CS585 is selective to activation of the IP receptor. (A) Aggregation of washed human platelets treated with vehicle or an IP receptor inhibitor (Ro 1138542, 5 μM), DP1 receptor inhibitor (MK-0542, 4 nM), or EP2 and EP4 receptor inhibitors (TG4-155, 2 μM and CJ-42794, 80 nM, respectively) before treatment with vehicle or CS585 (0.05-100 nM) stimulated with collagen (0.5 μg/mL; n = 4-5). Data represent mean ± SEM. Two-factor mixed-effects analysis with Tukey multiple comparisons test. Each condition is compared with its corresponding vehicle. (B) Representative aggregation curves for panel A. (C) Activation of integrin αIIbβ3, α-granule, and dense granule secretion of washed human platelets treated with the vehicle or an IP receptor inhibitor (Ro 1138542, 5 μM), DP1 receptor inhibitor (MK-0542, 4 nM), or EP2 and EP4 receptor inhibitors (TG4-155, 2 μM and CJ-42794, 80 nM) before treatment with the vehicle or CS585 (12.5-100 nM), stimulated with convulxin (25 ng/mL) (n = 4-5). The results are expressed as the relative mean (percentage of vehicle MFI) ± SEM. Two-factor mixed-effects analysis with Sidak multiple comparisons. Each condition is compared with the corresponding vehicle. (D) Expression of Ser157 pVASP (50 kDa) and GAPDH (37 kDa) in washed human platelets treated with vehicle or an IP receptor inhibitor (Ro 1138542, 5 μM), DP1 receptor inhibitor (MK-0542, 4 nM), or EP2 and EP4 receptor inhibitors (TG4-155, 2 μM and CJ-42794, 80 nM, respectively) before treatment with vehicle or CS585 (10 pM-100 nM; n = 4). Data represent mean ± SEM. Two-way ANOVA with Dunnett correction. Asterisks denote statistical differences between vehicle and treated groups. (E) Expression of Ser157 pVASP (50 kDa) and GAPDH (37 kDa) in washed platelets from WT and IP receptor-deficient (IP−/−) mice treated with vehicle, CS585 (1, 5, or 10 μM), 12-HETrE (10 or 25 μM), or forskolin (5 μM; n = 3; 2 mice pooled per n). ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001.
