ORIGINAL RESEARCH ARTICLE Isolation and characterization of platelet-derived extracellular vesicles

Background: Platelet-derived extracellular vesicles (EVs) participate, for example, in haemostasis, immunity and development. Most studies of platelet EVs have targeted microparticles, whereas exosomes and EV char-acterization under various conditions have been less analyzed. Studies have been hampered by the difficulty in obtaining EVs free from contaminating cells and platelet remnants. Therefore, we optimized an EV isolation protocol and compared the quantity and protein content of EVs induced by different agonists. Methods: Platelets isolated with iodixanol gradient were activated by thrombin and collagen, lipopolysac-charide (LPS) or Ca2 ionophore. Microparticles and exosomes were isolated by differential centrifugations. EVs were quantitated by nanoparticle tracking analysis (NTA) and total protein. Size distributions were deter-mined by NTA and electron microscopy. Proteomics was used to characterize the differentially induced EVs. Results: The main EV populations were 100250 nm and over 90 % were B500 nm irrespective of the acti-vation. However, activation pathways differentially regulated the quantity and the quality of EVs, which also formed constitutively. Thrombogenic activation was the most potent physiological EV-generator. LPS was a weak inducer of EVs, which had a selective protein content from the thrombogenic EVs. Ca2 ionophore generated a large population of protein-poor and unselectively packed EVs. By proteomic analysis, EVs were highly heterogeneous after the different activations and between the vesicle subpopulations