FIGURE 3.

Validation of optimum conditions for PAR1 and PAR4 antagonism. Whole blood was treated with vorapaxar (A, B) or BMS 986120 (C, D) at various concentrations and times as indicated. Blood was then stimulated with PAR1‐AP (10 μM) or PAR4‐AP (100 μM) for 10 minutes, stained with PAC1‐FITC and anti‐P‐selectin‐PE, and analyzed by flow cytometry. Vorapaxar inhibited PAR1‐AP–triggered platelet activation in a concentration‐ and time‐dependent manner, with no inhibition of PAR4‐AP‐triggered platelet activation (A, B). Conversely, BMS 986120 inhibited PAR4‐AP–triggered platelet activation in a concentration‐ and time‐dependent manner, with no inhibition of PAR1‐AP‐triggered platelet activation (C, D). Some weak enhancement of PAR1‐AP‐triggered platelet activation by BMS 986120 was noted. Concentrations and incubation time of antagonists for future experiments was chosen such that the primary target was completely inhibited but the other PAR was not affected. Data are mean ± standard error; n = 4 (*P < .05; **P < .01; ***P < .001 compared to vehicle‐treated control). FITC, fluorescein isothiocyanate; PAR, protease‐activated receptor