Activation of Rap1B by Gi family members in platelets

It has become increasingly appreciated that receptors coupled to Galpha(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 Gai family members in platelets. First, we show that platelets from mice lacking the Gai family member Galpha(z) (which couples to the alpha(2A) adrenergic receptor) are deficient in epinephrine-stimulated Rap1 activation. We also show that platelets from mice lacking Galpha(i2), which couples to the ADP receptor, P2Y12, exhibit reduced Rap1 activation in response to ADP. In contrast, platelets from mice that lack Galpha(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 Galpha(i)-coupled P2Y12 receptor and the Galpha(q)-coupled P2Y1 receptor. Galpha(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 Galpha(12)-/- mouse. Finally, using human platelets treated with selective inhibitors of phosphatidylinositol 3-kinase (PI3K) and mouse platelets selectively lacking the Goy-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(q)- and PI3K-dependent mechanisms in platelets and by G(q)- and Ca2+-dependent mechanisms, both of which may play a role in promoting platelet activation.