PI3-kinase is downstream of Rac1 activation, but it is not involved in cofilin dephosphorylation. (A) Left, Rac1 is upstream to PI3-kinase in thrombin-stimulated platelets. Rac1-GTP from resting platelets (lane 1) and platelets stimulated with thrombin for 5 seconds that were previously nontreated (lane 2) or pretreated with 100 nM wortmannin (lane 3) was pulled down and immunoblotted with anti-Rac1 antibody. Representative immunoblots showing Rac1 activation. Right, Rac1 activity is independent of P2Y₁₂ receptor activation. Rac1-GTP from resting platelets and platelets stimulated with thrombin for 5 seconds that were previously nontreated or pretreated with 2 μM AR-C69931MX, alternate lanes. Values are the mean ± SD for 4 independent experiments. (B) Inhibition of Akt (Ser473) phosphorylation by 300 μM NSC23766 (top) and 2 μM AR-C69931MX (bottom). Lysates of platelets stimulated with thrombin for 0, 30, and 120 seconds in the absence or presence of NSC23766 and AR-C69931MX (alternate lanes) were immunoblotted with anti–phospho (Ser473)–Akt antibody. Values are the mean ± SD for 4 independent experiments. (C) No inhibition of cofilin dephosphorylation by 100 nM wortmannin and 40 μM LY294002 in thrombin-stimulated platelets. Platelets lysates were immunoblotted with anti–phospho-cofilin antibody. Graphical representation of the cofilin phosphorylation in nontreated platelets (-■-) and platelets pretreated with wortmannin (-□-) and LY294002 (-▴-) during platelet stimulation with thrombin. Values are the mean + SD for 3 independent experiments.