A Gi-dependent Pathway Is Required for Activation of the Small GTPase Rap1B in Human Platelets

Paolo Lova,Simona Paganini,Fabiola Sinigaglia,Cesare Balduini,Mauro Torti

doi:10.1074/jbc.m111803200

Abstract

Stimulation of human platelets by cross-linking of the low affinity receptor for immunoglobulin, FcgammaRIIA, caused the rapid activation of the small GTPase Rap1B, as monitored by accumulation of the GTP-bound form of the protein. This process was totally dependent on the action of secreted ADP since it was completely prevented in the presence of either apyrase or creatine phosphate and creatine phosphokinase. Dose-dependent experiments revealed that the inhibitory effect of ADP scavengers was not related to the reduced increase of cytosolic Ca(2+) concentration in stimulated platelets. Activation of Rap1B induced by clustering of FcgammaRIIA was totally suppressed by AR-C69931MX, a specific antagonist of the G(i)-coupled ADP receptor P2Y12, but was not affected by blockade of the G(q)-coupled receptor, P2Y1. Similarly, direct stimulation of platelets with ADP induced the rapid activation of Rap1B. Pharmacological blockade of the P2Y1 receptor totally prevented ADP-induced Ca(2+) mobilization but did not affect activation of Rap1B. By contrast, prevention of ADP binding to the P2Y12 receptor totally suppressed activation of Rap1B without affecting Ca(2+) signaling. In platelets stimulated by cross-linking of FcgammaRIIA, inhibition of Rap1B activation by ADP scavengers could be overcome by the simultaneous recruitment of the G(i)-coupled alpha(2A)-adrenergic receptor by epinephrine. By contrast, serotonin, which binds to a G(q)-coupled receptor, could not restore activation of Rap1B. When tested alone, epinephrine was found to be able to induce GTP binding to Rap1B, whereas serotonin produced only a slight effect. Finally, activation of Rap1B induced by stimulation of the G(q)-coupled thromboxane A(2) receptor by was completely inhibited by ADP scavengers under conditions in which intracellular Ca(2+) mobilization was unaffected. Inhibition of -induced Rap1B activation was also observed upon blockade of the P2Y12 but not of the P2Y1 receptor for ADP. These results demonstrate that stimulation of a G(i)-dependent signaling pathway by either ADP of epinephrine is necessary and sufficient to activate the small GTPase Rap1B.

Highlights

Rap1 proteins are members of a family of small GTPases highly related to the product of the ras protooncogene
Even when platelet stimulation is promoted by the recruitment of receptors that are linked to tyrosine kinase-based signaling pathways rather than heterotrimeric G-proteins, such as in the case of Fc␥RIIA1 crosslinking, platelet responses largely depend on the activation of the Gi-coupled receptor P2Y12 by secreted ADP [23]
Activation of Rap1B in Human Platelets Stimulated by Cross-linking of Fc␥RIIA—Samples of gel-filtered platelets were placed in an aggregometer under constant stirring and treated with 2 ␮g/ml anti-Fc␥RIIA mAb IV.3 and 30 ␮g/ml sheep anti-mouse F(abЈ)2 fragments for increasing times

Summary

Introduction

Rap proteins are members of a family of small GTPases highly related to the product of the ras protooncogene. In the most recent studies, a mounting body of evidence indicates that platelet aggregation induced by many different agonists results from concomitant signaling through both Gqand Gi-coupled receptors. This concept has been initially developed upon studies on ADP-induced platelet activation. Even when platelet stimulation is promoted by the recruitment of receptors that are linked to tyrosine kinase-based signaling pathways rather than heterotrimeric G-proteins, such as in the case of Fc␥RIIA1 crosslinking, platelet responses largely depend on the activation of the Gi-coupled receptor P2Y12 by secreted ADP [23]. This results reveal a new link between heterotrimeric G-proteins of the Gi family and small GTPases of the Rap family and suggest a potential mechanism responsible for the potentiation of platelet activation by Gi-coupled receptors

Methods

Results

Conclusion