Labeling with GSAO and exposure of P-selectin distinguishes necrotic from apoptotic platelets. (A) Washed human platelets were incubated with 30 µM of the BH3 mimetic ABT-737 for 2 hours, and exposure of P-selectin and labeling with GSAO-AF647, control GSCA-AF647, polycaspase-FAM, or annexin V-PB were measured by flow cytometry. (B) Washed human platelets were incubated with increasing concentrations of ABT-737 for 2 hours, and labeling with GSAO-AF647 and exposure of P-selection were measured by flow cytometry. The percentage of GSAO⁺ platelets are graphed with respect to P-selectin exposure. (C) Washed human platelets were preincubated with 200 µM ZVADFMK or vehicle control for 15 minutes and then incubated with 30 µM ABT-737 for 2 hours (part F), or stimulated with thrombin (0.1 U/mL) and collagen (5 µg/mL) for 10 minutes (part G). Labeling with GSAO-AF647 and exposure of P-selection were measured by flow cytometry. (D) Washed human platelets from healthy donors (n = 5-11) were untreated or stimulated with thrombin (0.1 U/mL), collagen (5 µg/mL), or thrombin (0.1 U/mL) and collagen (5 µg/mL) for 10 min. Labeling with GSAO-AF647 and exposure of P-selection was measured by flow cytometry, and the results were expressed as GSAO⁺ platelets that are P-selectin⁻ or P-selection⁺. The error bars are mean ± standard deviation.