Activated platelets release two types of membrane vesicles: Microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules

Platelet activation leads to secretion of granule contents and to the forma tion of microvesicles by shedding of membranes from the cell surface. Recen tly, we have described small internal vesicles in multivesicular bodies (MV Bs) and alpha-granules, and suggested that these vesicles are secreted duri ng platelet activation, analogous to the secretion of vesicles termed exoso mes by other cell types. In the present study we report that two different types of membrane vesicles are released after stimulation of platelets with thrombin receptor agonist peptide SFLLRN (TRAP) or alpha-thrombin: microve sicles of 100 nm to 1 mu m, and exosomes measuring 40 to 100 nm in diameter , similar in size as the internal vesicles in MVBs and alpha-granules. Micr ovesicles could be detected by flow cytometry but not the exosomes, probabl y because of the small size of the latter. Western blot analysis showed tha t isolated exosomes were selectively enriched in the tetraspan protein CD63 . Whole-mount immune-electron microscopy (IEM) confirmed this observation. Membrane proteins such as the integrin chains alpha(llb)-beta(3) and beta(1 ), GPlb alpha, and P-selectin were predominantly present on the microvesicl es. IEM of platelet aggregates showed CD63(+) internal vesicles in fusion p rofiles of MVBs, and in the extracellular space between platelet extensions . Annexin-V binding was mainly restricted to the microvesicles and to a low extent to exosomes; Binding of factor X and prothrombin was observed to th e microvesicles but not to exosomes. These observations and the selective p resence of CD63 suggest that released platelet exosomes may have an extrace llular function other than the procoagulant activity, attributed to platele t microvesicles. (C) 1999 by The American Society of Hematology.