Nishinaga M, Ozawa T, Shimada K
Department of Cardiology, Jichi Medical School, Tochigi, Japan.
J Clin Invest. 1993 Sep;92(3):1381-6. doi: 10.1172/JCI116712.
Previous studies showed that homocysteine, a thrombo-atherogenic and atherogenic agent, inhibits an endothelial thrombomodulin-protein C anticoagulant pathway. We examined whether homocysteine might affect another endothelial anticoagulant mechanism; i.e., heparin-like glycosaminoglycan-antithrombin III interactions. Incubations of porcine aortic endothelial cell cultures with homocysteine reduced the amount of antithrombin III bound to the cell surface in a dose- and time-dependent fashion. The inhibitory effect was observed at a homocysteine concentration as low as 0.1 mM, and the maximal suppression occurred at 1 mM of homocysteine after 24 h. In contrast with a marked reduction in the maximal antithrombin III binding capacity (approximately 30% of control), the radioactivity of [35S]sulfate incorporated into heparan sulfate on the cell surface was minimally (< 15%) reduced. The cells remained viable after homocysteine treatment. Although neither net negative charge nor proportion in total glycosaminoglycans of cell surface heparan sulfate was altered by homocysteine treatment, a substantial reduction in antithrombin III binding capacity of heparan sulfate isolated from homocysteine-treated endothelial cells was found using both affinity chromatography and dot blot assay techniques. The antithrombin III binding activity of endothelial cells decreased after preincubation with 1 mM homocysteine, cysteine, or 2-mercaptoethanol; no reduction in binding activity was observed after preincubation with the same concentration 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 cells 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 thrombogenic property resulting from the homocysteine-induced endothelial cell perturbation, mediated by generation of hydrogen peroxide through alteration of the redox potential.
先前的研究表明，同型半胱氨酸作为一种促血栓形成和致动脉粥样硬化的因子，可抑制内皮血栓调节蛋白 - 蛋白C抗凝途径。我们研究了同型半胱氨酸是否可能影响另一种内皮抗凝机制，即肝素样糖胺聚糖 - 抗凝血酶III相互作用。用同型半胱氨酸孵育猪主动脉内皮细胞培养物，可使结合到细胞表面的抗凝血酶III量呈剂量和时间依赖性减少。在同型半胱氨酸浓度低至0.1 mM时即可观察到抑制作用，24小时后在1 mM同型半胱氨酸时出现最大抑制。与最大抗凝血酶III结合能力显著降低（约为对照的30%）相反，掺入细胞表面硫酸乙酰肝素的[35S]硫酸盐放射性仅有微小降低（<15%）。同型半胱氨酸处理后细胞仍保持活力。尽管同型半胱氨酸处理未改变细胞表面硫酸乙酰肝素的净负电荷或其在总糖胺聚糖中的比例，但使用亲和层析和斑点印迹分析技术发现，从同型半胱氨酸处理的内皮细胞中分离出的硫酸乙酰肝素的抗凝血酶III结合能力大幅降低。用1 mM同型半胱氨酸、半胱氨酸或2 - 巯基乙醇预孵育后，内皮细胞的抗凝血酶III结合活性降低；用相同浓度的蛋氨酸、丙氨酸或缬氨酸预孵育后，未观察到结合活性降低。这种巯基效应可能是由过氧化氢的产生引起的，因为用过氧化氢酶（而非超氧化物歧化酶）与同型半胱氨酸处理的内皮细胞一起孵育可防止这种降低，而铜可增强代谢产物的抑制作用。因此，我们的数据表明，抗凝硫酸乙酰肝素表达受抑制可能导致同型半胱氨酸诱导的内皮细胞紊乱所产生的血栓形成特性，这是由氧化还原电位改变导致过氧化氢生成介导的。
