Sequential regulation of the small GTPase Rap1 in human platelets

Rap1, a small GTPase of the Ras family, is ubiquitously expressed and parti cularly abundant in platelets. Previously we have shown that Rap1 is rapidl y activated after stimulation of human platelets with alpha-thrombin. For t his activation, a phospholipase C-mediated increase in intracellular calciu m is necessary and sufficient. Here we show that thrombin induces a second phase of Rap1 activation, which is mediated by protein kinase C (PKC). Inde ed, the PKC activator phorbol 12-myristate W-acetate induced Rap1 activatio n, whereas the PKC-inhibitor bisindolylmaleimide inhibited the second, but not the first, phase of Rap1 activation. Activation of the integrin alpha(I Ib)beta(3), a downstream target of PKC, with monoclonal antibody LIBS-6 als o induced Rap1 activation. However, studies with alpha(IIb)beta(3)-deficien t platelets from patients with Glanzmann's thrombasthenia type 1 show that alpha(IIb)beta(3) is not essential for Rap1 activation. Interestingly, indu ction of platelet aggregation by thrombin resulted in the inhibition of Rap 1 activation. This downregulation correlated with the translocation of Rap1 to the Triton X-100-insoluble, cytoskeletal fraction. We conclude that in platelets, or-thrombin induces Rap1 activation first by a calcium-mediated pathway independently of PKC and then by a second activation phase mediated by PKC and, in part, integrin alpha(IIb)beta(3). Inactivation of Rap1 is m ediated by an aggregation-dependent process that correlates with the transl ocation of Rap1 to the cytoskeletal fraction.