Evidence for a Glycoprotein IIb-IIIa- and Aggregation-independent Mechanism of Phosphatidylinositol 3′,4′-Bisphosphate Synthesis in Human Platelets

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.

The platelet cytoskeleton regulates the aggregation‐dependent synthesis of phosphatidylinositol 3,4‐bisphosphate induced by thrombin

“…This model is also supported by our results on the effect of cytochalasin D on PtdIns(3,4)P 2 production induced by the lectin ConA. It is known that this lectin is a potent stimulator of actin polymerization and cytoskeleton reorganization, and promotes the synthesis of PtdIns(3,4)P 2 through a mechanism totally independent of platelet aggregation [7]. Thus, while two mechanisms for PtdIns(3,4)P 2 production are operating in thrombin‐stimulated platelets, aggregation‐independent and aggregation‐dependent, only the former one takes place in ConA‐treated platelets.…”

“…To confirm the correlation between the aggregation‐dependent and the cytochalasin D‐sensitive synthesis of PtdIns(3,4)P 2 , we analyzed 32 P‐labeled platelets stimulated with the lectin ConA. We have previously demonstrated that ConA induces the accumulation of PtdIns(3,4)P 2 exclusively through a mechanism independent of integrin α IIb ‐β 3 and aggregation [7]. As shown in Fig.…”

“…This delayed but sustained production of PtdIns(3,4)P 2 has been related to platelet aggregation, because it is prevented by inhibitors of fibrinogen binding to integrin α IIb ‐β 3 and does not occur in platelets from patients affected by Glanzmann thrombasthenia [5,6]. However, the evidence that, in these conditions, the accumulation of PtdIns(3,4)P 2 is reduced but not abolished, and the observation that some agonists, like the lectin concanavalin A (ConA) or lysophosphatidic acid, can induce the synthesis of this lipid in the absence of aggregation [7,8] suggested the existence of a dual mechanism, aggregation‐independent and aggregation‐dependent, for PtdIns(3,4)P 2 accumulation in stimulated platelets.…”

Phosphatidylinositol 3,4,5-Trisphosphate Triggers Platelet Aggregation by Activating Ca2+ Influx

“…Substantial evidence indicates that the syntheses of PtdIns-(3,4,5)P 3 and PtdIns(3,4)P 2 are regulated via distinct mechanisms (24)(25)(26)(27)(28), and that these two D-3 phosphoinositides play different functional roles. For example, PtdIns(3,4)P 2 † This work was supported in part by National Institutes of Health Grant R01 GM53448 and by American Heart Association, Kentucky Affiliate, Grant KY-97-GS-26.…”

Clustering of Integrin αIIb-β3Differently Regulates Tyrosine Phosphorylation of pp72syk, PLCγ2 and pp125FAKin Concanavalin A-stimulated Platelets

“…The ability of ConA to induce, even in the absence of platelet aggregation, events that in thrombin-treated platelets are normally observed only during aggregation is a tipical feature of this lectin. We have recently demonstrated, for instance, that activation of the phosphatidylinositol 3-kinase, which is largely dependent on aggregation in thrombin-stimulated platelets, occurs through an aggregation-independent mechanism in ConA-treated platelets (30). This peculiarity of ConA may be due to the tetrameric binding properties of lectin, which induces topographic changes on the cell surface reproducing events occuring during aggregation.…”