Independent activation of Rac1-mediated and PKC-mediated pathways mediating secretion induced by thrombin. (A) No inhibition of Rac1 activation by the PKC inhibitor Gö6983 (2 μM) in thrombin-stimulated platelets. Rac1-GTP was pulled down from untreated platelets (−) and platelets pretreated with Gö6983 (+) and then stimulated with 0.5 U/mL thrombin. Representative immunoblots of pulled down Rac1-GTP and total Rac1. (B) Inhibition of PKC_δ (Thr505) phosphorylation by Gö6983, but not by the Rac1 inhibitor NSC23766. Platelet lysates of thrombin-stimulated platelets untreated or pretreated with Gö6983 (top) or NSC23766 (bottom) were immunoblotted with anti–phospho-PKC_δ (Thr505) antibody. Representative immunoblots for PKC_δ phosphorylation. (C) PDBu-induced secretion is independent of Rac1 activation. Platelets untreated (left) or pretreated with 300 μM NSC23766 or 2 μM Gö6983 were stimulated with 200 nM PDBu. Platelet aggregation and ATP secretion was monitored in lumi-aggregometer. Representative tracings for change in light transmission and ATP secretion (×0.5 gain) are shown.