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

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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.