Isolation of heparan sulfates with antithrombin III affinity and anticoagulant potency from BALB/c 3T3, B16.F10 melanoma, and cutaneous fibrosarcoma cell lines.
The heparan sulfates synthesized in vitro by three cell lines were isolated by proteolysis and preparative anion exchange chromatography and purified free of other glycosaminoglycans by selective enzymatic degradation. The isolates from the medium of BALB/c 3T3 fibroblasts, B16.F10 melanoma cells, and a cutaneous fibrosarcoma line, along with that from the detergent-extracted cell layer of the fibroblasts, were affinity-fractionated on columns of matrix-immobilized human antithrombin III. Each heparan sulfate contained subfractions with high affinity for the proteinase inhibitor, ranging from 3-34% of the starting material. The high affinity species possessed measurable anticoagulant activities by a clotting assay (6 to 30 units/mg). Since none of the lines were derived from cell types having any known biological role in vascular homeostasis, we suggest that anticoagulant activity of the glycosaminoglycan is a random property of its primary structure.
Piepkorn
M
M
Department of Medicine, University of Utah School of Medicine, Salt Lake City 84132.
Hovingh
P
P
Hentschel
W M
WM
D000818:Animals; D000990:Antithrombin III / Q000378:metabolism*; D001777:Blood Coagulation / Q000187:drug effects*; D002460:Cell Line; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D005347:Fibroblasts; D005354:Fibrosarcoma; D006025:Glycosaminoglycans / Q000302:isolation & purification* / Q000378:metabolism; D006497:Heparan Sulfate / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D008545:Melanoma; D051379:Mice; D008807:Mice, Inbred BALB C; D012878:Skin Neoplasms; D014407:Tumor Cells, Cultured
D000990:Antithrombin III; D006025:Glycosaminoglycans; D006497:Heparan Sulfate