INVOLVEMENT OF PROTEIN-TYROSINE KINASE P72(SYK) IN COLLAGEN-INDUCED SIGNAL-TRANSDUCTION IN PLATELETS

Previous studies have demonstrated that activation of platelets by col lagen results in a dramatic increase in tyrosine phosphorylation of se veral cellular proteins, including pp125(FAK), through the interaction of collagen with integrin alpha(2) beta(1) (GP Ia-IIa). In this study , we report that p72(syk) is a potential candidate for the protein-tyr osine phosphorylation event following collagen stimulation in porcine platelets. Washed platelets were stimulated with collagen and the acti vation of p72(syk) was assessed in an immunoprecipitation kinase assay . The activity of p72(syk) increased within 1 min, reached a maximum a t 5 min after stimulation by collagen, and the phosphorylation at tyro sine residues of p72(syk) in platelets also occurred in the same time course as the activation of p72(syk). Prior treatment of platelets wit h cytochalasin D to inhibit actin polymerization, or with aspirin and apyrase to inhibit the secondary reaction, or EGTA and the acetoxymeth yl ester of -bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid to chelate both extracellular and intracellular Ca2+, did not affect the activation of p72(syk) induced by collagen. Furthermore, herbimycin A , a potent protein tyrosine-kinase inhibitor, was capable of reducing collagen-evoked p72(syk) activation, Ca2+ mobilization and platelet ag gregation. These results suggest that upon stimulation by collagen p72 (syk) is physically activated by a process that is independent of the effects of Ca2+, ADP, and actin polymerization, and may participate in the regulation of Ca2+ mobilization mediated by collagen in platelets .