at room temperature, and refrigerated storage may provide a solution to this clinically relevant problem. Recently, we have shown that refrigerated storage of PLTs in apheresis-derived platelet concentrates (APCs) with a residual plasma content of approximately 35% is feasible and may extend the shelf life of GMP-produced APCs stored at 4°C for clinical use.

[0031] However, this approach is still not applicable in clinical practice, since platelets stored at 4°C showed higher expression of apoptotic markers compared to room temperature and were cleared more quickly in a humanized mouse model. In this study, we aimed to elucidate whether inhibition of apoptosis could result in better preservation of PLTs and maintain their function. We tested the effects of three different apoptosis inhibitors, namely a RhoA inhibitor (G04), a PKA inhibitor (forskolin), and a caspase-9 inhibitor (z-LEHD-fmk), to see if the loss in PLT viability and function could be restored.

[0032] Our results show that RhoA inhibitors could significantly improve PLT survival and restore function even at longer storage periods of 7-10 days. On the other hand, forskolin could partially improve PLT viability and reduce apoptosis, but no effect on PLT function was observed. Similarly, no significant positive effect was observed after treatment with the caspase-9 inhibitor z-LEHD-fmk.

[0033] Taken together, the data indicate that inhibition of apoptosis using the RhoA inhibitor G04 in apheresis-derived platelet concentrates, especially in combination with forskolin, represents a suitable alternative strategy for longer-term storage of platelets at 4°C.

[0034] Another aspect of the invention relates to a method of making a pharmaceutical composition according to the invention, comprising providing a sample of platelets that are stored refrigerated in a suitable storage medium and adding to said platelets at least one inhibitor of platelet apoptosis.

[0035] One preferred approach to prepare platelets is as apheresis-derived platelet concentrates collected using FENWAL's AMICUS (Amicus, Fresenius Kabi, Bad Homburg, Germany) and stored in plasma or PAS (SSP+, Macopharma, Langen, Germany) at a final plasma concentration of e.g. 35% at 4 °C and RT.

[0036] In preparation, citrate is the preferred anticoagulant for blood collection, since EDTA damages platelets and heparin alters their function. Although citrate allows the rapid production of platelet-rich plasma (PRP) with a high platelet yield, this method has certain disadvantages. To overcome these problems, a centrifugation technique was developed to separate and wash platelets from human or rodent blood anticoagulated with acid citrate dextrose (ACD). The cells are resuspended in a physiological buffer under well-defined conditions, in particular in the presence of plasma ionic calcium concentration (2 mM) and in the absence of clotting factors or other plasma components.

[0037] Preferred is a method according to the invention, wherein said platelets are derived from a mammalian donor, in particular a rodent donor, a humanized rodent donor or a human donor.

[0038] As above, preferred is a method according to the invention which comprises providing platelets as a platelet concentrate, such as autologous or apheresis platelet concentrate (APC), platelets in additive solution (PAS), or platelet rich plasma (PRP). Preferably, said PRP does not contain more than about 35% plasma. Autologous platelet concentrate (APC) APC or platelet rich plasma (PRP) are defined as a substrate which contains a platelet concentration above the basal level, i.e. above the level found in the peripheral blood of a mammal, e.g. a human.

[0039] Preferably, the apoptosis inhibitor added is a selective inhibitor targeting cold storage-induced apoptosis in platelets. Various references describe factors involved in cold storage-induced apoptosis in platelets (see above), such as 14-3-3ζ (Yan Y, Xie R, Ning Z., "14-3-3ζ Regulates the Platelet Apoptosis During Storage." Indian J Hematol Blood Transfus. 2020 Apr;36(2):324-329. doi:10.1007 / s12288-019-01229-z. Epub 2019 Nov 14. PMID: 32425384; PMCID: PMC7229142).

[0040] Preferably, the apoptosis inhibitor used in the method of the present invention is selected from the group of specific RhoA subfamily Rho GTPase inhibitors, specific phosphokinase A (PKA) activators, specific caspase-9 inhibitors, specific 14-3-3ζ activators, and combinations thereof (Shang X, et al., Small-molecule inhibitors targeting G-protein-coupled Rho guaninenucleotide exchange factors. Proc Natl Acad Sci US A. 2013 Feb 19;110(8):3155-60. doi: 10.1073 / pnas.1212324110. Epub 2013 Feb 4. PMID: 23382194; PMCID: PMC3581902; Evelyn CR, et al., al., "CCG-1423: a small-molecule inhibitor of RhoA transcriptional signaling." MolCancer Ther. 2007 Aug;6(8):2249-60. doi: 10.1158 / 1535-7163.MCT-06-0782. PMID:17699722). Further preferred examples are the RhoA inhibitors G04, forskolin, and z-LEHD-fmk. Particularly preferred are combinations, such as the RhoA inhibitors G04 and forskolin, and the RhoA inhibitors G04 and z-LEHD-fmk.

[0041] Next, another aspect of the present invention relates to a method for refrigerating a pharmaceutical composition comprising platelets, comprising the method according to the present invention and further comprising the step of refrigerating the pharmaceutical composition, wherein the refrigerated storage is at a temperature between about 0° C. and about 10° C., preferably between about 2° C. and about 5° C., more preferably at about 4° C. Storage in a normal refrigerator for about 7 to 10 days is preferred.

