Genetic and pharmacologic evidence that Rac1 GTPase is involved in regulation of platelet secretion and aggregation

Background: Ract GTPase, a member of the Ras-related Rho GTPase family, is the major Rac isoform present in platelets and has been shown to be involved in cell actin cytoskeleton reorganization and adhesion. Agonists that induce platelet secretion and aggregation also activate Rac1 GTPase, raising the possibility that Rac1 GTPase may be involved in regulation of platelet function. Objectives: To rigorously define the role of Rac I in platelet regulation. Methods: We have used a dual approach of gene targeting in mice and pharmacologic inhibition of Rac1 by NSC23766, a rationally designed specific small molecule inhibitor, to study the role of Rac1 in platelet function. Results: Platelets from mice as well as human platelets treated with NSC23766 exhibited a significant decrease in: (i) active Raci species and phosphorylation of the Rac effector, p21-activated kinase; (ii) expression of P-selectin and secretion of adenosine triphosphate induced by thrombin or U46619; and (iii) aggregation induced by adenosine 5'-diphosphate, collagen, thrombin and U46619, a stable analog of thromboxane A(2). NSC23766 did not alter the cAMP or cGMP levels in platelets. Consistent with the requirement of Rac 1 for normal platelet function, the bleeding times in Rac1(-/-) mice or mice given NSC23766 were significantly prolonged. Conclusions: Our data show that deficiency or inhibition of Rac1 GTPase blocks platelet secretion. The inhibition of secretion, at least in part, is responsible for diminished platelet aggregation and prolonged bleeding times observed in Rac1 knockout or Rac1 inhibitor-treated mice.