Inhibition of MYPT phosphorylation, LIMK-1 phosphorylation, LIMK-1 activity, and cofilin rephosphorylation by the Rho kinase inhibitor Y-27632 during thrombin-stimulated aggregation/secretion. Platelet samples stimulated by thrombin (0.5 U/mL) in the absence or presence of the Rho kinase inhibitor Y-27632 (20 μM) were immunoblotted with anti–phospho-MYPT, anti–phospho-LIMK-1/LIMK-2 (Thr508/505), and anti–phospho-cofilin antibodies. (A) Representative immunoblots showing concomitant inhibition of MYPT and LIMK-1 phosphorylation by Y-27632. (B) Immunoblot and graphic representation of the results of cofilin phosphorylation after thrombin stimulation of platelets in the absence (□) or presence of Y-27632 (▪). Values are mean + SD for 3 independent experiments. (C) LIMK-1 phosphorylation parallels LIMK-1 activity in thrombin-stimulated platelets. Inhibition by Y-27632. Platelets were preincubated with Y-27632 (20 μM) or solvent for 30 minutes and stimulated with thrombin in the presence of RGDS. LIMK-1 from resting and activated platelets (120 seconds) was immunoprecipitated. (Top) LIMK-1 immunoprecipitates (LIMK-1 IP) were blotted with anti–LIMK-1 antibody and anti–phospho-LIMK-1/LIMK-2 (Thr508/505) antibody. (Bottom) Immunoprecipitates were assayed for LIMK-1 activity using His-cofilin as substrate. Cofilin phosphorylation was measured by blotting the samples with anti–phospho-cofilin antibodies.