The Rac1-specific inhibitor NSC23766 blocks platelet aggregation, dense granule secretion, and Rac1 activation in platelets stimulated with thrombin. (A) Platelets untreated (0 μM) or pretreated with different concentrations of 30 to 300 μM NSC23766 for 5 minutes were stimulated with thrombin (0.1, 0.2, and 0.5 U/mL). NSC23766, dose-dependently inhibited thrombin-induced platelet aggregation (top) and dense granule secretion (bottom). Representative tracings for change in light transmission and ATP secretion (×0.05 gain) are shown, respectively. (B) Rac1 activation and inhibition by NSC23766 in thrombin-stimulated platelets. Top left, time course of Rac1 activation in 0.5 U/mL thrombin-stimulated platelets untreated (−) or pretreated with 300 μM NSC23766 (+). Platelets were stimulated in the presence of RGDS to inhibit aggregation. Representative immunoblots of pulled down Rac1-GTP and total Rac1. Bottom left, dose-dependent increase of Rac1 activation in platelets stimulated with increasing concentrations of thrombin (0.1, 0.2, and 0.5 U/mL) for 5 seconds. Representative immunoblots of pulled down Rac1-GTP and total Rac1 (left), and bar diagram exhibiting fold increase in Rac1 activation (right). Values are the mean + SD for 3 independent experiments. (C) Dose-dependent inhibition of 0.1 U/mL thrombin-induced Rac1 activation by NSC23766 (30 and 50 μM). Representative immunoblots of pulled down Rac1-GTP and total Rac1 from nonstimulated (−) and thrombin-stimulated (+) platelets. Values in mean-fold increase (± SD) obtained from 3 independent experiments are shown beneath the immunoblots. (D) Dose-dependent inhibition of 0.5 U/mL thrombin-induced Rac1 activation by 30 to 300 μM NSC23766. Top, representative immunoblots of pulled down Rac1-GTP and total Rac1 from nonstimulated (−) and thrombin-stimulated (+) platelets. Bottom, bar diagram showing inhibition of thrombin-induced Rac1 activation by NSC23766. Values are the mean + SD for 3 independent experiments.