[0042] Preferred is a method according to the invention, wherein said refrigerated storage of the pharmaceutical composition is for more than about 4 days, preferably for more than about 7 days, more preferably for more than about 10 days, most preferably for more than about 14 days. In the context of the present invention, it has been found that platelets stored at 4° C. in the presence of an apoptosis inhibitor have similar viability and functional properties compared to platelets stored at room temperature.

[0043] In the context of the present invention, the term "about" is intended to mean ±10% of a given value, unless otherwise indicated.

[0044] Another aspect of the present invention then relates to the pharmaceutical composition according to the present invention disclosed herein for use in the prevention and / or treatment of disease, preferably for use in platelet transfusion, more preferably for use in the treatment of sepsis, cancer, AIDS, leukemia, aplastic anemia, hypersplenism, surgery such as bone marrow transplantation, organ transplantation, or cardiopulmonary bypass, and / or radiation therapy.The attending physician can match each therapy to the pharmaceutical composition according to the present invention.This use may further comprise a step of testing the function of the desired PLT as disclosed above.

[0045] Another aspect of the present invention then relates to a method for treating or preventing a disease or condition related to, associated with, and / or caused by a deficiency of platelets, comprising administering to a subject in need of treatment or prevention an effective amount of a refrigerated pharmaceutical composition according to the present invention disclosed herein. Treatment or prevention may be in the case of sepsis, cancer, AIDS, leukemia, aplastic anemia, hypersplenism, bone marrow transplantation, organ transplantation, or surgery such as cardiopulmonary bypass, and / or radiation therapy in said subject, e.g., a human patient. The attending physician can match the respective therapy to the pharmaceutical composition according to the present invention. This use may further comprise a step of testing the function of the desired PLT as disclosed above.

[0046] In the studies underlying the present invention, the inventors investigated the effect of storage temperature and subsequent inhibition of apoptosis on the survival and function of PLTs during refrigerated storage of APCs.

[0047] Previously, we showed that storage of APCs at 4°C enhances the functionality of PLTs compared to storage at room temperature, thus significantly improving their clinical benefits. However, refrigerated storage of PLTs results in poor survival over longer storage periods. Interestingly, a significantly higher amount of apoptotic cells was observed with reduced viability, leading us to speculate that inhibition of apoptosis might improve PLT viability even over longer storage periods. We tested three different inhibitors of the apoptotic pathway and analyzed not only PLT viability but also function. These results demonstrate that the RhoA inhibitor G04 in particular significantly restores PLT viability and function.

[0048] Attempts to store APCs at 4°C were hampered by reduced survival of cold-stored PLTs. Consistently, in the context of the present invention, PLTs from APCs stored at 4°C consistently showed inferior survival curves compared to those stored at RT, confirming previous reports of accelerated clearance of cold-stored PLTs. [13、14] Data from animal models suggest that desialylation of GPIb is responsible for the accelerated clearance of cold-stored PLTs. [13、24] However, we found similar β-galactose exposure in RT- and refrigerated-stored PLTs, which is consistent with the results of a clinical study showing that reconstitution of sialylation in human PLTs does not prevent accelerated clearance of refrigerated-stored APCs.

[25] .

[0049] Interestingly, we found that cold storage induces phosphatidylserine exposure, indicating higher PLT apoptosis. Although the differences were not always significant, the trend was clear. This may indicate that the reduced in vivo survival of cold-stored PLTs is rather caused by an apoptosis-related mechanism. Indeed, cold storage has previously been shown to induce GPIbα clustering, making the receptor the initiation site for PLT apoptosis and macrophage recognition.

[26] It has been suggested that inhibition of prostacyclin reduces the changes in GPIb induced by cold storage.

[27] , which could be highlighted by the results. Of note, cold storage-induced apoptosis was not accompanied by a higher spontaneous expression of P-selectin or CD63, indicating that apoptosis and activation during PLT storage are mediated by two independent mechanisms. Thus, selective targeting of cold storage-induced apoptosis may provide a new approach to alleviate PLT storage damage. Indeed, it was recently reported that cold storage of PLTs enhances the binding of vWF to GPIbα, inducing an increase in intracellular calcium concentration and phosphatidylserine exposure, ultimately resulting in a rapid clearance of PLTs. The authors demonstrated that inhibition of this binding and the resulting downstream cascade, using a specific peptide that recognizes GPIbα, significantly increased the recovery and lifespan of cold-stored PLTs.

[28] Another study showed that a specific caspase-3 inhibitor significantly improved the functionality and viability of PLTs during 7 days of storage.

[29] Taken together with our results, inhibition of apoptosis appears to be a promising approach to mitigate the damage caused by cold storage.

[0050] Finally, we investigated the applicability of our findings to routine production and explored the possibility of extending the shelf life of PCs. To exclude the effect of different donors, we designed a follow-up study in which each donor donated APCs in duplicate in PAS containing 35% plasma. Each APC was stored at either RT or 4°C for up to 10 days. We found that PLTs from APCs collected and stored at 4°C under routine blood bank conditions maintained better functionality in terms of activation (granule release in response to TRAP) and aggregation compared to those stored at RT. These results correspond to those from the split design study. Taken together, a residual plasma content of about 35% is feasible to achieve refrigerated storage of PLTs in APCs for clinical use. Our results indicate the possibility of extending the shelf life of GMP-produced APCs stored at 4°C for clinical use by inhibiting apoptosis of PLTs.

