Platelet shape change is mediated by both calcium-dependent and -independent signaling pathways. Role of p160 Rho-associated coiled-coil-containing protein kinase in platelet shape change

Platelets undergo shape change upon activation with agonists. During shape change, disc-shaped platelets turn into spiculated spheres with protruding filopodia. When agonist-induced cytosolic CA(2+) increases were prevented using the cytosolic CA(2+) chelator, 5, 5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5, 5'-dimethyl-BAPTA), platelets still underwent shape change, although the onset was delayed and the initial rate was dramatically decreased. In the absence of cytosolic CA(2+), agonist-stimulated Myosin light chain phosphorylation was significantly inhibited. The Myosin light chain was maximally phosphorylated at 2 s in control platelets compared with 30 s in 5,5'-dimethyl-BAPTA-treated platelets. ADP, Thrombin, or U46619-induced CA(2+)-independent platelet shape change was significantly reduced by staurosporine, a nonselective kinase inhibitor, by the selective p160 Rho-associated coiled-coil-containing protein kinase inhibitor Y-27632, or by HA 1077. Both Y-27632 and HA 1077 reduced peak levels of ADP-induced platelet shape change and Myosin light chain phosphorylation in control platelets. In 5,5'-dimethyl-BAPTA-treated platelets, Y-27632 and HA 1077 completely abolished both ADP-induced platelet shape change and Myosin light chain phosphorylation. Our results indicate that CA(2+)/calmodulin-stimulated Myosin light chain kinase and p160 Rho-associated coiled-coil-containing protein kinase independently contribute to Myosin light chain phosphorylation and platelet shape change, through CA(2+)-sensitive and CA(2+)-insensitive pathways, respectively.