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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.

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Platelet extracellular vesicles (PEVs) have emerged as potential mediators in intercellular communication. PEVs exhibit several activities with pathophysiological importance and may serve as diagnostic biomarkers. Here, imaging and analytical techniques were employed to unveil morphological pathways of the release, structure, composition, and surface properties of PEVs derived from human platelets (PLTs) activated with the thrombin receptor activating peptide (TRAP). Based on extensive electron microscopy analysis, we propose four morphological pathways for PEVs release from TRAP-activated PLTs: (1) plasma membrane budding, (2) extrusion of multivesicular α-granules and cytoplasmic vacuoles, (3) plasma membrane blistering and (4) "pearling" of PLT pseudopodia. The PLT extracellular vesiculome encompasses ectosomes, exosomes, free mitochondria, mitochondria-containing vesicles, "podiasomes" and PLT "ghosts". Interestingly, a flow cytometry showed a population of TOM20LC3 PEVs, likely products of platelet mitophagy. We found that lipidomic and proteomic profiles were different between the small PEV (S-PEVs; mean diameter 103 nm) and the large vesicle (L-PEVs; mean diameter 350 nm) fractions separated by differential centrifugation. In addition, the majority of PEVs released by activated PLTs was composed of S-PEVs which have markedly higher thrombin generation activity per unit of PEV surface area compared to L-PEVs, and contribute approximately 60% of the PLT vesiculome procoagulant potency.

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