[0051] In summary, the present invention provides insight into the in vitro hemostatic function of cold-stored PLTs and suggests that inhibition of apoptosis in PLTs stored at 4°C may be an alternative method to extend the shelf life of PLTs and subsequently improve the current standard of care.

[0052] The present invention relates in particular to the following items:

[0053] Item 1: A pharmaceutical composition for refrigerated storage of platelets, comprising platelets in a suitable storage medium and at least one inhibitor of platelet apoptosis.

[0054] Item 2. The pharmaceutical composition according to Item 1, wherein the refrigerated storage is at a temperature between about 0°C and about 10°C, preferably between about 2°C and about 5°C, more preferably at about 4°C.

[0055] Item 3. The pharmaceutical composition according to Item 1 or 2, wherein the platelets are derived from a mammalian donor, particularly a human donor.

[0056] Item 4. The pharmaceutical composition according to any one of Items 1 to 3, comprising platelets as a platelet concentrate, such as autologous platelet concentrate or apheresis platelet concentrate (APC), platelets in additive solution (PAS), or platelet-rich plasma (PRP), wherein preferably the PRP does not contain more than about 35% plasma.

[0057] Item 5. The pharmaceutical composition according to any one of Items 1 to 4, wherein the apoptosis inhibitor is a selective inhibitor targeting refrigerated storage-induced apoptosis, and preferably the apoptosis inhibitor is selected from the group consisting of a specific RhoA subfamily Rho GTPase inhibitor, a RhoA inhibitor G04, a specific phosphokinase A (PKA) activator, forskolin, a specific caspase-9 inhibitor, z-LEHD-fmk, and combinations thereof, particularly a combination of a RhoA inhibitor G04 and forskolin.

[0058] Item 6. The pharmaceutical composition according to any one of Items 1 to 5, wherein the refrigerated-stored platelets exhibit a platelet (PLT) viability (%) of greater than about 50%, preferably greater than about 55%, and more preferably greater than about 60% on day 10.

[0059] Item 7. The pharmaceutical composition according to any one of Items 1 to 6, wherein the refrigerated-stored platelets exhibit apoptosis (%) of less than about 40%, preferably less than about 35%, on day 10.

[0060] Item 8. The pharmaceutical composition according to any one of Items 1 to 7, wherein the refrigerated-stored platelets exhibit a PLT aggregation (%) of greater than about 60%, preferably greater than about 65%, on the 7th day.

[0061] Item 9. A method for producing the pharmaceutical composition according to any one of items 1 to 8, comprising providing a sample of platelets stored refrigerated in a suitable storage medium, and adding at least one platelet apoptosis inhibitor to the platelets.

[0062] Item 10. The method according to item 9, wherein the platelets are derived from a mammalian donor, particularly a human donor.

[0063] Item 11. The method according to item 9 or 10, wherein the platelets are concentrated platelets, such as autologous or apheresis platelets (APC), platelets in additive solution (PAS), or platelet-rich plasma (PRP), or platelet concentrates derived therefrom, wherein preferably the PRP does not contain more than about 35% plasma.

[0064] Item 12. The method according to any one of Items 9 to 11, wherein the apoptosis inhibitor is a selective inhibitor targeting refrigerated storage-induced apoptosis, and preferably, the apoptosis inhibitor is selected from the group consisting of a specific RhoA subfamily Rho GTPase inhibitor, a RhoA inhibitor G04, a specific phosphokinase A (PKA) activator, forskolin, a specific caspase-9 inhibitor, z-LEHD-fmk, and combinations thereof, particularly a combination of a RhoA inhibitor G04 and forskolin.

[0065] Item 13. The method according to any one of Items 9 to 12, further comprising a step of refrigerating the pharmaceutical composition, wherein the refrigerated storage is at a temperature between about 0°C and about 10°C, preferably between about 2°C and about 5°C, more preferably at about 4°C.

[0066] Item 14. The method according to Item 13, wherein the refrigerated storage of the pharmaceutical composition is for more than about 4 days, preferably more than about 7 days, more preferably more than about 10 days, and most preferably more than about 14 days.

[0067] Item 15. The pharmaceutical composition according to any one of Items 1 to 8, for use in the prevention and / or treatment of a disease, preferably for use in platelet transfusion.

[0068] Item 16. A method for treating or preventing a disease or condition related to, associated with, and / or caused by a deficiency of platelets, comprising administering to a subject in need of such treatment or prevention an effective amount of a refrigerated pharmaceutical composition according to the present invention disclosed herein.

[0069] Item 17. The method of item 16, wherein the disease or condition is selected from sepsis, cancer, AIDS, leukemia, aplastic anemia, hypersplenism, bone marrow transplant, organ transplant, or surgery such as cardiopulmonary bypass, and / or radiation therapy in the subject, e.g., a human patient.

[0070] The invention may relate to the following further specific embodiments.

[0071] In one preferred of such further specific embodiments, platelets refrigerated in a pharmaceutical composition of the invention exhibit increased adhesion capacity compared to platelets refrigerated in a pharmaceutical composition not containing an apoptosis inhibitor (preferably compared to otherwise the same or similar conditions), In this embodiment, the apoptosis inhibitor is preferably a RhoA inhibitor, most preferably a RhoA inhibitor as described herein above, such as the RhoA inhibitor G04.

