G_12/13 signaling is required for thrombin- and thrombin receptor-activating peptide-induced Akt phosphorylation. (A) YM-254890 does not inhibit G_12/13-induced platelet shape change. Aspirin-treated, washed human platelets preincubated with 10 μM Y-27632 or 50 nM YM-2548890 were stimulated with agonists as noted, and platelet aggregation was measured as described in “Materials and methods.” The arrows indicate the addition of agonists and antagonists. Ten micromolar MRS-2179 and 1 μM AR-C69931MX was added to samples prior to stimulation with agonists. Tracings are representative of 3 experiments from 3 different donors. (B) Effect of YM-254890 on 2-MeSADP-induced Akt phosphorylation. Washed human platelets were stimulated with 1 μM 2-MeSADP in the presence or absence of reagents as indicated. Platelet proteins were separated by SDS-PAGE, Western blotted, and probed with anti-phospho-Akt (Ser⁴⁷³) antibody. (C) Selective G_12/13 signaling restores PAR agonist-induced Akt phosphorylation in the presence of G_i/G_z signaling. Washed human platelets preincubated in the absence and presence of 50 nM YM-254890 were stimulated at 37°C for 5 minutes with either SFLLRN (10 μM), AYPGKF (500 μM), or thrombin (0.5 unit/mL). The addition of 1 μM 2-MeSADP or 10 μM epinephrine was made as indicated. Samples were separated by SDS-PAGE, Western blotted, and probed with anti-phospho-Akt (Ser⁴⁷³) antibody. Results are representative of 3 experiments.