The Anti-Platelet Effects Of Apocynin Are Not Mediated By Inhibition Of Nadph Oxidase

The methoxy‐substituted catechol, apocynin is a powerful inhibitor of NADPH oxidase. Recently apocynin has been shown to prevent the aggregation of platelets in response to agonists such as collagen and thrombin. We examined whether NADPH oxidase plays a role in collagen‐induced aggregation, elucidating the mechanism(s) of action of apocynin on this enzyme complex. Platelets were isolated from 10 week‐old male C57BL6/J and Nox2‐deficient (Nox2 −/− ) mice to assess platelet aggregation (aggregometry) and NADPH oxidase expression (immunoblotting for Nox2). Collagen (1‐30 μg/ml) caused a concentration‐dependent increase in platelet aggregation (maximum response ~35.35 ± 3.18%; n=6). This response was inhibited by apocynin (P<0.05, n=6), albeit at higher concentrations (=100µM). Immunoblotting with an anti‐Nox2 antibody from platelets isolated from wild‐type mice revealed strong bands at 58kDa, 65kDa and 91kDa. Collagen was as effective at eliciting aggregation in platelets from Nox2 −/− and wild‐type mice. Moreover, the superoxide scavengers, SOD (300U/ml) and PEG‐SOD (300U/ml) had no effect on platelet aggregation in response to collagen. These data suggest that while platelets express NADPH oxidase, it is not responsible for mediating collagen‐induced aggregation. Apocynin's anti‐platelet actions are likely to be due to an off target effect of the drug which remains to be determined. (NHMRC 384136)