Thrombin-induced activation of RhoA in platelet shape change.

Thrombin-induced activation of RhoA and its involvement in the regulation of myosin II light chain(20) phosphorylation (MLC-P) in alpha-toxin permeabilized platelets was investigated. Permeabilized platelets, expressing normal levels of P-selectin, displayed a Ca(2+)-dependent increase in shape change and MLC-P. Thrombin activated RhoA as measured by a rhotekin-binding assay within 30 s of stimulation under conditions of constant Ca(2+). Under the same conditions and timecourse, thrombin or GTPgammaS induced an increase in MLC-P and platelet shape change which was not dependent on an increase in Ca(2+). The thrombin- and GTPgammaS-induced MLC-P in constant Ca(2+) was inhibited by the addition of Y27632, a Rho-kinase inhibitor. This study directly demonstrates that thrombin can activate RhoA in platelets in a timecourse compatible with a role in increasing MLC-P and shape change (not involving an increase in Ca(2+)). This is also Rho-kinase-dependent.