Calpain inhibition prevents platelet microvesiculation, caspase processing, and apoptotic substrate cleavage, but not PS externalization. Platelets were preincubated with vehicle (lanes 1 and 2), ZVAD-fmk (25 μmol/L; lane 3) or calpeptin (25 μmol/L; lane 4) for 30 minutes at room temperature and then treated with buffer (lane 1) or A23187 (lanes 2 through 4) for 15 minutes at 37°C. Microvesicles were then isolated from platelet supernatants as described in Materials and Methods and analyzed by Western blotting (10-μL aliquots/lane) with anti-gpIIb antibodies (A). The graph in (A) shows the extent of microvesiculation as assessed by gpIIb densitometry (arbitrary units). In (B), plasma membrane PS externalization was determined by annexin V-FITC binding and FACS analysis. (C) Whole-cell lysates analyzed by Western blotting with antibodies against caspase-9, caspase-3, gelsolin, and PKCδ (50 μg protein/lane). (D) Inhibition of 100 nmol/L purified calpain and caspase-3 by ZVAD-fmk (•) and calpeptin (▪). Error bars represent the SEM, N = 4.