[0072] In another preferred further specific embodiment, the number of adherent PLTs is increased by at least 30%, at least 45%, at least 60% after at least 4 days of refrigerated storage in a pharmaceutical composition of the invention compared to platelets after at least 4 days of refrigerated storage in a pharmaceutical composition not containing an apoptosis inhibitor.

[0073] In one further particular embodiment, which may be preferred, the number of adherent PLTs is increased by at least 65%, at least 90%, at least 115% after at least 7 days of refrigerated storage in a pharmaceutical composition of the invention compared to platelets after at least 7 days of refrigerated storage in a pharmaceutical composition not containing an apoptosis inhibitor.

[0074] The adhesion capacity in these further specific embodiments preferably refers to the number of seeded PLTs that adhere to the fibrinogen-coated coverslip relative to the number of PLTs seeded on the fibrinogen-coated coverslip. The adhesion capacity in further specific embodiments can be determined under the following experimental conditions: 100 mg / mL fibrinogen is coated on the coverslip and PLTs (1×10 per mL) are plated on the coverslip. 8 PLTs) are seeded onto these coverslips and incubated for 1 h with 0.1 M thrombin receptor activating peptide (TRAP). The adherent cells are then fixed with 4% paraformaldehyde for 20 min at RT.

[0075] In one additional, and possibly preferred, particular embodiment, the apoptosis inhibitor is selected from compounds that do not adversely affect functional parameters of PLTs selected from cell adhesion, thrombin generation, and clot formation, which parameters can be determined in comparison to a control under the conditions shown in the experiments of Figures 7, 8, and 9 herein.

[0076] The present invention will now be further described in the following examples with reference to the accompanying drawings, without however being limited thereto. For the purposes of the present invention, all references cited herein are hereby incorporated by reference in their entirety. [Brief description of the drawings]

[0077] [Figure 1] FIG. 1 shows that the RhoA inhibitor G04 results in increased viability and reduced apoptosis during longer APC storage. [Diagram 2] FIG. 1 shows that refrigerated storage in the presence of the Rho inhibitor G04 rescues loss of delta granule secretion and PLT aggregation. [Figure 2-1] (same as above) [Diagram 3]FIG. 1 shows that forskolin treatment partially restores survival of PLTs and reduces apoptosis of cold-stored APCs. [Figure 4] FIG. 1 shows that the PKA inhibitor forskolin does not affect the decline in PLT function during longer refrigerated storage periods. [Figure 4-1] (same as above) [Diagram 5] FIG. 1 shows that the caspase-9 inhibitor z-LEHD-fmk partially restores viability of PLTs and reduces apoptosis in cold-stored APCs. [Figure 6] FIG. 1 shows that the caspase-9 inhibitor z-LEHD-fmk does not affect the loss of PLT function over the longer term. [Figure 7] FIG. 1 shows that the RhoA inhibitor G04 restores PLT adhesion and has no effect on thrombin generation and clot formation. [Figure 8] FIG. 1 shows that forskolin treatment does not impair PLT adhesion, thrombin generation, and clot formation. [Figure 9] FIG. 1 shows that the caspase-9 inhibitor z-LEHD-fmk does not affect the function of PLTs during refrigerated storage. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0078] Materials and Methods Preparation of apheresis platelet concentrates (APC) APCs were collected from healthy volunteers according to the German hemotherapy guidelines: 10 individuals donated 2 units of APCs, which were collected using FENWAL AMICUS (Amicus, FreseniusKabi, Bad Homburg, Germany) and stored in plasma or PAS (SSP+, Macopharma, Langen, Germany) at 4°C and RT at a final plasma concentration of 35%.

[0079] Treatment of APCs with apoptosis inhibitors Appropriate concentrations for the inhibitors were determined by titrating different concentrations before the experiment. Immediately after collection, APCs were stored for 1 h without stirring. APCs were further divided into different culture bags under aseptic conditions and stored in sterile oxygen-permeable bags (Fresenius Kabi AG, Bad Homburg, Germany). APCs were treated with 150 μM of the RhoA inhibitor G04, 0.75 μM of the phosphokinase A (PKA) activator forskolin, or 40 μM of the caspase-9 inhibitor z-LEHD-fmk. All inhibitors were diluted in DMSO, so DMSO (600 μM) was used as a vehicle control to exclude any solvent-mediated effects. APCs without any added were used as a negative control. After 2 h of treatment at room temperature, all samples except the RT control were stored at 4 °C with constant gentle stirring. All samples were activated with the thrombin receptor activating peptide TRAP (20 μM, HART Biologicals) before measurements.

[0080] Determination of apoptosis PLTs from APCs were washed, resuspended in 1 mM CaCl2, and stained with Annexin V-FITC (Beckman Coulter) for 60 min at RT. Freshly isolated PLTs were incubated with 10 μM ionomycin (Abcam, Cambridge, UK) and used as a positive control.

[0081] Platelet activation and granule release PLT (10 × 10 per mL) 6(cells) were incubated with TRAP6 (20 μM) for 30 min at 37 °C and fixed with 4% PFA for 20 min at RT. Expression of CD62P (CLBThromb / 6, Beckman Coulter) and CD63 (CLBGran / 12, Beckman Coulter) was determined by FC. Conformational changes in the glycoprotein (GP) IIb / IIIa complex upon activation by TRAP were detected by PAC-1 antibody (BD Bioscience) using FC.

