Heparin reacts with and inactivates nitric oxide

Abstract

Although heparin is a well-known anticoagulant, in some cases it promotes a prothrombotic state and does so through both antibody-dependent and antibody-independent platelet activation. In this study, heparin was found to reverse the antiplatelet effect of an NO donor, S-nitroso-glutathione (SNO-Glu), with an EC50 of 1.8 U/mL. Ultraviolet/visible spectral analysis and the Griess assay showed that increasing heparin concentrations on a dose-dependent basis eliminated acidified NOx species. Since heparin is a heterogeneous mixture of glycosaminoglycans, the effects of six different heparin disaccharides were compared with various substitutions on the hexose rings to determine which functional group(s) of the polysaccharide interact with acidified NOx. Among the six disaccharides tested, only types I-S and II-S had the effect, suggesting that the sulfamino-group at the C2 position of the glucosamine moiety was critical for the elimination of acidified NOx species. Mass spectrometry experiments gave results consistent with these observations, indicating that only the I-S and II-S heparin disaccharides were modified upon treatment with NaNO2/HCl. Negative-ion electrospray ionization MS and tandem MS analyses of the native compounds and their deuterium-labeled analogs confirmed that the reaction products from nitrosation of these N-sulfated disaccharides had eliminated the C2-sulfamino-moiety and replaced it with methoxide derived from the solvent. Participation of the 6-sulfato-substituent appears to facilitate the elimination reaction. These data show that heparin can impair the antiplatelet properties of nitric oxide by interacting with the nitrosating species, and suggest that heparin-like glycosaminoglycans may interact with endothelium-derived nitric oxide in vivo to regulate the bioactivity of this important antiplatelet and vasorelaxant substance.