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The ligand-affinity molecular cloning of endothelial cell anticoagulant heparin-like compounds

内皮细胞抗凝类肝素化合物的配体亲和分子克隆

基本信息

批准号：
04454270
负责人：
SHIMADA Kazuyuki
金额：
$ 4.1万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

项目摘要

The purpose of this project was originally a molecular cloning of the core protein of anticoagulantly active heparan sulfate proteoglycans (HSPG) on the surface of vascular endotherial cells using a ligand-affinity technique. We found that cells were not bound to the solid-phase antithrombin III with a high affinity enough for cell sorting. During this study, its cloning was reported by other invesigators. Their results suggest that core proteins of anticoagulant HSPG are not different from those of non-anticoagulant HSPG.Then, what is the exact mechanism of the synthesis of anticoagulant glycosaminoglycans (GAG) in endothelial cells? Core proteis may not be involved in this specific metabolic regulation. In order to answer this question, we developed a unique model in which anticoagulant (i.e., antithrombin III-affinity) HSPG is specifically lacking, whereas overall HSPG metabolism is not altered. Homocysteine, a thrombo-atherogenic agent specifically inhibited anticoagulant HSPG.This is mediated by free radical generation via SH-derived redox reaction. Furthermore, we found that the metabolic inhibition of endothelial NO,which has a free radical scavenging activity, markedly reduced the anticoagulant HSPG on endothelial cells. This was demonstrared to be accompanied by an increase in intracellular hydroperoxide using fluorescent probes. These results indicate that the synthesis of anticoagulant HSPG may be regulated by intracellular free radical activities. Endothelium-derived relaxant factor, NO,may have a novel antithrombotic activity by playing an anticoagulant role of the vascular endothelium.

该项目的目的最初是利用配体亲和技术将具有抗凝活性的硫酸乙酰肝素蛋白聚糖（HSPG）核心蛋白分子克隆到血管内皮细胞表面。我们发现细胞与固相抗凝血酶 III 的结合力不足以进行细胞分选。在这项研究期间，其他研究人员报告了它的克隆。他们的研究结果表明，抗凝HSPG的核心蛋白与非抗凝HSPG的核心蛋白没有什么不同。那么，内皮细胞中合成抗凝糖胺聚糖（GAG）的确切机制是什么呢？核心蛋白质可能不参与这种特定的代谢调节。为了回答这个问题，我们开发了一个独特的模型，其中特别缺乏抗凝剂（即抗凝血酶 III 亲和力）HSPG，而整体 HSPG 代谢没有改变。同型半胱氨酸是一种血栓致动脉粥样硬化剂，可特异性抑制抗凝剂 HSPG。这是由 SH 衍生的氧化还原反应产生自由基介导的。此外，我们发现具有自由基清除活性的内皮NO的代谢抑制显着降低了内皮细胞的抗凝HSPG。使用荧光探针证明这伴随着细胞内氢过氧化物的增加。这些结果表明抗凝剂HSPG的合成可能受到细胞内自由基活性的调节。内皮源性松弛因子NO可能通过发挥血管内皮的抗凝作用而具有新型的抗血栓活性。