Shimada K, Ozawa T
Department of Medicine and Geriatrics, Kochi Medical School, Japan.
Arteriosclerosis. 1987 Nov-Dec;7(6):627-36. doi: 10.1161/01.atv.7.6.627.
The interaction between antithrombin III and heparinlike glycosaminoglycan molecules present on the vascular surface seems to play a significant role in the regulation of coagulation. We have tested the hypothesis that altered synthesis of glycosaminoglycans by endothelial cells could influence this interaction by using 4-methylumbelliferyl-beta-D-xyloside for metabolic perturbation of glycosaminoglycan production. Incubation of purified porcine 125I-antithrombin III with cultured porcine aortic endothelial cells demonstrated specific, time-dependent, saturable binding of this protease inhibitor to the endothelial cell surface with antithrombin III concentration at half-maximal binding of approximately 40 nM. This binding was displaced by heparin and was completely abolished by selective removal of heparan sulfate from cells with heparitinase, indicating that antithrombin III binds to heparan sulfate on the surface of endothelial cells. Incubation of cell cultures with beta-D-xyloside resulted in a reduction of maximum antithrombin III binding by approximately 65% with little alteration in binding affinity. beta-D-Xyloside did not affect the cellular growth or morphology. Reduction of the binding after exposure to various concentrations (33 to 500 microM) of xyloside occurred in parallel with the decrease in incorporation of both 35S-sulfate and 3H-glucosamine into cell surface heparan sulfate. Whereas the size of heparan sulfate chains derived from the cell surface was not altered by xyloside treatment, they appeared to have slightly less net negative charge and a significantly reduced proportion of the molecule with high affinity for antithrombin III in the presence of xyloside. On the other hand, secretion of free chondroitin sulfate (and dermatan sulfate) chains into the medium was markedly increased (16-fold) in the presence of xyloside, accompanied by a smaller increase in secretion of free heparan sulfate chains. There was a good correlation between conditions with decreased antithrombin III binding and an inhibition of the endothelial cell-mediated acceleration of thrombin inactivation by antithrombin III. These results suggest that beta-D-xyloside caused a dose-dependent decrease in production as well as some subtle structural alterations of cell-surface-associated heparan sulfate, which could serve as binding sites for antithrombin III on the endothelial cells and mediate enhancing the anticoagulant activity of this protein. This system may offer a potentially useful model to investigate the possible mechanisms responsible for the development of a procoagulant state involving these endothelial macromolecules.
抗凝血酶III与血管表面存在的类肝素糖胺聚糖分子之间的相互作用似乎在凝血调节中发挥着重要作用。我们通过使用4-甲基伞形酮基-β-D-木糖苷对糖胺聚糖产生进行代谢扰动，来检验内皮细胞糖胺聚糖合成改变可能影响这种相互作用的假说。将纯化的猪125I-抗凝血酶III与培养的猪主动脉内皮细胞一起孵育，结果显示这种蛋白酶抑制剂与内皮细胞表面存在特异性、时间依赖性、可饱和的结合，抗凝血酶III浓度在半最大结合时约为40 nM。这种结合可被肝素取代，并且通过用肝素酶从细胞中选择性去除硫酸乙酰肝素可使其完全消除，这表明抗凝血酶III与内皮细胞表面的硫酸乙酰肝素结合。用β-D-木糖苷孵育细胞培养物导致抗凝血酶III最大结合减少约65%，而结合亲和力几乎没有改变。β-D-木糖苷不影响细胞生长或形态。暴露于各种浓度（33至500 μM）的木糖苷后结合减少与35S-硫酸盐和3H-葡萄糖胺掺入细胞表面硫酸乙酰肝素的减少同时发生。虽然木糖苷处理未改变源自细胞表面的硫酸乙酰肝素链的大小，但在木糖苷存在下，它们似乎净负电荷略少，且对抗凝血酶III具有高亲和力的分子比例显著降低。另一方面，在木糖苷存在下，游离硫酸软骨素（和硫酸皮肤素）链向培养基中的分泌显著增加（16倍），同时游离硫酸乙酰肝素链的分泌也有较小增加。抗凝血酶III结合减少的情况与抗凝血酶III对内皮细胞介导的凝血酶失活加速作用的抑制之间存在良好的相关性。这些结果表明，β-D-木糖苷导致细胞表面相关硫酸乙酰肝素的产生呈剂量依赖性减少以及一些细微的结构改变，而硫酸乙酰肝素可作为抗凝血酶III在内皮细胞上的结合位点并介导增强该蛋白的抗凝活性。该系统可能为研究涉及这些内皮大分子的促凝状态发展的可能机制提供一个潜在有用的模型。
