All buffers and antibodies were filtered twice through 0.2 mm membrane (Millipore) filters for MV analysis. Platelet free plasma (PFP) was prepared by double centrifugation at 30006g for 15 min. PFP was diluted (15 mL PFP+85 mL HEPES/HANKS' buffer) and incubated with 4 mL FITC-conjugated annexin-V and PE-conjugated CD41 or CD45 antibodies for 30 min in dark, at which time 350 mL HANKS' balanced salts buffer (pH 7.4) with 2.5 mM CaCl 2 was added. Matched isotype control antibodies were used to set threshold and exclude nonspecific binding. MV were quantified based on counts of calibration beads (TruCOUNT TM beads) added immediately to the samples prior to analysis by flow cytometry (FACSCanto TM , BD Biosciences, San Jose, CA) as previously described [25, 26, 31] . In this study, MV were defined as events of ,1 mm in diameter using size calibration beads and positive for annexin-V and platelet-and leukocyte-specific markers.