Platelet apoptosis, but not platelet activation, depends on caspase activation. Washed human platelets were incubated in HBS with ABT-737 (100nM) for 2 hours or A23187 (10μM), CRP-XL (2 μg/mL), or TRAP (100μM) for 15 minutes with or without the caspase inhibitor z-VAD.fmk (50μM). (A) Loss of mitochondrial membrane potential was assessed by staining with TMRE (50nM) before analysis by flow cytometry. Data are mean ± SD (n = 4). (B) Release of mitochondrial cytochrome c was assessed by lysis in 0.05% digitonin and differential centrifugation. The heavy membrane fraction (HM) containing mitochondria and the supernatant (SN) containing cytosol were analyzed for cytochrome c content by Western blotting. (C) Release of mitochondrial cytochrome c and loss of mitochondrial membrane potential after exposure to ABT-737 (100nM) for 2 hours were assessed by immunohistochemistry and staining with TMRE (red, top panels) and anti-CD41/FITC (green, top panels), or anti–cytochrome c (green, bottom panels) and anti–active-caspase-3 (red, bottom panels). Bar represents 5 μm. Note that control cells display spreading on glass slides, whereas ABT-737-treated cells do not. (D) Cleavage of caspases and caspase targets (fodrin, gelsolin) was analyzed by Western blotting. cl indicates cleaved caspase fragments. (E) Apoptosis was assessed by PS externalization and staining with annexin V/FITC before analysis by flow cytometry. Data are mean ± SD (n = 4).