Characterization of procoagulant extracellular vesicles and platelet membrane disintegration in DMSO‐cryopreserved platelets

Abstract

BackgroundFreezing is promising for extended platelet (PLT) storage for transfusion. 6% DMSO cryopreserved PLTs (CPPs) are currently in clinical development. CPPs contain significant amount of platelet membrane vesicles (PMVs). PLT-membrane changes and PMV release in CPP are poorly understood, and haemostatic effects of CPP PMVs are not fully elucidated. This study aims to investigate PLT-membrane alterations in CPPs and provide comprehensive characterization of CPP PMVs, and their contribution to procoagulant activity (PCA) of CPPs.MethodsCPPs and corresponding liquid-stored PLTs (LSPs) were characterized by flow cytometry (FC), fluorescence polarization (FP), nanoparticle tracking analysis (NTA), electron microscopy (SEM, TEM), atomic force microscopy (AFM) and thrombin-generation (TG) test.ResultsSEM and TEM revealed disintegration and vesiculation of the PLT-plasma membrane and loss of intracellular organization in 60% PLTs in CPPs. FP demonstrated that 6% DMSO alone and with freezing–thawing caused marked increase in PLT-membrane fluidity. The FC counts of annexin V-binding PMVs and CD41a+ PMVs were 68- and 56-folds higher, respectively, in CPPs than in LSPs. The AFM and NTA size distribution of PMVs in CPPs indicated a peak diameter of 100 nm, corresponding to exosome-size vesicles. TG-based PCA of CPPs was 2- and 9-folds higher per PLT and per volume, respectively, compared to LSPs. Differential centrifugation showed that CPP supernatant contributed 26% to CPP TG-PCA, mostly by the exosome-size PMVs and their TG-PCA was phosphatidylserine dependent.ConclusionsMajor portion of CPPs does not show activation phenotype but exhibits grape-like membrane disintegration with significant increase of membrane fluidity induced by 6% DMSO alone and further aggravated by freezing–thawing process. DMSO cryopreservation of PLTs is associated with the release of PMVs and marked increase of TG-PCA, as compared to LSPs. Exosome-size PMVs have significant contribution to PCA of CPPs.

Quality Assessment of Established and Emerging Blood Components for Transfusion

“…Thrombin generation in platelet concentrates can be assessed using calibrated automated thrombinography (CAT) [170]. PS and tissue factor, both of which are present in high amounts on the membrane of cryopreserved platelets and their microparticles, have the potential to trigger thrombin generation [115, 171, 172]. Thus thrombin generation is useful for measuring the procoagulant activity of cryopreserved platelets and reveals that cryopreserved platelets and platelet microparticles are also more procoagulant than fresh, liquid-stored platelets in terms of the amount of thrombin they generate and the lag time for thrombin generation [115, 171].…”

“…PS and tissue factor, both of which are present in high amounts on the membrane of cryopreserved platelets and their microparticles, have the potential to trigger thrombin generation [115, 171, 172]. Thus thrombin generation is useful for measuring the procoagulant activity of cryopreserved platelets and reveals that cryopreserved platelets and platelet microparticles are also more procoagulant than fresh, liquid-stored platelets in terms of the amount of thrombin they generate and the lag time for thrombin generation [115, 171]. However, the influence of the solution used for reconstitution after thawing must be taken into consideration, as non-plasma-based reconstitution solutions may not provide sufficient tissue factor for detectable thrombin generation [115].…”

“…Given that cryopreserved platelets are known to be activated, it would be expected that CD62P expression would be high on cryopreserved platelets. However, there are variations in what has been observed by different groups examining cryopreserved platelets, whereby some have observed high expression (>60%) [138, 171], some have observed partial activation with 20–40% CD62P expression [162, 165], and others have observed low CD62P expression [145, 174, 175]. This variation may arise due to differences in the platelets themselves, differences arising from use of different antibody clones, staining protocols, or instrument settings on different flow cytometers.…”

“…Cryopreserved platelets also express high levels of PS and shed more PS-positive platelet microparticles when compared to fresh liquid-stored platelets. Annexin V binding to PS may therefore be a better indicator of platelet activation in cryopreserved platelets, as it is consistently reported as high by many groups [115, 145, 158, 171]. …”

Pre‐clinical development of a cryopreservable megakaryocytic cell product capable of sustained platelet production in mice

“…Even in the face of such cell losses, cMK product remained fully functional and generated PLTs in vivo in a manner identical to that of the fresh product. Cryopreservation, however, may lead to changes in cell surface receptors expression and increased procoagulant activity . hPLTs produced after infusion of cMK were slightly activated at baseline but so were those produced after infusion of the fresh product.…”