title: Cloned bovine aortic endothelial cells synthesize anticoagulantly active heparan sulfate proteoglycan.
abstract: Cloned bovine aortic endothelial cells were cultured with [35S]Na2SO4 and proteolyzed extensively with papain. Radiolabeled heparan sulfate was isolated by DEAE-Sephacel chromatography. The mucopolysaccharide was then affinity fractionated into two separate populations utilizing immobilized antithrombin. The heparan sulfate, which bound tightly to the protease inhibitor, represented 0.84% of the mucopolysaccharide mass, accounted for greater than 99% of the initial anticoagulant activity, and exhibited a specific activity of 1.16 USP units/10(6) 35S-cpm. However, the heparan sulfate that interacted minimally with the protease inhibitor constituted greater than 99% of the mucopolysaccharide mass, represented less than 1% of the starting biologic activity, and possessed a specific anticoagulant potency of less than 0.0002 USP unit/10(6) 35S-cpm. An examination of the disaccharide composition of the two populations revealed that the high-affinity heparan sulfate contained a 4-fold or greater amount of GlcA----GlcN-SO3-3-O-SO3 (where GlcA is glucuronic acid), which is a marker for the antithrombin-binding domain of commercial heparin, as compared with the depleted material. Cloned bovine aortic endothelial cells were incubated with [35S]Na2SO4 as well as tritiated amino acids and completely solubilized with 4 M guanidine hydrochloride and detergents. The double-labeled proteoglycans were isolated by DEAE-Sephacel, Sepharose CL-4B, and octyl-Sepharose chromatography. These hydrophobic macromolecules were then affinity fractionated into two separate populations utilizing immobilized antithrombin. The heparan sulfate proteoglycans which bound tightly to the protease inhibitor represented less than 1% of the starting material and exhibited a specific anticoagulant activity as high as 21 USP units/10(6) 35S-cpm, whereas the heparan sulfate proteoglycan that interacted weakly with the protease inhibitor constituted greater than 99% of the starting material and possessed a specific anticoagulant potency as high as 0.02 USP unit/10(6) 35S-cpm. The high-affinity heparan sulfate proteoglycan is responsible for more than 85% of the anticoagulant activity of the cloned bovine aortic endothelial cells. Binding studies conducted with 125I-labeled antithrombin demonstrated that these biologically active proteoglycans are located on the surface of cloned bovine aortic endothelial cells.
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000302:isolation & purification* / Q000494:pharmacology; D000991:Antithrombins / Q000378:metabolism; D001011:Aorta / Q000378:metabolism; D001665:Binding Sites; D001808:Blood Vessels / Q000378:metabolism*; D002417:Cattle; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D002846:Chromatography, Affinity; D002999:Clone Cells; D004727:Endothelium / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006025:Glycosaminoglycans / Q000096:biosynthesis*; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D009844:Oligosaccharides / Q000032:analysis; D011509:Proteoglycans / Q000096:biosynthesis*
chemical_list: D000925:Anticoagulants; D000991:Antithrombins; D011508:Chondroitin Sulfate Proteoglycans; D006025:Glycosaminoglycans; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D007457:Iodine Radioisotopes; D009844:Oligosaccharides; D011509:Proteoglycans