INTRACELLULAR CALCIUM-DEPENDENT ACTIVATION OF P72(SYK) IN PLATELETS

We previously demonstrated that thrombin-induced activation of p72(syk) was independent of intracellular Ca2+ elevation in platelets. However, our previous studies also demonstrated that activation of platelets by ionophore A23187 results in a dramatic increase in tyrosine phosphorylation of several cellular proteins. In the present study, we investigated the effect of Ca2+ elevation on the activity of p72(syk). When washed porcine platelets were stimulated with ionophore A23187 and the activity of p72(syk) was assessed by means of an immunoprecipitation kinase assay, A23187 caused a time- and dose-dependent increase in the specific activity of p72(syk). In addition, pretreatment of platelets with both aspirin and ADP scavengers or chelation of extracellular Ca2+ by EGTA had no effect on the A23187-induced activation of p72(syk). These results indicate that A23187-induced activation of p72(syk) is independent of the formation of endoperoxide/thromboxage A(2), released ADP and extracellular Ca2+, suggesting the existence of a novel pathway for activation of p72(syk). Furthermore, evidence is presented which indicates a synergistic effect of A23187 and thrombin on the activation of p72(syk), and an inhibitory effect of pretreatment with phorbol 12-myristate 13-acetate, a protein kinase C activator, on the activation of p72(syk) induced by either A23187 or thrombin.