Cloned bovine aortic endothelial cells synthesize anticoagulantly active heparan sulfate proteoglycan

J. A. Marcum, D. H. Atha, L. M S Fritze, P. Nawroth, David Stern, R. D. Rosenberg

Research output: Contribution to journalArticle

238 Citations (Scopus)

Abstract

Cloned bovine aortic endothelial cells were cultured with [35S]Na2SO4 and proteolyzed extensively with papain. Radiolabeled heparin sulfate was isolated by DEAE-Sephacel chromatography. The mucopolysaccharide was then affinity fractionated into two separate populations utilizing immobilized antithrombin. The heparin sulfate, which bound tightly to the protease inhibitor, represented 0.84% of the mucopolysaccharide mass, accounted for > 99% of the initial anticoagulant activity, and exhibited a specific activity of 1.16 USP units/106 35S-cpm. However, the heparan sulfate that interacted minimally with the protease inhibitor constituted > 99% of the mucopolysaccharide mass, represented < 1% of the starting biologic activity, and possessed a specific anticoagulant potency of < 0.0002 USP unit/106 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 < 1% of the starting material and exhibited a specific anticoagulant activity as high as 21 USP units/ 106 35S-cpm, whereas the heparan sulfate proteoglycan that interacted weakly with the protease inhibitor constituted > 99% of the starting material and possessed a specific anticoagulant potency as high as 0.02 USP unit/106 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.

Original languageEnglish (US)
Pages (from-to)7507-7517
Number of pages11
JournalJournal of Biological Chemistry
Volume261
Issue number16
StatePublished - 1986
Externally publishedYes

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Heparan Sulfate Proteoglycans
Endothelial cells
Glycosaminoglycans
Anticoagulants
Antithrombins
Endothelial Cells
Protease Inhibitors
Sulfates
Heparin
Heparitin Sulfate
Papain
Proteoglycans
Chromatography
Population

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Marcum, J. A., Atha, D. H., Fritze, L. M. S., Nawroth, P., Stern, D., & Rosenberg, R. D. (1986). Cloned bovine aortic endothelial cells synthesize anticoagulantly active heparan sulfate proteoglycan. Journal of Biological Chemistry, 261(16), 7507-7517.

Cloned bovine aortic endothelial cells synthesize anticoagulantly active heparan sulfate proteoglycan. / Marcum, J. A.; Atha, D. H.; Fritze, L. M S; Nawroth, P.; Stern, David; Rosenberg, R. D.

In: Journal of Biological Chemistry, Vol. 261, No. 16, 1986, p. 7507-7517.

Research output: Contribution to journalArticle

Marcum, JA, Atha, DH, Fritze, LMS, Nawroth, P, Stern, D & Rosenberg, RD 1986, 'Cloned bovine aortic endothelial cells synthesize anticoagulantly active heparan sulfate proteoglycan', Journal of Biological Chemistry, vol. 261, no. 16, pp. 7507-7517.
Marcum, J. A. ; Atha, D. H. ; Fritze, L. M S ; Nawroth, P. ; Stern, David ; Rosenberg, R. D. / Cloned bovine aortic endothelial cells synthesize anticoagulantly active heparan sulfate proteoglycan. In: Journal of Biological Chemistry. 1986 ; Vol. 261, No. 16. pp. 7507-7517.
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abstract = "Cloned bovine aortic endothelial cells were cultured with [35S]Na2SO4 and proteolyzed extensively with papain. Radiolabeled heparin sulfate was isolated by DEAE-Sephacel chromatography. The mucopolysaccharide was then affinity fractionated into two separate populations utilizing immobilized antithrombin. The heparin sulfate, which bound tightly to the protease inhibitor, represented 0.84{\%} of the mucopolysaccharide mass, accounted for > 99{\%} of the initial anticoagulant activity, and exhibited a specific activity of 1.16 USP units/106 35S-cpm. However, the heparan sulfate that interacted minimally with the protease inhibitor constituted > 99{\%} of the mucopolysaccharide mass, represented < 1{\%} of the starting biologic activity, and possessed a specific anticoagulant potency of < 0.0002 USP unit/106 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 < 1{\%} of the starting material and exhibited a specific anticoagulant activity as high as 21 USP units/ 106 35S-cpm, whereas the heparan sulfate proteoglycan that interacted weakly with the protease inhibitor constituted > 99{\%} of the starting material and possessed a specific anticoagulant potency as high as 0.02 USP unit/106 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.",
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AU - Marcum, J. A.

AU - Atha, D. H.

AU - Fritze, L. M S

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AU - Stern, David

AU - Rosenberg, R. D.

PY - 1986

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N2 - Cloned bovine aortic endothelial cells were cultured with [35S]Na2SO4 and proteolyzed extensively with papain. Radiolabeled heparin sulfate was isolated by DEAE-Sephacel chromatography. The mucopolysaccharide was then affinity fractionated into two separate populations utilizing immobilized antithrombin. The heparin sulfate, which bound tightly to the protease inhibitor, represented 0.84% of the mucopolysaccharide mass, accounted for > 99% of the initial anticoagulant activity, and exhibited a specific activity of 1.16 USP units/106 35S-cpm. However, the heparan sulfate that interacted minimally with the protease inhibitor constituted > 99% of the mucopolysaccharide mass, represented < 1% of the starting biologic activity, and possessed a specific anticoagulant potency of < 0.0002 USP unit/106 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 < 1% of the starting material and exhibited a specific anticoagulant activity as high as 21 USP units/ 106 35S-cpm, whereas the heparan sulfate proteoglycan that interacted weakly with the protease inhibitor constituted > 99% of the starting material and possessed a specific anticoagulant potency as high as 0.02 USP unit/106 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.

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