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Abstract
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.
Highlights
The biochemical pathways and the regulatory mechanisms leading to the production of the 3-phosphorylated phosphoinositides are not completely understood
In human platelets stimulated with thrombin, the Ptdlns 3-kinase was found associated with the tyrosine kinases pp60src, pp5gfyn, and pp120F AK (6, 7), and the accumulation of Ptdlns(3,4)P2 was reduced by preincubation of the cells with the tyrosine kinase inhibitor tyrphostin (B)
The HPLC profile of the deacylated lipids from 32P-labeled platelets stimulated with concanavalin A (ConA) was very similar to that from thrombin-stimulated platelets, indicating that, like thrombin, ConA induced both phospholipase C activation and accumulation of Ptdlns(3,4)P2' We observed a small amount of GroPlns(3)P in resting platelets, which did not significantly change upon treatment with the agonists
Summary
Vol 270, No 22, Issue of June 2, pp. 13179-13185, 1995 Printed in U.S.A. Evidence for a Glycoprotein Ilb-Illa- and Aggregation-independent Mechanism of Phosphatidylinositol 3',4'-Bisphosphate Synthesis in Human Platelets*. 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 Ptdlns(3,4)P2 These results demonstrate the existence of a novel mechanism of Ptdlns(3,4)P2 synthesis in human platelets, which is independent of glycoprotein lIb-IlIa and aggregation, but requires clustering of membrane glycoproteins. A strongly reduced accumulation of PtdIns(3,4)P2 in response to thrombin was observed in platelets from patients affected by Glanzmann thrombasthenia, which lack GP Ilb-Illa and fail to aggregate (3) These evidences indicate a regulatory role of GP lIb-IlIa and platelet aggregation on the synthesis of Ptdlns(3,4)P2'. We show that ConA stimulates the accumulation of Ptdlns(3,4)P2 in human platelets and that this effect does not require fibrinogen binding to GP lIb-IlIa and platelet aggregation but is dependent on the lectin-induced clustering ofmembrane glycoproteins
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