[0082] Platelet aggregation PLT function was analyzed by FC and light transmission PLT aggregation assay (LTA) using a four-channel aggregometer (Labitec, Ahrensburg, Germany).Collagen (8 μg / ml), ristocetin (1.5 μM) (both Moelab, Langenfeld, Germany), or ADP (80 μM, Nycomed, Konstanz, Germany) were used as inducers.

[0083] Testing of refrigerated stored APCs in mice Cold-stored APCs treated with 150 μM of the RhoA inhibitor G04 will be tested using a mouse model to analyze their survival and function in vivo. Inhibitor pre-treated samples will be collected on days 7 and 10 and transfused into NOD / SCID mice. PLT recovery and clearance kinetics will be compared to untreated controls. Furthermore, the mechanism of PLT degradation will be investigated.

[0084] statistical analysis Statistical analysis was performed using GraphPad Prism 7 (La Jolla, USA). T-tests were used to analyze normally distributed results. Nonparametric tests were used when data did not follow normal distribution as assessed by the D'Agostino and Pearson omnibus normality test. Between-group comparisons were performed using Wilcoxon rank sum tests and Fisher's exact tests with categorical variables. A p-value of less than 0.05 was considered to represent statistical significance.

[0085] Refrigerated storage in combination with the RhoA inhibitor G04 results in increased viability and reduced apoptosis over longer storage periods We studied the effect of the RhoA inhibitor G04 on platelet viability and apoptosis in refrigerated stored APCs. APCs were incubated with or without 150 μM G04 at 4° C. with constant gentle agitation. A significant increase in viability was observed in G04-treated PLTs at day 7 (FIG. 1A) (% viable cells 89.13 vs. 80.42, p=0.040), which further improved at day 10 (60.82 vs. 33.07, p=0.0023). In confirmation of this trend, G04 treatment also significantly reduced apoptosis in PLTs at day 4 (% apoptotic cells 9.3 vs. 17.6, p=0.0400) and even more so at day 10 (% apoptotic cells 33 vs. 60.1, p=0.0020) (FIG. 1B). A similar trend was observed in measurements of mitochondrial transmembrane potential on days 7 and 10 (ΔΨm MFI 17.88 vs. 10.34, p=0.0131 and 11.02 vs. 3.95, p=0.034, respectively) (Figure 1C).

[0086] Refrigerated storage in combination with the Rho inhibitor G04 reverses the loss of delta granule secretion and improves PLT function Following the significant improvement of PLT survival by the addition of G04, we analyzed further effects on PLT function. A significant increase in CD63 fold expression (Figure 2B) was observed on days 4 (CD63 MFI 6.38 vs. 2.32, p=0.0215), 7 (CD63 MFI 2.98 vs. 1.25, p=0.035), and 10 (CD63 MFI 3.97 vs. 1.24, p=0.049) after treatment of PLTs with G04. Analysis of PAC-1 (Figure 2C) showed a significant increase on day 7 (PAC1 MFI 8.82 vs. 2.45, p=0.0047), but not on days 4 and 10. Similarly, enhanced PLT aggregation (Figure 2D) was observed in PLTs treated with G04 at day 7 (maximum aggregation (%) 67.33 vs. 21.98, p=0.0186). No significant differences were observed in the levels of CD62 expression and PLT shrinkage rates.

[0087] Effect of PKA inhibition using forskolin on cold-stored APCs Furthermore, we tested the efficacy of forskolin, a clinically approved PKA inhibitor, on platelet viability and apoptosis in refrigerated stored APCs. APCs were incubated with and / or without 0.75 μM forskolin at 4° C. with gentle agitation. A significant increase in viability was observed in forskolin-treated PLTs at day 7 (% viable cells 87.1 vs. 73.4, p=0.0416) and day 10 (58.95 vs. 44.46, p=0.044) (FIG. 3A). In confirmation of this trend, forskolin treatment also significantly reduced apoptosis in PLTs at day 7 (% apoptotic cells 9.3 vs. 17.6, p=0.0400) and even more so at day 10 (% apoptotic cells 33 vs. 60.1, p=0.0020) (FIG. 3B). A similar trend was observed in measurements of mitochondrial transmembrane potential on days 7 and 10 (ΔΨm MFI 17.88 vs. 10.34, p=0.0131 and 11.02 vs. 3.95, p=0.034, respectively) (Figure 3C).

[0088] Effect of PKA inhibition on the function of cold-stored APCs Refrigerated APCs incubated with forskolin were also analyzed for their functional parameters. No significant effects were observed in any of the parameters utilized in our experiments. There was a trend toward increased expression of PAC-1 after treatment with forskolin, but this was not significant. A slight enhancement of aggregation was observed at day 4, which was also observed in untreated APCs.

[0089] Effect of caspase-9 inhibition on cold-stored APCs: Along with G04 and forskolin, we also included z-LEHD-fmk, a known inhibitor of the caspase-9 pathway with a demonstrated role in platelet apoptosis. z-LEHD-fmk also improved PLT viability and function of cold-stored PLTs. An increase in PLT viability (Figure 5A) was observed at day 10 after treatment with z-LEHD-fmk (viable cells (%) 60.38 vs. 41.04, p=0.0336). Analysis of apoptosis (Figure 5B) revealed a decrease in apoptotic cells at days 7 (apoptotic cells (%) 9.85 vs. 21.69, p=0.038) and 10 (apoptotic cells (%) 39.00 vs. 61.12, p=0.0136) after treatment. Mitochondrial transmembrane potential measurements (Figure 5C) showed an increase at day 10 (ΔΨm MFI 6.26 vs. 3.34, p=0.0472).

