HOMOCYSTEINE, A THROMBOGENIC AGENT, SUPPRESSES ANTICOAGULANT HEPARAN-SULFATE EXPRESSION IN CULTURED PORCINE AORTIC ENDOTHELIAL-CELLS

Previous studies showed that homocysteine, a thromboatherogenic and at herogenic agent, inhibits an endothelial thrombomodulin-protein C anti coagulant pathway. We examined whether homocysteine might affect anoth er endothelial anticoagulant mechanism; i.e, heparin-like glycosamino- glycan-antithrombin III interactions. Incubations of porcine aortic en dothelial cell cultures with homocysteine reduced the amount of antith rombin III bound to the cell surface in a dose- and time-dependent fas hion. The inhibitory effect was observed at a homocysteine concentrati on as low as 0.1 mM, and the maximal suppression occurred at 1 mM of h omocysteine after 24 h. In contrast with a marked reduction in the max imal antithrombin Ill binding capacity (approximately 30% of control), the radioactivity of [S-35]sulfate incorporated into heparan sulfate on the cell surface was minimally (< 15%) reduced. The cells remained viable after homocysteine treatment. Although neither net negative cha rge nor proportion in total glycosaminoglycans of cell surface heparan sulfate was altered by homocysteine treatment, a substantial reductio n in antithrombin III binding capacity of heparan sulfate isolated fro m homocysteine-treated endothelial cells was found using both affinity chromatography and dot blot assay techniques. The antithrombin III bi nding activity of endothelial cells decreased after preincubation with 1 mM homocysteine, cysteine, or 2-mercaptoethanol; no reduction in bi nding activity was observed after preincubation with the same concentr ation of methionine, alanine, or valine. This sulfhydryl effect may be caused by generation of hydrogen peroxide, as incubation of catalase, but not superoxide dismutase, with homocysteine-treated endothelial c ells prevented this reduction, whereas copper augmented the inhibitory effects of the metabolite. Thus, our data suggest that the inhibited expression of anticoagulant heparan sulfate may contribute to the thro mbogenic property resulting from the homocysteine-induced endothelial cell perturbation, mediated by generation of hydrogen peroxide through alteration of the redox potential.