Evidence for iNOS-dependent peroxynitrite production in diabetic platelets

Aims/hypothesis. The aim of the present study was twofold. Firstly, to dete rmine whether diabetic platelets produce more peroxynitrite than normal pla telets and secondly to correlate the peroxynitrite production with the intr aplatelet induction of the inducible isoform of nitric oxide-synthase. Methods. Intraplatelet peroxynitrite production was monitored with dichloro fluorescin acetate with a combination of confocal microscopy and steady-sta te fluorescence. The platelets were probed for the induction of the inducib le-nitric oxide-synthase by western immunoblotting. Results. In the presence of extracellular L-arginine (100 mu mol/l), platel ets from subjects with Type I (insulin-dependent) diabetes displayed about 5 times higher fluorescence than those from control subjects. To determine whether inducible-nitric oxide-synthase was the source of peroxynitrite, di chlorofluorescein production was quantified as a function of L-arginine as well as nitric oxide-synthase inhibitors, in platelets from control subject s, subjects with Type I diabetes and subjects with Type II (non-insulin-dep endent) diabetes mellitus, Platelets from subjects with Type I yielded abou t sevenfold and those from Type II about threefold larger amounts of L-argi nine/nitric oxide-synthase-dependent dichlorofluorescein fluorescence than those from control subjects. The platelets were then immunologically probed for inducible-nitric oxide-synthase, which has previously been implicated in peroxynitrite production and detected in megakaryocytes of subjects with coronary heart disease. Western immunoblots of intraplatelet proteins indi cated that the inducible-nitric oxide-synthase was absent in control subjec ts. Platelets from both Type I and Type II diabetic subjects, however, cont ained inducible-nitric oxide-synthase. Conclusion/interpretation. Inducible-nitric oxide-synthase-derived peroxyni trite is a source of platelet damage in diabetes.