GSAO marks functionally procoagulant platelets. (A-B) Washed human platelets were stimulated with thrombin (0.1 U/mL) or thrombin (0.1 U/mL) and collagen (5 µg/mL), and phosphatidylserine externalization was assessed by annexin V binding. The mean fluorescence intensity of annexin V binding to GSAO⁻ and GSAO⁺ platelets is shown in B. Flow plots and bars are representative of n ≥ 3 separate experiments. (C) Platelet-rich plasma was recalcified and stimulated with thrombin (1 U/mL) and collagen (5 µg/mL) with fibrin polymerization inhibition. FXa on the platelet surface was detected using the small molecule inhibitor, Glu-Gly-Arg chloromethyl ketone-fluorescein isothiocyanate. Of the FXa⁺ platelets, 93% were co-labeled with GSAO. (D) Washed human platelets were stimulated with thrombin (0.1 U/mL) or thrombin (0.1 U/mL) and collagen (5 µg/mL), and their procoagulant potential was assessed using the Calibrated Automated Thrombogram. Correlation between peak thrombin time and the percentage of GSAO⁺ platelets in the preparation (n = 18). The dotted line is the nonlinear least squares fit of the data to a single exponential (r² = 0.77; P < .001). (E) Dylight 649-conjugated anti-platelet CD42b antibody, GSAO-AF546, and AF488-conjugated anti-fibrin antibody were injected into the murine circulation and thrombus initiated in the cremaster muscle arterioles by FeCl₃ injury. Images are represented as cross-sectional orthogonal views of single confocal plane separately displaying GSAO and fibrin signal (left) and platelet signal (right). Fibrin signal preferentially localized with the GSAO⁺ platelets (representative image, n = 9 images in 3 independent mice). (F) 3D reconstruction of an occlusive thrombus.