[0090] Functional analysis of cold-stored APCs incubated with z-LEHD-fmk revealed no significant effect on the parameters of PLTs included in the present invention. A trend towards decreased PLT aggregation was observed 4 days after treatment, but this was not significant (Figure 6C).

[0091] The results are summarized in Tables 1 and 2.

[0092] Table 1: Summary of results, viability and apoptosis of APCs stored at 4°C. [Table 1]

[0093] Table 2: Summary of results, PLT aggregation of APCs stored at 4 °C [Table 2]

[0094] Further studies on the effects of the apoptosis inhibitors G04, forskolin, and caspase-9 z-LEHD-fmk on PLT functionality We further investigated the effect of apoptosis inhibitors on the in vitro hemostatic function of cold-stored platelets. As shown in the data below, the RhoA inhibitor G04 is able to restore cell function even after 7 days of storage. Furthermore, the PKA activator forskolin and the caspase-9 inhibitor z-LEHD-fmk showed no adverse effect on the functionality of PLTs during cold storage.

[0095] Materials and Methods Platelet adhesion Coverslips (Corning, New York, USA) were coated with 100 mg / mL fibrinogen (SigmaAldrich, Munich, Germany). After 4 and 7 days of storage, PLTs (1 × 10 per mL) were cultured. 8 PLTs) were seeded onto coverslips and incubated with TRAP (0.1 mM, HartBiologicals, Hartlepool, UK) for 1 h at room temperature (RT). Adherent cells were fixed with 4% paraformaldehyde for 20 minutes (min) at RT. Images were acquired from five different microscope fields / coverslip (100x magnification, Olympus IX73, Tokyo, Japan).

[0096] Thrombin generation Thrombin generation was quantified using a fluorescence-based assay performed using a calibrated automated thrombogram system (CAT, Stago, Maastricht, The Netherlands). Fluorescence signals were acquired over 60 min using a 110 excitation at 390 nm / emission filter set at 460 nm.

[0097] clot formation Clot formation was analyzed using a six-channel thromboelastometer device (Clot Pro, Enicor, Germany) and parameters were calculated automatically by dedicated software.

[0098] result Refrigerated storage in combination with a RhoA inhibitor restores PLT adhesion and leaves thrombin generation and clot formation unaffected. We analyzed the adhesion ability of refrigerated PLTs after 4 and 7 days of storage. A significant increase in the number of adherent cells was observed after PLTs were treated with G04 (Figure 7A). Furthermore, at each storage time point, comparable thrombin generation (AUC, area under the curve) was detected in the presence or absence of RhoA inhibitors (Figure 7B). Similarly, no significant effect on clot formation (MCF, maximum clot firmness) of refrigerated PLTs incubated with G04 was observed (Figure 7C).

[0099] Effect of forskolin on the functionality of refrigerated stored PLTs Furthermore, the functionality of PLTs stored at 4°C in combination with forskolin was analyzed. No significant effect was observed on all parameters, namely cell adhesion (Figure 8A), thrombin generation (Figure 8B), and clot formation (Figure 8C), compared to PLTs stored in buffer. A trend towards a decrease in clot formation was observed on days 1, 4, and 7 after treatment with forskolin, but this was not statistically significant (Figure 8C).

[0100] Effect of caspase-9 inhibitors on the function of PLTs during refrigerated storage Functional investigation of cold-stored PLTs incubated with caspase-9 inhibitors revealed no significant effects in any of the parameters tested in our experiments compared to untreated cells at each storage time point (Figures 9A, 9B, and 9C).

[0101] summary These new findings confirm that inhibition of apoptosis, especially by RhoA inhibitors, is a promising and suitable alternative strategy for longer storage of PLTs at 4° C. A summary of the results is shown in Table 3.

[0102] Table 3: Summary of results, effects of G04, forskolin, and z-LEHD-fmk on PLT function. [Table 3]

