Nitroxyl (HNO) displays pharmacological and therapeutic actions distinct from those of its redox sibling nitric oxide (NO(•)). It remains unclear, however, whether the vasoprotective actions of HNO are preserved in disease. The ability of the HNO donor isopropylamine NONOate (IPA/NO) to induce vasorelaxation, its susceptibility to tolerance development, and antiaggregatory actions were compared with those of a clinically used NO(•) donor, glyceryl trinitrate (GTN), in hypercholesterolemic mice. The vasorelaxant and antiaggregatory properties of IPA/NO and GTN were examined in isolated carotid arteries and washed platelets, respectively, from male C57BL/6J mice [wild-type (WT)] maintained on either a normal diet (WT-ND) or high fat diet (WT-HFD; 7 wk) as well as apolipoprotein E-deficient mice maintained on a HFD (ApoE(-/-)-HFD; 7 wk). In WT-ND mice, IPA/NO (0.1-30 μmol/l) induced concentration-dependent vasorelaxation and inhibition of collagen (30 μg/ml)-stimulated platelet aggregation, which was predominantly soluble guanylyl cyclase/cGMP dependent. Compared with WT-HFD mice, ApoE(-/-)-HFD mice displayed an increase in total plasma cholesterol levels (P < 0.001), vascular (P < 0.05) and platelet (P < 0.05) superoxide (O(2)(·-)) production, and reduced endogenous NO(•) bioavailability (P < 0.001). Vasorelaxant responses to both IPA/NO and GTN were preserved in hypercholesterolemia, whereas vascular tolerance developed to GTN (P < 0.001) but not to IPA/NO. The ability of IPA/NO (3 μmol/l) to inhibit platelet aggregation was preserved in hypercholesterolemia, whereas the actions of GTN (100 μmol/l) were abolished. In conclusion, the vasoprotective effects of IPA/NO were maintained in hypercholesterolemia and, thus, HNO donors may represent future novel treatments for vascular diseases.

Apocynin, or a (myelo)peroxidase-derived product thereof, is a powerful inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Apocynin has also been shown to prevent aggregation of platelets in response to agonists such as collagen and thrombin. The aims of this study were to establish whether NADPH oxidase activity is required for aggregation of murine platelets to collagen and other agonists and whether the anti-aggregatory effects of apocynin are due to an inhibitory action against this enzyme. Washed platelets were isolated from male C57BL6 (wild-type), Nox2-deficient (Nox2(-/y )), and p47phox-deficient (p47phox(-/-)) mice for assessment of aggregation and NADPH oxidase subunit (Nox2, p47phox) expression. Collagen and U46619 elicited aggregation of murine platelets, and these responses were inhibited by apocynin at concentrations ≥100 μM. By contrast, aggregations to a direct protein kinase C activator, phorbol-12,13-dibutyrate, were insensitive to apocynin. Immunoblotting of platelet protein homogenates from wild-type mice with anti-Nox2 or p47phox antibodies revealed strong bands at 58 and 50 kDa, respectively. While expression of these immunoreactive bands was greatly diminished in platelets from Nox2(-/y ) and p47phox(-/-) mice, collagen still elicited aggregations that were similar to those observed in platelets from wild-types. Moreover, apocynin was an equally effective inhibitor of aggregation in platelets from all three mouse strains. In conclusion, these data suggest that NADPH oxidase-derived reactive oxygen species play no role in the aggregation response of washed murine platelets to collagen. Thus, our observation that apocynin is a powerful inhibitor of platelet aggregation raises further questions about the selectivity of this drug as an NADPH oxidase inhibitor.

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)