Caspase-9 promotes megakaryocyte and platelet apoptosis. (A) Loss of caspase-9 inhibits activation of downstream caspase-3/7 in megakaryocytes. FLC-derived megakaryocytes were treated with staurosporine (STS), etoposide (Etop.), ABT-737, or ethanol (EtOH) or DMSO vehicle controls for 5 hours. Lysates were prepared and assayed for their ability to cleave the specific substrate for caspase-3/7 (z-DEVD-pNA). Data represent the mean ± SD; n = 3-4 livers per genotype (except EtOH and DMSO vehicle controls, n = 2); **P < .01, ***P < .001. (B) Loss of caspase-9 inhibits activation of downstream caspase-3/7 following Bcl-2 antagonism. Lysates were prepared and assayed for their ability to cleave specific substrates for caspase-9 (z-LEHD-pNA) or caspase-3/7 (z-DEVD-pNA). Data represent the mean ± SD; n = 3 mice per genotype (except wild type + Q-VD-OPh, n = 1). (C) Comparable attenuation of apoptosis in Casp9^−/− platelets and those lacking Bak/Bax. Washed platelets were treated with varying amounts of ABT-737 for the time indicated. ATP content, proportional to light signal output, was determined by luminescence. Data represent the mean ± SD; n = 3 mice per genotype; *P < .05, **P < .01. (D) Loss of caspase-9 perturbs PS externalization. Washed platelets were treated with varying amounts of ABT-737 for 90 minutes (left panel) or 360 minutes (right panel), or the calcium ionophore A23187, and stained with annexin V before flow cytometric analysis. Data represent the mean ± SD, n = 3-5 mice per genotype; *P < .05, ***P < .001.