Abstract
Agonist induced generation of reactive oxygen species (ROS) by NADPH oxidases (NOX) enhances platelet aggregation and hence the risk of thrombosis. RhoA and Rac1 GTPases are involved in ROS generation by NOX in a variety of cells, but their roles in platelet ROS production remain unclear. In this study we used platelets from RhoA and Rac1 conditional knockout mice as well as human platelets treated with Rhosin and NSC23767, rationally designed small molecule inhibitors of RhoA and Rac GTPases, respectively, to better define the contributions of RhoA and Rac1 signaling to ROS generation and platelet activation. Treatment of platelets with Rhosin inhibited: (a) U46619 induced activation of RhoA; (b) phosphorylation of p47phox, a critical component of NOX; (c) U46619 or thrombin induced ROS generation; (d) phosphorylation of myosin light chain (MLC); (e) platelet shape change; (f) platelet spreading on immobilized fibrinogen; and (g) release of P-selectin, secretion of ATP and aggregation. Conditional deletion of RhoA or Rac1 gene inhibited thrombin induced ROS generation in platelets. Addition of Y27632, a RhoA inhibitor, NSC23766 or Phox-I, an inhibitor of Rac1-p67phox interaction, to human platelets blocked thrombin induced ROS generation. These data suggest that: (a) RhoA/ROCK/p47phox signaling axis promotes ROS production that, at least in part, contributes to platelet activation in conjunction with or independent of the RhoA/ROCK mediated phosphorylation of MLC; and (b) RhoA and Rac1 differentially regulate ROS generation by inhibiting phosphorylation of p47phox and Rac1-p67phox interaction, respectively.
Highlights
Blood platelets play a critical role in atherothrombosis [1]
Based on the reports that RhoA/ ROCK mediated phosphorylation of p47phox, the organizing subunit of NADPH oxidases (NOX), regulates reactive oxygen species (ROS) generation and ROS in turn directly activates RhoA, we investigated the possibility that RhoA regulates platelet activation by affecting ROS generation
Since ROS generation in platelets stimulated by diverse agonists has been shown by flow cytometry [20] and a number of reports have shown that agonist induced ROS generation in platelets involves NADPH oxidase (NOX) [20, 44,45,46]
Summary
Blood platelets play a critical role in atherothrombosis [1]. Following a rupture of an atherosclerotic plaque or a vascular injury platelets come in contact with sub-endothelial extracellular matrix and undergo rapid activation that results in cytoskeletal reorganization, platelet shape change, granular secretion, aggregation and thrombus formation. Agonist induced generation of reactive oxygen species (ROS) including superoxide anion (O-2) and hydrogen peroxide (H2O2) enhance platelet aggregation and the risk of thrombosis [14, 15]. Two isoforms of NOX, namely NOX1 and NOX2 and their regulatory subunits p22phox, p47phox and p67phox, have been characterized in platelets [14, 15, 17, 18] and recent reports have confirmed that ROS activities play a role in regulation of platelet activation [18,19,20,21]. Delaney et al [18] have recently reported that ROS generation by NOX leads to platelet secretion and aggregation via the Syk/phospholipase Cγ2/calcium signaling pathway
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