Rho kinase–dependent activation of LIMK-1 without stimulation of cofilin phosphorylation during shape change. (A, top) LIMK-1 but not LIMK-2 is expressed in platelets. Resting platelets, platelets stimulated for 30 and 60 seconds with thrombin, and endothelial-cell (EC) lysates were immunoblotted with specific anti–LIMK-1 and anti–LIMK-2 antibodies. (Bottom) Identification of cofilin in its unphosphorylated and phosphorylated states in resting platelets. Resting platelet lysates were subjected to isoelectric focusing (IEF) electrophoresis and subsequently immunoblotted with anti-cofilin antibody. Top band is the more basic, dephosphorylated form of cofilin and bottom band is the more acidic, phosphorylated form of cofilin. (B) LIMK-1 and cofilin phosphorylation during platelet shape change induced by thrombin (0.075 U/mL). Graphic representation of the result for LIMK-1 and cofilin phosphorylation. Values are the mean + SD for 3 independent experiments. (C) Effect of Y-27632 (20 μM) on LIMK-1 phosphorylation and cofilin phosphorylation in resting platelets and during thrombin (0.075 U/mL)–induced shape change. (Left) Representative immunoblots of platelets blotted with anti–phospho-LIMK-1/LIMK-2 (Thr508/505), anti–LIMK-1, anti–P-cofilin (Ser3) and anti-cofilin antibodies. (Right) Bar diagram showing cofilin phosphorylation of nontreated (□) and Y-27632-treated (▪) platelets in control and after thrombin stimulation (120 seconds). Values for cofilin phosphorylation in resting platelets and activated platelets are mean + SD of 8 and 4 independent experiments, respectively. *Statistically significant; P < .05 with respect to nontreated control.