Abstract 20: RhoA GTPase Regulates Reactive Oxygen Species--Mediated Platelet Activation
Rho family GTPases and reactive oxygen species (ROS) are known to be involved in platelet activation. Although Rac1, a Rho member, has been shown to be critical for ROS generation by NADPH oxidase (NOX) and platelet activation, RhoA, the founding member of the Rho family, has not been suspected in ROS generation and associated platelet activation. In this study we investigated the effects of Rhosin, a rationally designed small molecule that specifically binds to RhoA at the site required for its activation by Rho-GEF and thereby prevents activation of RhoA (Chem. Biol. 19, 699-710, 2012) on ROS generation and platelet activation to better understand the role of RhoA in platelet signaling. Addition of Rhosin to washed human platelets two minutes prior to stimulation with U46619, a stable analog of TXA2, inhibited (a) activation of RhoA but not of Rac1 or Cdc42, (b) ROS generation, and (c) phosphorylation of p47phox, a critical component of the NOX complex. In other experiments, U46619 induced ROS generation in platelets from RhoA+/+ but not in RhoA-/- mice. Treatment of platelets with Rhosin inhibited U46619-induced (a) phosphorylation of myosin light chain (MLC) in the presence or absence of aspirin and apyrase, (b) platelet shape change, filopodia formation and spreading on immobilized fibrinogen, (c) release of P-selectin, secretion of ATP and aggregation, and (d) phosphorylation of ERK and p38-MAPK, and Akt. These findings suggest that RhoA induced ROS generation contributes to platelet activation via phosphorylation of p38-MAPK, ERK and Akt. Rhosin also inhibited thrombin induced ROS generation, platelet shape change, secretion, aggregation and clot retraction. Washing of platelets after incubation with Rhosin abolished the inhibitory effects of Rhosin on platelet activation. Administration of Rhosin to mice prolonged tail bleeding times. These data suggest that: (a) agonist-RhoA/ROCK mediated phosphorylation of p47phox plays a critical role in ROS generation which in turn induces platelet activation in conjunction with or independent of the RhoA/ROCK mediated phosphorylation of MLC; and (b) targeting RhoA by rationally designed small molecule inhibitors offers a novel approach for developing safer and more effective antithrombotic agent.
Author Disclosures: H. Akbar: None. X. Duan: None. S. Saleem: None. A.K. Davis: None. Y. Zheng: None.
- © 2014 by American Heart Association, Inc.