title: Presence and function of chondroitin-4-sulfate on recombinant human soluble thrombomodulin.
abstract: We constructed a human soluble thrombomodulin (sTM) expression vector using the RSV promoter. Recombinant sTM (rsTM) was expressed in CHO cells and was recovered from culture medium by ion exchange chromatography. Two active fractions, designated as rsTM alpha (low salt elution) and rsTM beta (high salt elution), were detected and further purified by immunoaffinity chromatography. Purified rsTM beta contained bound chondroitin-4-sulfate as judged by HPLC detection of the chondroitinase ABC and AC I digestion product, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose. The apparent Kd values for thrombin of alpha and beta were 7.4 and 1.4 nM respectively. RsTM beta was more effective at inhibition of thrombin clotting activity and had antithrombin III-dependent anticoagulant activity which was not possessed by rsTM alpha. Both anticoagulant activities were lost after chondroitinase treatment of rsTM beta.
authors.affiliation: Research Institute, Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Nawa
authors.forename: K
authors.initials: K
authors.lastname: Sakano
authors.forename: H
authors.initials: H
authors.lastname: Fujiwara
authors.forename: Y
authors.initials: Y
authors.lastname: Sato
authors.forename: N
authors.initials: N
authors.lastname: Sugiyama
authors.forename: T
authors.initials: T
authors.lastname: Teruuchi
authors.forename: M
authors.initials: M
authors.lastname: Iwamoto
authors.forename: Y
authors.initials: Y
authors.lastname: Marumoto
mesh_terms: D000818:Animals; D002460:Cell Line; D002807:Chondroitin / Q000031:analogs & derivatives*; D002808:Chondroitin Lyases; D002809:Chondroitin Sulfates / Q000302:isolation & purification* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D008970:Molecular Weight; D010957:Plasmids; D011956:Receptors, Cell Surface / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D018179:Receptors, Thrombin; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013917:Thrombin / Q000378:metabolism; D014162:Transfection
chemical_list: D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D002807:Chondroitin; D002809:Chondroitin Sulfates; D013917:Thrombin; D002808:Chondroitin Lyases