[0103] References cited 1. Rosenfeld BA,Herfel B, Faraday N, Fuller A, Braine H: Effects of storage time on quantitative and qualitative platelet function after transfusion.Anesthesiology 1995;83:1167-1172. 2. Brecher ME, Blajchman MA, Yomtovian R, Ness P, AuBuchon JP: Addressing the risk of bacterial contamination of platelets within the United States: a history tohelp illuminate the future. Transfusion 2013;53:221-231. 3. Dumont LJ,Gulliksson H, van der Meer PF, et al.: Interruption of agitation of plateletconcentrates: a multicenter in vitro study by the BEST Collaborative on theeffects of shipping platelets. Transfusion 2007;47:1666-1673. 4. Seghatchian J,Krailadsiri P: The platelet storage lesion. Transfus Med Rev 1997;11:130-144. 5. Capocelli KE,Dumont LJ: Novel platelet storage conditions: additive solutions, gas, andcold. Curr Opin Hematol 2014;21:491-496. 6. Devine DV, SerranoK: The platelet storage lesion. Clin Lab Med 2010;30:475-487. 7. Currie LM, HarperJR, Allan H, Connor J: Inhibition of cytokine accumulation and bacterial growthduring storage of platelet concentrates at 4 degrees C with retention of invitro functional activity. Transfusion 1997;37:18-24. 8. Sandgren P,Hansson M, Gulliksson H, Shanwell A: Storage of buffy-coat-derived platelets inadditive solutions at 4 degrees C and 22 degrees C: flow cytometry analysis ofplatelet glycoprotein expression. Vox Sang 2007;93:27-36. 9. Reddoch KM,Pidcoke HF, Montgomery RK, et al.: Hemostatic function of apheresis plateletsstored at 4 degrees C and 22 degrees C. Shock 2014;41 Suppl 1:54-61. 10. Pidcoke HF,Spinella PC, Ramasubramanian AK, et al.: Refrigerated platelets for thetreatment of acute bleeding: a review of the literature and reexamination ofcurrent standards. Shock 2014;41 Suppl 1:51-53. 11. Montgomery RK,Reddoch KM, Evani SJ, Cap AP, Ramasubramanian AK: Enhanced shear-inducedplatelet aggregation due to low-temperature storage. Transfusion2013;53:1520-1530. 12. Lee DH, BlajchmanMA: Novel treatment modalities: new platelet preparations and substitutes. Br JHaematol 2001;114:496-505. 13. Hoffmeister KM,Felbinger TW, Falet H, et al.: The clearance mechanism of chilled bloodplatelets. Cell 2003;112:87-97. 14. Murphy S, GardnerFH: Effect of storage temperature on maintenance of plateletviability--deleterious effect of refrigerated storage. N Engl J Med1969;280:1094-1098. 15. Pidcoke HF,McFaul SJ, Ramasubramanian AK, et al.: Primary hemostatic capacity of wholeblood: a comprehensive analysis of pathogen reduction and refrigeration effectsover time. Transfusion 2013;53 Suppl 1:137S-149S. 16. Jobes D, Wolfe Y,O'Neill D, et al.: Toward a definition of "fresh" whole blood: an invitro characterization of coagulation properties in refrigerated whole bloodfor transfusion. Transfusion 2011;51:43-51. 17. Valeri CR:Circulation and hemostatic effectiveness of platelets stored at 4 C or 22 C:studies in aspirin-treated normal volunteers. Transfusion 1976;16:20-23. 18. Sweeney J,Kouttab N, Holme S, Kurtis J, Cheves T, Nelson E: Storage of platelet-richplasma-derived platelet concentrate pools in plasma and additive solution.Transfusion 2006;46:835-840. 19. Slichter SJ,Corson J, Jones MK, et al.: Exploratory studies of extended storage ofapheresis platelets in a platelet additive solution (PAS). Blood2014;123:271-280. 20. Hornsey VS,McColl K, Drummond O, et al.: Extended storage of platelets in SSP plateletadditive solution. Vox Sang 2006;91:41-46. 21. Thiele T, PohlerP, Kohlmann T, et al.: Tolerance of platelet concentrates treated withUVC-light only for pathogen reduction--a phase I clinical trial. Vox Sang2015;109:44-51. 22. Bakchoul T,Fuhrmann J, Chong BH, Bougie D, Aster R, Subcommittee on Platelet I:Recommendations for the use of the non-obese diabetic / severe combinedimmunodeficiency mouse model in autoimmune and drug-induced thrombocytopenia:communication from the SSC of the ISTH. J Thromb Haemost 2015;13:872-875. 23. Fuhrmann J, JouniR, Alex J, et al.: Assessment of human platelet survival in the NOD / SCID mousemodel: technical considerations. Transfusion 2016;56:1370-1376. 24. Jansen AJ,Josefsson EC, Rumjantseva V, et al.: Desialylation accelerates plateletclearance after refrigeration and initiates GPIbalphametalloproteinase-mediated cleavage in mice. Blood 2012;119:1263-1273. 25. Wandall HH,Hoffmeister KM, Sorensen AL, et al.: Galactosylation does not prevent the rapidclearance of long-term, 4 degrees C-stored platelets. Blood 2008;111:3249-3256. 26. van der Wal DE,Du VX, Lo KS, Rasmussen JT, Verhoef S, Akkerman JW: Platelet apoptosis bycold-induced glycoprotein Ibalpha clustering. J Thromb Haemost2010;8:2554-2562. 27. van der Wal DE,Verhoef S, Schutgens RE, Peters M, Wu Y, Akkerman JW: Role of glycoproteinIbalpha mobility in platelet function. Thromb Haemost 2010;103:1033-1043. 28. Chen W, DruzakSA, Wang Y, et al.: Refrigeration-Induced Binding of von Willebrand FactorFacilitates Fast Clearance of Refrigerated Platelets. Arterioscler Thromb VascBiol 2017;37:2271-2279. 29. Shiri R, Yari F,Ahmadinejad M, Vaeli S, Tabatabaei MR: The caspase-3 inhibitor (peptideZ-DEVD-FMK) affects the survival and function of platelets in plateletconcentrate during storage. Blood Res 2014;49:49-53. 30. Slichter SJ,Bolgiano D, Corson J, et al.: Extended storage of buffy coat plateletconcentrates in plasma or a platelet additive solution. Transfusion2014;54:2283-2291.

