The Rho kinase inhibitor Y-27632 inhibits shape change, F-actin increase, and MYPT phosphorylation induced by thrombin. Platelets were incubated with Y-27632 (20 μM) or solvent (H2O) for 30 minutes and then stimulated by thrombin (0.075 U/mL). (A) Inhibition of shape change by Y-27632. Shape change was measured by the decrease in light transmission, (left) and by confocal fluorescence microscopy of platelets stained for F-actin with Alexa Fluor 546 phalloidin (right). Bar, 10 μm. (B) Effect of Y-27632 on F-actin content during shape change. Values are mean + SD of 4 independent experiments. *Statistically significant; P < .05 with respect to time (0 seconds). †Significance between control (□) and Y-27632 (▪)–treated unstimulated platelets. (C, left) Platelet lysates were immunoblotted with anti–phospho-Thr853-MYPT antibody. Graphic representation of the result of MYPT phosphorylation in thrombin (0.075 U/mL)–stimulated platelets, evaluated by densitometry. Values are mean + SD of 3 experiments with platelets from different donors. (Right) Representative Western blot of MYPT phosphorylation in the absence and presence of Y-27632.
