Hepatic Xanthine Oxidoreductase Sustains Antithrombotic Nitric Oxide Signalling Through the Nitrate-Nitrite-Nitric Oxide Pathway

Background: Tonic endothelium-derived nitric oxide (NO) suppresses platelet activation physiologically, and its loss underlies the thrombotic risk of endothelial dysfunction. Inorganic nitrate and nitrite provide an alternative NO source, and xanthine oxidoreductase (XOR) is a candidate nitrite reductase, but whether endogenous XOR sustains platelet NO signalling in vivo, and its source is unknown. Methods: Dietary nitrate (15 mmol/L KNO3) was given to eNOS-/- and ApoE-/- mice. XOR was interrogated pharmacologically (allopurinol) and genetically (global Xdh+/- and hepatocyte-specific XOR knockout, HXOR KO). Haemostasis and thrombosis were assessed by tail bleeding and intravital microscopy imaging of FeCl3-induced mesenteric arterial thrombosis, alongside aggregometry, flow cytometry, platelet VASPSer239 phosphorylation, cGMP and ozone chemiluminescence-based analysis of nitrate and nitrite. Results: Dietary nitrate raised plasma nitrate and nitrite levels in all genotypes. In eNOS-/- and ApoE-/- mice, it prolonged bleeding time and normalised thrombus burden. In ApoE-/- mice nitrate treatment improved vasorelaxation, without altering XOR or eNOS expression. Allopurinol suppressed nitrite reductase activity in liver and plasma but not aorta, elevated plasma nitrite, shortened bleeding time and reduced platelet P-VASPSer239 expression. Xdh+/- mice were spontaneously prothrombotic, showed reduced P-VASPSer239 and exaggerated calcium mobilisation, and were refractory to dietary nitrate treatment despite equivalent nitrite elevation. XOR was undetectable in platelets. HXOR KO mice phenocopied global deficiency, with blunted nitrite-induced vasorelaxation but preserved acetylcholine and spermine-NO responses, reduced P-VASPSer239, enhanced aggregation and shortened bleeding time. Conclusions: Hepatic XOR sustains platelet NO-cGMP signalling and thromboresistance through an inter-organ, endocrine-like axis. Dietary nitrate restores antithrombotic protection when endothelial NO generation fails, whereas XOR inhibition removes physiological tonic antithrombotic signal.