GSAO labels a subpopulation of activated platelets. (A) Structure of GSAO and site of conjugation of AF647, Oregon Green, or biotin. (B) Washed human platelets were stimulated with calcium ionophore (1 µM) and activation and plasma membrane compromise measured by flow cytometry. Activated platelets were detected from surface elaboration of P-selectin and plasma membrane compromise by labeling with GSAO-AF647 or negative control GSCA-AF647. (C) Washed human platelets were left unstimulated or stimulated with thrombin (0.1 U/mL), collagen (5 µg/mL), or thrombin (0.1 U/mL) and collagen (5 µg/mL), and exposure of P-selectin and labeling with GSAO-AF647 were measured by flow cytometry. (D) Platelet stimulation with both thrombin and collagen results in significantly more labeling with GSAO-AF647 than with either agonist alone. (E) Washed human platelets were stimulated with collagen or collagen-related peptide, and labeling with GSAO-AF647 was measured by flow cytometry.