Abstract 58: Differential Signaling by CRP-Rac1-NOX1 and Thrombin-Rac1-NOX2 Axes Regulates Ros Generation and Platelet Activation

Abstract

Agonist induced generation of reactive oxygen species (ROS) enhances platelet aggregation and hence the risk of thrombosis. Rac1 GTPase has been shown to be critical in ROS generation by both NOX1 and NOX2. However, its role in agonist induced ROS generation in platelets remains to be determined. We have shown previously that gene targeting or inhibition of Rac1 GTPase inhibits platelet secretion and aggregation. In this study we tested our hypothesis that Rac1 regulates platelet activation, at least in part, by regulating ROS generation through the NOX enzymes by using Rac1 and NOX2 gene targeted mice and/or pharmacologic inhibitors. ROS generation was determined by flow cytometry in dcf-da loaded washed platelets. Collagen-related peptide (CRP) or thrombin induced ROS generation in a time- and a concentration-dependent manner. A two minute pre-incubation of platelets with NSC23766 (3-30 μM), a Rac GTPases inhibitor, or Phox-I (1-10 μM), an inhibitor of NOX2 that blocks Rac1-p67 phox interaction, diminished CRP or thrombin induced ROS generation, ATP secretion and aggregation. However, treatment of platelets with NSC23766 or Phox-I did not inhibit phosphorylation of p47 phox , a critical subunit of NOX. Addition of CRP or thrombin induced significantly less ROS generation in platelets from Rac1 -/- , as compared to Rac1 +/+ , mice. Addition of CRP generated significantly less ROS and exhibited diminished aggregation in NOX2 -/- platelets than NOX2 +/+ platelets. A two minute incubation of platelets with ML171 (0.1 -1.0 μM), a selective inhibitor of NOX1, inhibited CRP induced but not thrombin induced ROS generation, ATP secretion and aggregation. Addition of a combination of ML171 and NSC23766, Phox-I and NSC23766 or ML171and Phox-I to platelets did not result in additive inhibitory effect on CRP induced ROS generation. Together, these results show for the first time that Rac1 GTPase: (a) is essential for agonist induced ROS generation in platelets; (b) inhibits ROS generation by inhibiting Rac1-p67 phox interaction but not activation of p47 phox ; (c) is involved in ROS generation by both NOX1 and NOX2. The differential signaling of CRP activated Rac1-NOX1-ROS and thrombin-stimulated Rac1-NOX2-ROS play an important role in platelet secretion and aggregation.