title: Identification of P2Y12-dependent and -independent mechanisms of glycoprotein VI-mediated Rap1 activation in platelets.
abstract: Glycoprotein (GP) VI is a critical platelet collagen receptor, yet the steps involved in GPVI-mediated platelet activation remain incompletely understood. Because activation of Rap1, an abundant small guanosine triphosphatase (GTPase) in platelets, contributes to integrin alpha(IIb)beta(3) activation, we asked whether and how GPVI signaling activates Rap1 in platelets. Here we show that platelet Rap1 is robustly activated upon addition of convulxin, a GPVI-specific agonist. Using a reconstituted system in RBL-2H3 cells, we found that GPVI-mediated Rap1 activation is dependent on FcRgamma but independent of another platelet collagen receptor, alpha(2)beta(1). Interestingly, GPVI-mediated Rap1 activation in human platelets is largely dependent on adenosine diphosphate (ADP) signaling through the P2Y(12) and not the P2Y(1) receptor. However, experiments with specific ADP receptor antagonists and platelets from knockout mice deficient in P2Y(1) or the P2Y(12)-associated G-protein, Galphai(2), indicate that human and murine platelets also have a significant P2Y(12)-independent component of GPVI-mediated Rap1 activation. The P2Y(12)-independent component is dependent on phosphatidylinositol 3-kinase and is augmented by epinephrine-mediated signaling. P2Y(12)-dependent and -independent components are also observed in GPVI-mediated platelet aggregation, further supporting a role for Rap1 in aggregation. These results define mechanisms of GPVI-mediated platelet activation and implicate Rap1 as a key signaling protein in GPVI-induced platelet signaling.
authors.affiliation: Department of Pharmacology, Center for Thrombosis and Hemostasis and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, 27599, USA.
authors.forename: Mark K
authors.initials: MK
authors.lastname: Larson
authors.forename: Hong
authors.initials: H
authors.lastname: Chen
authors.forename: Mark L
authors.initials: ML
authors.lastname: Kahn
authors.forename: Anne M
authors.initials: AM
authors.lastname: Taylor
authors.forename: Jean-Etienne
authors.initials: JE
authors.lastname: Fabre
authors.forename: Richard M
authors.initials: RM
authors.lastname: Mortensen
authors.forename: Pamela B
authors.initials: PB
authors.lastname: Conley
authors.forename: Leslie V
authors.initials: LV
authors.lastname: Parise
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001792:Blood Platelets / Q000201:enzymology / Q000502:physiology*; D003435:Crotalid Venoms / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004837:Epinephrine / Q000502:physiology; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000097:blood / Q000172:deficiency; D006801:Humans; D038982:Integrin alpha2beta1 / Q000097:blood; D037181:Lectins, C-Type; D008565:Membrane Proteins; D051379:Mice; D018345:Mice, Knockout; D019869:Phosphatidylinositol 3-Kinases / Q000097:blood; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000097:blood / Q000172:deficiency; D058919:Purinergic P2 Receptor Antagonists; D017452:Receptors, IgG / Q000097:blood; D018048:Receptors, Purinergic P2 / Q000097:blood / Q000172:deficiency / Q000502:physiology*; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; D015398:Signal Transduction; D020827:rap1 GTP-Binding Proteins / Q000097:blood*
chemical_list: D003435:Crotalid Venoms; D038982:Integrin alpha2beta1; D037181:Lectins, C-Type; D008565:Membrane Proteins; C546382:P2RY1 protein, human; C548576:P2RY12 protein, human; C546383:P2ry1 protein, mouse; C548577:P2ry12 protein, mouse; D010980:Platelet Membrane Glycoproteins; D011518:Proto-Oncogene Proteins; D058919:Purinergic P2 Receptor Antagonists; D017452:Receptors, IgG; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; C098341:platelet membrane glycoprotein VI; C028890:convulxin; D000244:Adenosine Diphosphate; D019869:Phosphatidylinositol 3-Kinases; C498015:GNAI2 protein, human; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C498016:Gnai2 protein, mouse; D020827:rap1 GTP-Binding Proteins; D004837:Epinephrine