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

Background and PurposeNADPH 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 ApproachThe 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 ResultsSuperoxide 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 ImplicationsTaken 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.