[0104] Drawing translation Figure 1 % Viable cells(Calcein) storage time(days) buffer buffer % Apoptotic cells (Annexin) Figure 2 Fold IncreaseCD62 (MFI with / without TRAP) storage time(days) buffer buffer Fold IncreaseCD63 (MFI with / without TRAP) Fold IncreasePAC-1 (MFI with / without TRAP) max.aggregation (%) Maximum aggregation (%) shrunk PLTs (% of population) Figure 3 % Viable cells(Calcein) storage time(days) buffer buffer forskolin % Apoptotic cells (Annexin) Figure 4 Fold IncreaseCD62 (MFI with / without TRAP) storage time(days) buffer buffer forskolin Fold IncreaseCD63 (MFI with / without TRAP) Fold IncreasePAC-1 (MFI with / without TRAP) max.aggregation (%) Maximum aggregation (%) shrunk PLTs (% of population) Figure 5 % Viable cells(Calcein) storage time(days) buffer buffer % Apoptotic cells (Annexin) Figure 6 Fold IncreaseCD62 (MFI with / without TRAP) storage time(days) Fold IncreaseCD63 (MFI with / without TRAP) buffer buffer max.aggregation (%) Maximum aggregation (%) shrunk PLTs (% of population) Figure 7 Number of adherent cells / field Number of adherent cells per field Storage time(days) buffer buffer Figure 8 Number of adherent cells / field Number of adherent cells per field Storage time(days) buffer buffer forskolin Figure 9 Number of adherent cells / field Number of adherent cells per field Storage time(days) buffer buffer

Claims

1. A pharmaceutical composition for refrigerated storage of platelets, comprising platelets in a suitable storage medium and at least one inhibitor of platelet apoptosis.

2. 2. The pharmaceutical composition of claim 1, wherein the refrigerated storage is at a temperature between about 0° C. and about 10° C., between about 2° C. and about 5° C., or at about 4° C.

3. 2. The pharmaceutical composition of claim 1, wherein the platelets are derived from a mammalian donor or a human donor.

4. The pharmaceutical composition of claim 1, comprising platelets as platelet concentrates, autologous or apheresis platelet concentrates (APC), platelets in additive solution (PAS), or platelet-rich plasma (PRP); or 2. The pharmaceutical composition of claim 1, comprising platelets as PRP, wherein the PRP does not contain more than about 35% plasma.

5. The pharmaceutical composition according to claim 1, wherein the apoptosis inhibitor is a selective inhibitor that targets refrigeration-induced apoptosis; or 2. The pharmaceutical composition of claim 1, wherein the apoptosis inhibitor is a selective inhibitor targeting refrigeration-induced apoptosis, wherein the apoptosis inhibitor is selected from the group consisting of specific RhoA subfamily Rho GTPase inhibitors, RhoA inhibitor G04, specific phosphokinase A (PKA) activators, forskolin, specific caspase-9 inhibitors, z-LEHD-fmk, and combinations thereof; 2. The pharmaceutical composition of claim 1, wherein the apoptosis inhibitor is a selective inhibitor that targets refrigerated storage-induced apoptosis, wherein the apoptosis inhibitor is a combination of RhoA inhibitor G04 and forskolin.

6. 2. The pharmaceutical composition of claim 1, wherein the refrigerated stored platelets exhibit a percent platelet (PLT) viability of greater than about 50%, or greater than about 55%, or greater than about 60% at 10 days.

7. 2. The pharmaceutical composition of claim 1, wherein the refrigerated stored platelets exhibit apoptosis (%) of less than about 40%, or less than about 35% on day 10.

8. 2. The pharmaceutical composition of claim 1, wherein the refrigerated stored platelets exhibit a PLT aggregation (%) of greater than about 60%, or greater than about 65% on day 7.

9. 13. A method of making the pharmaceutical composition of claim 1, comprising providing a sample of platelets that is stored refrigerated in a suitable storage medium and adding at least one platelet apoptosis inhibitor to the platelets.

10. 10. The method of claim 9, wherein the platelets are derived from a mammalian donor or a human donor.

11. 10. The method of claim 9, wherein the platelets are platelet concentrates, autologous or apheresis platelet concentrates (APC), platelets in additive solution (PAS), or platelet rich plasma (PRP), or derived therefrom; or 10. The method of claim 9, wherein the platelets are PRP, wherein the PRP does not contain more than about 35% plasma.

12. 10. The method of claim 9, wherein the apoptosis inhibitor is a selective inhibitor that targets cold storage-induced apoptosis; or 10. The method of claim 9, wherein the apoptosis inhibitor is a selective inhibitor targeting cold storage-induced apoptosis, wherein the apoptosis inhibitor is selected from the group of specific RhoA subfamily Rho GTPase inhibitors, RhoA inhibitor G04, specific phosphokinase A (PKA) activators, forskolin, specific caspase-9 inhibitors, z-LEHD-fmk, and combinations thereof; or 10. The method of claim 9, wherein the apoptosis inhibitor is a selective inhibitor that targets cold storage-induced apoptosis, wherein the apoptosis inhibitor is a combination of RhoA inhibitor G04 and forskolin.

13. 13. The method of any one of claims 9 to 12, further comprising storing the pharmaceutical composition under refrigeration, wherein the refrigerated storage is at a temperature between about 0°C and about 10°C, between about 2°C and about 5°C, or at about 4°C.

14. 14. The method of claim 13, wherein the refrigerated storage of the pharmaceutical composition is for more than about 4 days, or for more than about 7 days, or for more than about 10 days, or for more than about 14 days.

15. The pharmaceutical composition according to any one of claims 1 to 8 for use in the prevention and / or treatment of a disease or for use in platelet transfusion.

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