title: Activation of Rap1B by G(i) family members in platelets.
abstract: It has become increasingly appreciated that receptors coupled to G(alpha)(i) family members can stimulate platelet aggregation, but the mechanism for this has remained unclear. One possible mediator is the small GTPase, Rap1, which has been shown to contribute to integrin activation in several cell lines and to be activated by a calcium-dependent mechanism in platelets. Here, we demonstrate that Rap1 is also activated by G(alpha)(i) family members in platelets. First, we show that platelets from mice lacking the G(alpha)(i) family member G(alpha)(z) (which couples to the alpha(2A) adrenergic receptor) are deficient in epinephrine-stimulated Rap1 activation. We also show that platelets from mice lacking G(alpha)(i2), which couples to the ADP receptor, P2Y12, exhibit reduced Rap1 activation in response to ADP. In contrast, platelets from mice that lack G(alpha)(q) show no decrease in the ability to activate Rap1 in response to epinephrine but show a partial reduction in ADP-stimulated Rap1 activation. This result, combined with studies of human platelets treated with ADP receptor-selective inhibitors, indicates that ADP-stimulated Rap1 activation in human platelets is dependent on both the G(alpha)(i)-coupled P2Y12 receptor and the G(alpha)(q)-coupled P2Y1 receptor. G(alpha)(i)-dependent activation of Rap1 in platelets does not appear to be mediated by enhanced intracellular calcium release because no increase in intracellular calcium concentration was detected in response to epinephrine and because the calcium response to ADP was not diminished in platelets from the G(alpha)(i2)-/- mouse. Finally, using human platelets treated with selective inhibitors of phosphatidylinositol 3-kinase (PI3K) and mouse platelets selectively lacking the G(beta)(gamma)-activated form of his enzyme (PI3Kgamma), we show that G(i)-mediated Rap1 activation is PI3K-dependent. In summary, activation of Rap1 can be stimulated by G(alpha)(i)- and PI3K-dependent mechanisms in platelets and by G(q)- and Ca(2+)-dependent mechanisms, both of which may play a role in promoting platelet activation.
authors.affiliation: Department of Medicine, Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Donna
authors.initials: D
authors.lastname: Woulfe
authors.forename: Hong
authors.initials: H
authors.lastname: Jiang
authors.forename: Richard
authors.initials: R
authors.lastname: Mortensen
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.forename: Lawrence F
authors.initials: LF
authors.lastname: Brass
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000502:physiology; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D004837:Epinephrine / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D018048:Receptors, Purinergic P2 / Q000502:physiology; D058925:Receptors, Purinergic P2Y12; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; C548576:P2RY12 protein, human; C548577:P2ry12 protein, mouse; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D019869:Phosphatidylinositol 3-Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020827:rap1 GTP-Binding Proteins; D000262:Adenylyl Cyclases; D004837:Epinephrine