Specific RAP isoforms regulate granule secretion, RAC1 activation, and TxA₂generation. Dose response studies of thrombin-induced (A) or convulxin-induced (B) P-selectin exposure (anti-CD62P binding by flow cytometry; clone RB40.34; BD Biosciences). Data shown are mean fluorescence intensity (MFI) ± SEM of control (Rap1a^fl/flRap1b^fl/flPf4-Cre⁻; black), Rap1a-mKO (Rap1a^fl/flRap1b^wt/wtPf4-Cre⁺; green), Rap1b-mKO (Rap1a^wt/wtRap1b^fl/flPf4-Cre⁺; orange), and Rap1a/b-mKO (Rap1a^fl/flRap1b^fl/flPf4-Cre⁺; red) platelets (n = 6). (C-D) RAC1-GTP pull-down assay. (C) RAC1 activation was determined by pull-down assay in platelets of the indicated genotype stimulated for 0, 3, or 10 minutes with convulxin (Cvx). Total RAC1 was determined as loading control. Western blot images are representative of 3 independent experiments. (D) The ratio of RAC1-GTP over RAC1 band intensity for the 3 experiments is shown as fold increase over resting. (E-F) TxB₂-generation assay. (E) Washed platelets from control, Rap1a-mKO, Rap1b-mKO, and Rap1a/b-mKO mice were stimulated in standard aggregometry (data not shown) with low doses of Cvx (LD Cvx; 0.16 µg/mL). After 0, 3, and 10 minutes of stimulation, samples were withdrawn to measure the levels of TxB₂, the stable product of TxA₂. (F) In similar experimental conditions, Rap1a/b-mKO and Caldaggef1^−/−P2y12^−/− platelets were stimulated for 10 minutes with low-dose Cvx (LD Cvx; 0.16 µg/mL) or high-dose Cvx (HD Cvx; 1.6 µg/mL). *P < .05, **P < .01, ***P < .001. ns, not significant.