Cytochrome c and dATP initiate sequential caspase-9 and caspase-3 activation in platelet cytosolic extracts. Platelet cytosolic extracts derived from hospital concentrates (final concentration, 5 mg protein/mL) were treated with buffer or cytochrome c (10 μmol/L) and dATP (1 mmol/L) and then analyzed by SDS-PAGE and Western blotting (35 μg protein/lane). (A) Procaspase-9 processing in response to cytochrome c and dATP. (B) The blot from (A) was stripped and reprobed with anti–caspase-3 antibodies. At the indicated times after incubation with buffer (▪) or cytochrome c and dATP (▴), aliquots (75 μg protein) of cell-free reactions were analyzed for activity against DEVD-pNA (C). (A) and (B) are representative of 3 independent experiments. In (C), error bars represent the SEM, N = 3.