Isolation and characterization of platelet‐derived extracellular vesicles

Abstract

BackgroundPlatelet-derived extracellular vesicles (EVs) participate, for example, in haemostasis, immunity and development. Most studies of platelet EVs have targeted microparticles, whereas exosomes and EV characterization 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, lipopolysaccharide (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 determined by NTA and electron microscopy. Proteomics was used to characterize the differentially induced EVs.ResultsThe main EV populations were 100–250 nm and over 90% were <500 nm irrespective of the activation. 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.ConclusionsAlthough platelets constitutively release EVs, vesiculation can be increased, and the activation pathway determines the number and the cargo of the formed EVs. These activation-dependent variations render the use of protein content in sample normalization invalid. Since most platelet EVs are 100–250 nm, only a fraction has been analyzed by previously used methods, for example, flow cytometry. As the EV subpopulations could not be distinguished and large vesicle populations may be lost by differential centrifugation, novel methods are required for the isolation and the differentiation of all EVs.

Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes

“…Integrin α2B (ITGA2B or CD41) was specifically pulled down with CD9. This integrin was previously shown to be enriched in medium-sized EVs released by platelets (19). Therefore, the subpopulation of CD9-single positive sEVs probably forms at the PM and early endocytic locations.…”

“…To obtain these results, it was necessary to combine several separation procedures and to follow a nonbiased approach, rather than a molecule-or EV subtype-targeted one. Some groups have recently performed comparative proteomic analyses of medium-sized and small EVs (with or without floatation into a gradient) from platelets (19) or tumor cells (26)(27)(28)(29). Another group has compared the expression of a few surface proteins on B lymphoma-EVs immunoprecipitated with either CD9, CD63, or CD81 (30).…”

Extracellular vesicle isolation and characterization: toward clinical application

“…34-37 and Figure 1). Other vesicle types, such as apoptotic bodies (50-2000 nm) that are released by cells undergoing apoptosis (38), blood-derived vesicles (130-500 nm) that are released upon platelet activation ("platelet dust") (39)(40)(41), and autophagosomes, (42) will not be covered in this review.…”

Human plasma platelet‐derived exosomes: effects of aspirin

“…In contrast, platelet release of plasma membrane-derived, cytosol-containing 150-to 1000-nm-diameter microvesicles is strikingly enhanced by many stimuli, physicochemical stresses, and apoptosis (3). Furthermore, in contrast to the predominantly phospholipid content of microvesicles, platelet exosomal cargoes may include diverse cytokines, chemokines, growth factors, coagulation factors, lipoproteins, and other lipids, as well as several types of RNA (1)(2)(3). Platelet exosome membrane proteins also reflect those of their platelet source, including the constitutively expressed glycoprotein GPIb, as well as GPVI, aIIbb3, CD40 ligand, and P-selectin from activated platelets (1,3,4).…”