A selective role for phosphatidylinositol 3,4,5-trisphosphate in the G_i-dependent activation of platelet Rap1B

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 Feγ receptor IIA (FcγRIIA) in the presence of ADP scavengers. Moreover, elevation of the intracellular cAMP concentration did not impair the G_i-dependent 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 FcγRIIA, by stimulation of the P2Y₁₂ receptor for ADP, or by epinephrine. However, in platelets from PI3Kγ-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 PI3Kγ 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)P₃), but not PtdIns(3,4)P₂, although both lipids were found to support phosphorylation of Akt. Moreover, PtdIns(3,4,5)P₃ was able to relieve the inhibitory effect of apyrase on FcγRIIA-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)P₃, but not PtdIns(3,4)P₂, and that this process may contribute to potentiation of platelet aggregation.