Diluted (1:100) whole blood (100 mL) was incubated with PEconjugated platelet-and leukocyte-specific antibodies (CD41 and CD45, respectively), or separately with annexin V-FITC (binding to phosphatidylserine) for 30 min, after which 1% paraformaldehyde (400 mL) was added. Matched fluorescein conjugated isotype control antibodies were used simultaneously staining to set the threshold and exclude nonspecific binding. Interactions between cell (platelets or leukocytes) and cell-derived MV and PS expression on both platelets and leukocytes were analyzed by flow cytometry (FACSCalibur TM and FACSCanto TM , BD Biosciences, San Jose, CA). Platelets were identified by forward and side scatter and with fluorescein conjugated CD41 antibody until 20,000 events gated for each sample, respectively ( Figure 1A ).