|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1991 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1990 : ¥1,400,000 (Direct Cost : ¥1,400,000)
It is thought that the non-thrombogenic properties of blood vessels are partially regulated by anticoagulantly active heparin-like compoundson the luminal surface of vascular endotherial cells. Heparan sulfate proteoglycans(HSPGS)are also known to be located along the abluminal side of the endothelium, that is, basement membrane or extracellular matrix(ECM)of endothelial cells. Does ECM contain HSPG which interact with antithrombin III (AT III)? If so, how abundant are they, as compared with those on the luminal surface? To answer this question, we have studied the interaction of ^<125>I -labeled AT III with cultured porcine aortic endothelial cells to localize the cellular site of anticoagulantly active HSPGS. ECM, prepared from endothelial cells cultured on plastic dishes, by removing the cells with nonenzymatic methods, specifically bound ^<125>I-AT III. The amount of AT III bound to ECM was approximately 40% of that bound to the intact cell. The binding was efficiently displaced by
heparan sulfate, and almost completely abolished by pretreatment of ECM with Flavobacterium heparitinase. ECM associated HSPGs apparently represented approximately 40% of HSPGs associated with intact cell, in parallel with the binding experiments. Approximately 15-20% of ECM-associated ^<35>S-glycosaminoglycans was bound to AT III affinity column with high affinity. ECM accelerated inactivation of thrombin by AT III. Based upon the above data, we conclude that approximately at least a half of anticoagulantly active HSPG is located in the ECM, that is, abluminal side of cultured aortic endothelial cells. The physiological significance of this finding remains to be determined.
In the next series of experiments, ECM was solubilized and applied onto the DEAE-Sephacell chromatography. Proteoheparan sulfate was eluted first at approximately 0.35 M NaCl followed by chondroitin sulfates. This fraction was concentrated and was subjected to SDS-PAGE(3-11%). Only after digestions with heparitinase, the protein band was migrated into separating gel plate, showing an apparent molecular weight of>200, 000. Further purifications are currently underway. Less