A Selective Role for Phosphatidylinositol 3,4,5-Trisphosphate in the Gi-dependent Activation of Platelet Rap1B

Abstract

The small GTP-binding protein Rap1B is activated in human platelets upon stimulation of a G i -dependent signaling pathway. In this work, we found that inhibition of platelet adenylyl cyclase by dideoxyadenosine or SQ22536 did not cause activation of Rap1B and did not restore Rap1B activation in platelets stimulated by cross-linking of Fc␥ receptor IIA (Fc␥RIIA) in the presence of ADP scavengers. Moreover, elevation of the intracellular cAMP concentration did not impair the G idependent activation of Rap1B. Two unrelated inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, totally prevented Rap1B activation in platelets stimulated by cross-linking of Fc␥RIIA, by stimulation of the P2Y 12 receptor for ADP, or by epinephrine. However, in platelets from PI3K␥-deficient mice, both ADP and epinephrine were still able to normally stimulate Rap1B activation through a PI3K-dependent mechanism, suggesting the involvement of a different isoform of the enzyme. Moreover, the lack of PI3K␥ did not prevent the ability of epinephrine to potentiate platelet aggregation through a G i -dependent pathway. The inhibitory effect of wortmannin on Rap1B activation was overcome by addition of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ), but not PtdIns(3,4)P 2 , although both lipids were found to support phosphorylation of Akt. Moreover, PtdIns(3,4,5)P 3 was able to relieve the inhibitory effect of apyrase on Fc␥RIIA-mediated platelet aggregation. We conclude that stimulation of a G i -dependent signaling pathway causes activation of the small GTPase Rap1B through the action of the PI3K product PtdIns(3,4,5)P 3 , but not PtdIns(3,4)P 2 , and that this process may contribute to potentiation of platelet aggregation.Rap1B is a small GTP-binding protein highly expressed in human platelets (1). In resting cells, it is mainly located at the membrane, but it translocates to the cytosol upon phosphorylation by protein kinase A (2). In activated platelets, Rap1B rapidly interacts with the reorganized actin-based cytoskeleton (3). As other GTPases, Rap1B is activated by binding of GTP. Platelet stimulation by different agonists, such as thrombin, collagen, and ADP, induces the rapid binding of GTP to Rap1B (4, 5). An increase in the intracellular Ca 2ϩ concentration in stimulated platelets has been shown to be sufficient to promote Rap1B activation, and specific Ca 2ϩ /calmodulin-sensitive guanine nucleotide exchange factors for Rap1B have been identified (5, 6). We (7) and others (8) have recently described a new pathway for Rap1B activation that is initiated by stimulation of membrane G i -coupled receptors and that is independent of intracellular Ca 2ϩ increases. In fact, the sole binding of ADP to the P2Y 12 receptor, as well as the interaction of epinephrine with the ␣ 2A -adrenergic receptor, is sufficient to trigger Rap1B activation. Moreover, we have found that agonists that activate platelets through stimulation of G q -coupled receptors, such as the thromboxane A 2 analog U46619, or through stimu...

P2 receptors and platelet function

“…One important intracellular pathway which regulates G i -dependent integrin αIIbβ3 activation is constituted by phosphoinositide 3-kinase (PI 3-K) [56, [67][68][69][70]. PI 3-K isoform p110β regulates integrin activation through a classical lipid kinase-dependent mechanism, involving the small GTPase Rap1 and/or the serine-threonine protein kinase B/Akt (PKB/Akt) [71][72][73][74][75][76], whereas p110γ appears to regulate integrin principally through a non-catalytic signalling mechanism [77,78]. Whether other PI3K class I isoforms such as the p110α or PI3K class II or III isoforms, which are highly expressed in blood platelets, play a role in integrin αIIbβ3 activation remains to be determined.…”

Contribution of Protease-activated Receptors 1 and 4 and Glycoprotein Ib-IX-V in the Gi-independent Activation of Platelet Rap1B by Thrombin

“…Intracellular Ca 2ϩ and protein kinase C have been proposed to mediate agonist-induced Rap1B activation in platelets (10,11). However, more recently it has been reported that platelet Rap1B can also be activated by stimulation of members of the G i family of heterotrimeric G-proteins in the absence of a Ca 2ϩ increase or protein kinase C stimulation (12)(13)(14)(15). For instance, the ␣ 2A -adrenergic receptor, which couples to G␣ z in platelets, mediates epinephrine-induced activation of Rap1B (12,13).…”

Differential proteomic analysis of platelets suggested target-related proteins in rabbit platelets treated with Rhizoma Corydalis

“…Ras-related protein Rap-1b (an abundant small GTPase), which is the predominant Rap 1 isoform and the most abundant Ras family member in platelets, has been reported to activate downstream GPCRs and upstream integrin a IIb b 3 . Predominantly, ADP stimulates Rap 1b activation mainly via a calcium-independent pathway to activate downstream Gai-coupled P2Y12 receptor, and also activates phosphoinositide 3-kinase and its lipid product phosphatidylinositol 3,4,5-triphosphate (Lova et al 2002;Crittenden et al 2004;Chrzanowska-Wodnicka et al 2005). Hence, the expression of Ras-related protein Rap-1b down regulated after treated with RC is corresponded to CalDAG-GEFI (calcium-DAG-GEF/RasGRP protein family) and PI3K signalling pathways.…”