Tyrosine kinases and phosphoinositide metabolism in thrombin-stimulated human platelets

In this study we have examined the implication of tyrosine kinase activities in aggregation, 5-hydroxytryptamine secretion and mainly phosphoinositide metabolism in response to human platelet stimulation by thrombin. Using the potent tyrosine kinase inhibitor tyrphostin AG-213, we have observed a significant inhibition of aggregation and 5-hydroxytryptamine release; however, this percentage inhibition was lower at high thrombin concentrations. On the other hand, tyrphostin treatment of metabolically 32P-labelled platelets significantly inhibited the thrombin-dependent accumulation of PtdIns(3,4)P2, which involves at least a PtdIns 3-kinase and/or a PtdIns3P 4-kinase, whereas the synthesis of phosphatidic acid (PtdOH), a good reflection of the phospholipase C (PLC) activation in platelets, was partially blocked. Inositol phosphate production was also inhibited by about 40% when tyrphostin-treated platelets were stimulated with thrombin. In addition, we show by Western-blot analysis that PLC gamma 1, as well as the regulatory subunit (p85) of the PtdIns 3-kinase, were present in the anti-phosphotyrosine immunoprecipitate isolated from thrombin-stimulated platelets. Furthermore, tyrphostin treatment clearly decreased the PLC gamma 1 and p85 contents in such an anti-phosphotyrosine immunoprecipitate. Our results provide the first evidence for a direct or indirect regulation of PtdIns(3,4)P2 accumulation and PLC gamma 1 activity by tyrosine phosphorylation during thrombin stimulation of human platelets.