Evidence for a glycoprotein IIb-IIIa- and aggregation-independent mechanism of phosphatidylinositol 3′,4′-bisphosphate synthesis in human platelets

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.