In thrombin-stimulated platelets (XiibP3 integrin engagement triggers both phosphatidylinositol 3',4'-6wphosphate synthesis and calpain activation. We checked the possible involvement of calpains in phosphatidylinositol 3-kinase signalling pathway using a cell permeant specific inhibitor of calpains, calpeptin. In conditions where thrombin-induced platelet aggregation and secretion were not impaired, we found a dosedependent inhibition of phosphatidylinositol 3,4-6wphosphate synthesis by calpeptin from 50 jig/ml. Moreover, pretreatment of platelets by both calpeptin and the peptide RGDS, an inhibitor of fibrinogen binding to activated an b p 3 integrin, did not induce additive effects on phosphatidylinositol 3,4-/>/vphosphate inhibition. Finally, the p85 regulatory subunit of phosphatidylinositol 3-kinase was still translocated to the cytoskeleton in calpeptintreated platelets. These data indicate that calpains are involved in the regulation of a 1Ib p 3 integrin-dependent phosphatidylinositol 3-kinase signalling pathway.

The synthesis of phosphatidylinositol 3',4'-bisphosphate (PtdIns(3,4)P2) in 32P-labeled human platelets induced by the tetrameric lectin concanavalin A and the physiological agonist thrombin were compared. Like thrombin, concanavalin A stimulated a time-dependent accumulation of PtdIns(3,4)P2, which reached maximal levels after 5 min of stimulation. However, while synthesis of PtdIns(3,4)P2 induced by thrombin was dependent on platelet aggregation, the production of PtdIns(3,4)P2 induced by concanavalin A was unchanged when aggregation was prevented by the omission of stirring or when fibrinogen binding to platelets was inhibited by the tetrapeptide RGDS. Accumulation of PtdIns(3,4)P2 was not observed in platelets stimulated with succinyl-concanavalin A, a dimeric derivative of the lectin that binds to the same receptors on the platelet surface but does not promote clustering of membrane glycoproteins. The synthesis of PtdIns(3,4)P2 induced by concanavalin A was also independent of the membrane glycoprotein IIb-IIIa, as normal accumulation of this lipid was observed in platelets from two patients affected by Glanzmann thrombasthenia. In contrast, thrombin showed a strongly reduced ability to stimulate PtdIns(3,4)P2 production in thrombasthenic platelets. Although concanavalin A was able to induce association of the regulatory subunit of the phosphatidylinositol 3-kinase with tyrosine-phosphorylated proteins, the tyrosine kinase inhibitor tyrphostin AG-213 did not inhibit the lectin-induced synthesis of PtdIns(3,4)P2. These results demonstrate the existence of a novel mechanism of PtdIns(3,4)P2 synthesis in human platelets, which is independent of glycoprotein IIb-IIIa and aggregation, but requires clustering of membrane glycoproteins. As clustering events occur during platelet aggregation promoted by physiological agonists, this new mechanism may also be involved in the aggregation-dependent production of PtdIns(3,4)P2 in thrombin-stimulated platelets.