NAD(P)H oxidase–dependent platelet superoxide anion release increases platelet recruitment

Platelets, although not phagocytotic, have been suggested to release O2−. Since O2−-producing reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases can be specifically activated by certain agonists and are found in several nonphagocytotic tissues, we investigated whether such an enzyme is the source of platelet-derived O2−. We further studied which agonists cause platelet O2−release and whether platelet-derived O2− influences thrombus formation in vitro. Collagen, but not adenosine 5′-diphosphate (ADP) or thrombin, increased O2− formation in washed human platelets. This was a reduced nicotinamide adenine dinucleotide (NADH)–dependent process, as shown in platelet lysates. Consistent with a role of a platelet, NAD(P)H oxidase expression of its subunits p47phox and p67phoxand inhibition of platelet O2− formation by diphenylene-iodoniumchloride (DPI) and by the specific peptide-antagonist gp91ds-tat were observed. Whereas platelet-derived O2− did not influence initial aggregation, platelet recruitment to a preformed thrombus following collagen stimulation was significantly attenuated by superoxide dismutase (SOD) or DPI. It was also inhibited when ADP released during aggregation was cleaved by the ectonucleotidase apyrase. ADP in supernatants of collagen-activated platelets was decreased in the presence of SOD, resulting in lower ADP concentrations available for recruitment of further platelets. Exogenous O2−increased ADP- concentrations in supernatants of collagen-stimulated platelets and induced irreversible aggregation when platelets were stimulated with otherwise subthreshold concentrations of ADP. These results strongly suggest that collagen activation induces NAD(P)H oxidase–dependent O2− release in platelets, which in turn enhances availability of released ADP, resulting in increased platelet recruitment.