High PS exposure induced by ABT737 depends on elevated intracellular Ca²⁺. (A-F) Washed platelets were stimulated by 10 μM ABT737 in the absence of CaCl₂ or after loading with dimethyl-BAPTA (+ BAPTA) in the presence of 2 mM CaCl₂. Representative flow cytometry histograms (A-B) and dot plots (C-D) of AF647-annexin (Anx) A5 binding after 1-h treatment with ABT737. Markers M1, M2, and M3 indicate fractions of platelets with no, moderate, or high Anx A5 binding, respectively. (E-F) Distribution of platelet populations over M1, M2, and M3 during treatment in time. (G) Platelets were loaded with the intracellular Ca²⁺ probe Fluo-4 and stimulated with ABT737 (10 μM, 1 h) in the presence of 1 mM CaCl₂. Fluo-4 fluorescence was determined for the M1, M2, and M3 fractions. Dotted line represents maximal Fluo-4 fluorescence obtained with 10 μM ionomycin, as positive control. (H) Anx A5 binding of platelets stimulated by ABT737 in the presence of 1 mM CaCl₂ or 1 mM EGTA after 1 to 3 h. (I) Caspase activity (arbitrary fluorescence units/min) of ABT737-stimulated platelets in the presence of 1 mM CaCl₂ or 1 mM EGTA. Mean ± SEM (n = 3-6); *P < .05.