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

该项目最初的目的是利用配基亲和技术克隆血管内皮细胞表面抗凝活性硫酸乙酰肝素蛋白多糖(HSPG)的核心蛋白。我们发现细胞不能与具有足够高亲和力的固相抗凝血酶III结合，以进行细胞分选。在这项研究中，它的克隆被其他调查者报道。他们的结果表明，抗凝剂HSPG的核心蛋白与非抗凝剂HSPG的核心蛋白没有区别。那么，内皮细胞合成抗凝剂糖胺聚糖(GAG)的确切机制是什么？核心蛋白可能不参与这种特定的代谢调节。为了回答这个问题，我们开发了一种独特的模型，在该模型中，特定的抗凝血剂(即抗凝血酶III亲和力)HSPG缺乏，而总体HSPG的代谢没有改变。同型半胱氨酸是一种血栓形成试剂，能特异性地抑制抗凝剂HSPG，这是通过SH衍生的氧化还原反应产生自由基来调节的。此外，我们还发现，具有清除自由基活性的内皮NO的代谢抑制显著减少了对内皮细胞的抗凝剂HSPG。这被证明伴随着使用荧光探针的细胞内过氧化氢的增加。这些结果表明，抗凝剂HSPG的合成可能受细胞内自由基活性的调节。内皮衍生的松弛因子，NO可能通过发挥血管内皮细胞的抗凝作用而具有新的抗血栓活性。

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.