The novel NOX inhibitor 2-acetylphenothiazine impairs collagen-dependent thrombus formation in a GPVI-dependent manner.

Background and Purpose NADPH oxidases ( NOXs) contribute to platelet activation by a largely unknown mechanism. Here, we studied the effect of the novel NOX inhibitor 2-acetylphenothiazine (2- APT) on human platelet functional responses and intracellular signaling pathways. Experimental Approach The generation of superoxide ions was assessed by single cell imaging on adhering platelets using dihydroethidium ( DHE), while other reactive oxygen species ( ROS) were detected with 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate ( CM- H2- DCFDA). Whole blood thrombus formation, washed platelet aggregation, integrin α IIbβ3 inside-out signalling, Syk phosphorylation and PKC activation were analysed to understand the functional consequences of NOX inhibition by 2- APT in platelets. Key Results Superoxide ion generation stimulated by platelet adhesion on collagen and fibrinogen was significantly inhibited by 2- APT in concentration-dependent manner ( IC50 = 306 n M and 227 n M, respectively), whereas cumulative ROS accumulation was not affected by this pharmacological agent. 2- APT also abolished collagen-dependent whole blood thrombus formation and washed platelet aggregation in response to collagen but not thrombin. The activation of integrin α IIbβ3 and PKC in response to the GPVI-specific agonist collagen-related peptide ( CRP) was significantly reduced, whereas the same responses to thrombin were not significantly affected by 2- APT. Finally, Syk activation in response to collagen but not thrombin was inhibited by 2- APT. Conclusions and Implications Taken together, our results suggest that 2- APT attenuates GPVI-specific signalling and is a novel inhibitor of collagen-induced platelet responses. Therefore, NOXs could represent a novel target for the discovery of anti-thrombotic drugs. [ABSTRACT FROM AUTHOR]