Heparinlike molecules have been shown to be important in maintaining, in part, the non-thrombogenic properties of the vascular endothelium. However, little is known concerning the native structure of anticoagulantly active heparinlike proteoglycans from endothelial cells. To examine these issues, cultured endothelial cells will be isolated from macrovascular and microvascular sources, and double-labeled proteoglycans will be extracted from these cellular elements utilizing guanidine and purified employing gel filtration and ion exchange chromatography. The anticoagulant activity of the proteoglycans will be quantitated utilizing purified human thrombin, human antithrombin, and a specific radioimmunoassay for the enzyme-inhibitor complex. The heparinlike nature of these macromolecules will be established by destroying the biologic activity with purified Flavobacterium heparinase. The heparinlike proteoglycans will be fractionated into two distinct populations employing antithrombin-Sepharose. The bound as well as the unbound proteoglycans will be deglycosylated, and peptide maps and partial amino acid sequence of the N-terminal region of core proteins will be examined. The protein cores of the anticoagulantly active will be injected into rabbits, and the antisera specific for the biologically active macromolecule will be obtained. The antisera will be incubated with cultured rat endothelial cells and perfused through the rat hindlimb circulation. The anatomical location of the biologically active proteoglycans within the microvasculature and the macrovasculature as well as their cellular location on the plasma membrane, cell junctions, or coated pits will be assessed by light and electron microscopic analyses utilizing a ferritin-conjugated second antibody. The number of glycosaminoglycan chains per anticoagulantly active proteoglycan will be determined by estimating the molecular weights of the intact proteoglycan, the core protein, and the individual mucopolysaccharides. The number of antithrombin binding domains within a single polysaccharide chain will be assessed by quantitating the amount of unique tetrasaccharides and disaccharides in the polysaccharide chain responsible for inhibitor binding. To examine the percentage of glycosaminoglycan chains which exhibit anticoagulant activity in the affinity fractionated proteoglycan, the free chains will be subjected to an additional fractionation with antithrombin. Alterations of heparinlike proteoglycan biosynthesis by endothelial cells could result in arterial as well as venous thrombotic disease in humans.
