Hypoxic potentiation of nitrite effects in human vessels and platelets

Previous studies in non-human blood vessels and in platelets have demonstrated that under hypoxic conditions release of NO from nitrite (NO ₂^-) is potentiated by deoxyhaemoglobin. In the current study, we characterized hypoxic potentiation of NO₂^- effects in human vasculature and platelets in vitro, addressing underlying mechanisms. The vasodilator efficacy of NO₂^-, in comparison with glyceryl trinitrate (GTN), was evaluated in vitro, using segments of human saphenous vein. Under hypoxic conditions, there was a leftward shift of the NO₂^- concentration-response curve (EC ₅₀: 22 μM in hyperoxia vs 3.5 μM in hypoxia; p < 0.01), but no significant potentiation of GTN effect. In the presence of red blood cells, hypoxic potentiation of NO₂^- vasodilator effect was accentuated. In whole blood samples and platelet-rich plasma (PRP) we assessed inhibition of platelet aggregation by NO₂^- (1 mM), in comparison with that of sodium nitroprusside (SNP, 10 μM). In individual subjects (n = 37), there was a strong correlation (r = 0.75, p < 0.0001) between anti-aggregatory effects of NO₂^- and SNP in whole blood, signifying that resultant sGC activation underlies biological effect and responses to NO₂^- are diminished in the presence of NO resistance. In PRP, the effects of NO₂^- were less pronounced than in whole blood (p = 0.0001), suggesting an important role of Hb (within RBCs) in the bioconversion of NO₂^- to NO. Inhibition of platelet aggregation by NO₂^- was almost 3-fold greater in venous than in arterial blood (p < 0.0001), and deoxyHb concentration directly correlated (r = 0.69, p = 0.013) with anti-aggregatory response. Incremental hypoxia applied to venous blood samples (in hypoxic chamber) caused a progressive increase in both deoxyHb level and anti-aggregatory effect of NO₂^-. When subjects inhaled a 12% O₂ mixture for 20 min, there was a 3-fold rise in blood deoxyHb fraction (p < 0.01). In PRP, response to NO₂^- also increased under hypoxia, and was further enhanced (p < 0.01) by deoxyHb. Furthermore, deoxyHb exerted significant anti-aggregatory effects even in the absence of added NO₂^-, suggesting a role for endogenous NO₂^-. The results of this work provide further mechanistic insights into hypoxic potentiation of vasodilator and anti-aggregatory actions of NO₂^-. In human saphenous veins and blood, the balance of evidence suggests differential rates of NO release from NO₂ ^- (largely modulated by deoxyHb) as the fundamental mechanism.