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 αIIbβ3 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 nM and 227 nM, 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 αIIbβ3 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.