Structural analysis of platelet fragments and extracellular vesicles produced by apheresis platelets during storage.

Platelets (PLTs) for transfusion can be stored up to 7 days at room temperature. Apheresis PLTs quality decreases over storage time, which affects PLT hemostatic functions. Here we characterized the membranous particles produced by PLT storage lesion (PSLPs) including degranulated PLTs, PLT ghosts, membrane fragments, and extracellular membrane vesicles (PEVs). The PSLPs generated in apheresis platelet units were analyzed at day 1, 3, 5, and 7 of RT storage. A differential centrifugation and a sucrose density gradient were used to separate PSLP populations. PSLPs were characterized using scanning and transmission electron microscopy (EM), atomic force microscopy (AFM), flow cytometry (FC) and nanoparticle tracking analysis (NTA). PSLPs have different morphologies and a broad size distribution; FC and NTA showed that the concentration of small and large PSLPs increases with storage time. Density gradient separated 3 PSLP populations: a) degranulated PLTs, PLT ghosts and large PLT fragments; b) PEVs originated from PLT activation and organelles released by necrotic PLTs, and c) PEV ghosts. Majority of PLPs exposed phosphatidyl serine and induced a thrombin generation. PSLPs contained extracellular mitochondria and were positive for autophagosome marker LC3. PSLPs encompass degranulated PLTs, PLT ghosts, large PLT fragments, large and dense PEVs and low-density PEV ghosts. While the activation-related PSLPs are released particularly during early stage of storage (day 1 -3), the release of apoptosis and necrosis related PSLPs prevails after that. No elevation of LC3 and TOM20 positive PLPs indicates that the increase of extracellular mitochondria during APU storage is not associated with PLT mitophagy.