Abstract. Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (Ptdlns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29 % of the p85o~ regulatory subunit of phosphoinositide 3-kinase (Ptdlns 3-kinase) and is accompanied by a significant increase in Ptdlns 3-kinase activity in this subcellular fraction. Actually, Ptdlns(3,4)P2 accumulation and Ptdlns 3-kinase, pp60 ~-s'~, and p125 eA~ translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of Ptdlns(3,4)P2 and the relocalization of Ptdlns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85o~ was detected in anti-phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85a. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85et with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with Ptdlns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85tx with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125 rAK. In addition, we show that Ptdlns 3-kinase is significantly activated by the p125 rAK proline-rich sequence binding to the src homology 3 domain of p85a subunit. This interaction may represent a new mechanism for Ptdlns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas linked to the actin filaments play a critical role in signaling events that occur upon ligand engagement of o~ro/~3 integrin and platelet aggregation evoked by thrombin.T HE accumulation of phosphatidylinositol 3,4-bisphosphate (Ptdlns(3,4)P2) m and Ptdlns(3,4,5)P3 in thrombin-stimulated human platelets is now well established (16,24,38,41) and seems to be regulated differently (38). However, the exact mechanism of activation of phosphoinositide 3-kinase (Ptdlns 3-kinase) in these ceils is still obscure.

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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.