Nitric oxide degradation of heparin and heparan sulphate.

NO is a bioactive free radical produced by NO synthase in various tissues including vascular endothelium. One of the degradation products of NO is HNO2, an agent known to degrade heparin and heparan sulphate. This report documents degradation of heparin by cultured endothelial-cell-derived as well as exogenous NO. An exogenous narrow molecular-mass preparation of heparin was recovered from the medium of cultured endothelial cells using strong-anion exchange. In addition, another narrow molecular-mass preparation of heparin was gassed with exogenous NO under argon. Degradation was evaluated by gel-filtration chromatography. Since HNO2 degrades heparin under acidic conditions, the reaction with NO gas was studied under various pH conditions. The results show that the degradation of exogenous heparin by endothelial cells is inhibited by NO synthase inhibitors. Exogenous NO gas at concentrations as low as 400 p.p.m. degrades heparin and heparan sulphate. Exogenous NO degrades heparin at neutral as well as acidic pH. Endothelial-cell-derived NO, as well as exogenous NO gas, did not degrade hyaluronan, an unrelated glycosaminoglycan that resists HNO2 degradation. Peroxynitrite, a metabolic product of the reaction of NO with superoxide, is an agent that degrades hyaluronan; however, peroxynitrite did not degrade heparin. Thus endothelial-cell-derived NO is capable of degrading heparin and heparan sulphate via HNO2 rather than peroxynitrite. These observations may be relevant to various pathophysiological processes in which extracellular matrix is degraded, such as bone development, apoptosis, tissue damage from inflammatory responses and possible release of growth factors and cytokines.

Degradation of hyaluronan by peroxynitrite.

Treatment of high-molecular-weight hyaluronan (HA) with peroxynitrite at neutral pH (ONOO-/ONOOH) results in altered mobility on agarose gel electrophoresis, as well as reduced limiting viscosity number. Both effects are consistent with a reduction in HA molecular weight. HA is protected from peroxynitrite attack to varying extents by addition of alternate target molecules. Thiourea is extremely effective as a protective agent, dimethyl sulfoxide is moderately effective, while sodium benzoate and mannitol are slightly effective. A similar pattern of protection is observed when HA is degraded by hydroxyl radical generated by a metal ion/hydrogen peroxide system. On the basis of these observations, peroxynitrite is proposed to have hydroxyl radical-like activity in degrading HA.