Abstract 196: NADPH Oxidase Isoforms NOX1 and NOX2 Differentially Regulate GPVI- and Non-GPVI-Dependent ROS Generation and Platelet Activation
Reactive oxygen species (ROS) play an important role in platelet activation. Although diverse biochemical reactions contribute to ROS generation, NADPH oxidases (NOX) have emerged as critical sources of agonist induced ROS generation in platelets. Platelets express two NOX isoforms, NOX1 and NOX2. In this study we investigated the effects of ML171 (0.1-1 μM), a selective NOX1 inhibitor (ACS Chem Biol. 5, 981-993, 2010), and Phox-I (1-10 μM), a rationally designed small molecule inhibitor of NOX2 (Chem. Biol. 19, 699-710, 2012), to define the roles of NOX1 and NOX2 in ROS-mediated platelet activation by diverse agonists. A two-minute pre-incubation of washed human platelets with ML171 or Phox-I inhibited CRP, a GPVI agonist, induced ROS generation, ATP secretion and platelet aggregation. In contrast, only platelets treated with Phox-I, but not ML171, inhibited CRP induced binding of PAC-1 to platelets and expression of P-selectin. Treatment of platelets with Phox-I, but not ML-171, inhibited thrombin or U46619, a stable analog of TXA2, induced ROS generation, P-selectin expression, ATP secretion and aggregation. Both Phox-I and ML171 inhibited CRP induced phosphorylation of myosin light chain (MLC) and Akt. However, only Phox-I inhibited thrombin induced phosphorylation of MLC or Akt. Treatment of platelets with Phox-I or ML171 only minimally or partially inhibited CRP induced phosphorylation of p38-MAPK or ERK. In other experiments, incubation of platelets with varying concentrations of both ML171 and Phox-I did not exhibit additive effect on CRP induced ROS generation. These results imply that ROS generation by these NOX isoforms may be sequential. These data suggest that NOX1 is specifically involved in GPVI mediated, whereas NOX2 is involved in GPVI- and non-GPVI-dependent, signals in ROS generation and platelet activation.
Author Disclosures: H. Akbar: None. X. Duan: None. S. Saleem: None. A.K. Davis: None. Y. Zheng: None.
- © 2014 by American Heart Association, Inc.