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J Biol Chem, Vol. 274, Issue 40, 28293-28300, October 1, 1999

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

Benjamin Z. S. Paul, James L. Daniel^§, and Satya P. Kunapuli^§¶

From the Departments of  Pharmacology and ^¶ Physiology and the ^§ Sol Sherry Thrombosis Research Center, Temple University Medical School, Philadelphia, Pennsylvania 19140

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 Ca²⁺ increases were prevented using the cytosolic Ca²⁺ 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 Ca²⁺, 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 Ca²⁺-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 Ca²⁺/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 Ca²⁺-sensitive and Ca²⁺-insensitive pathways, respectively.

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