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 Fcgamma receptor IIA (FcgammaRIIA) 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 FcgammaRIIA, by stimulation of the P2Y(12) receptor for ADP, or by epinephrine. However, in platelets from PI3Kgamma-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 PI3Kgamma 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 FcgammaRIIA-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.
Highlights
The small GTP-binding protein Rap1B is activated in human platelets upon stimulation of a Gi-dependent signaling pathway
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
Gi-dependent Activation of Rap1B Does Not Require Inhibition of Adenylyl Cyclase—We have previously demonstrated that stimulation of a Gi-dependent pathway by epinephrine or by binding of ADP to the P2Y12 receptor is sufficient to trigger activation of the small GTPase Rap1B and that Rap1B activation promoted by cross-linking of Fc␥RIIA is completely dependent on the stimulation of the Gi-coupled P2Y12 receptor by secreted ADP (7)
Summary
Fc␥RIIA, Fc␥ receptor IIA; PI3K, phosphatidylinositol 3-kinase; PAPS, adenosine 3Ј-phosphate 5Ј-phosphosulfate; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(3,4)P2, phosphatidylinositol 3,4-bisphosphate; mAb, monoclonal antibody; RBD, Rap-binding domain; GST, glutathione S-transferase; RIPA, radioimmune precipitation assay. The recent finding that stimulation of a Gi-coupled receptor is sufficient to trigger activation of Rap1B suggests that this small GTPase could be involved in potentiation of platelet aggregation. We have investigated the mechanism of Rap1B activation downstream of stimulation of Gi-coupled receptors in an attempt to reveal a correlation between this small GTPase and the potentiation of agonist-induced platelet activation. We have found that inhibition of adenylyl cyclase is not sufficient to stimulate GTP binding to Rap1B. We provide evidence suggesting that activation of Rap1B downstream of PI3K is associated with the.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
