title: Purification and characterization of two forms of soluble thrombomodulin from human urine.
abstract: We have isolated and characterized two forms of soluble thrombomodulin from human urine. The purification procedure consisted of ultrafiltration, chromatography on DEAE-Sepharose, affinity chromatography on diisopropyl-phosphate-thrombin and/or monoclonal anti-thrombomodulin IgG affigel followed by reverse-phase HPLC. An active soluble form of thrombomodulin was purified 1600-fold from 34-l urine. The purified protein migrated as a doublet, with molecular mass 76/72 kDa under reducing conditions and 63/57 kDa under non-reducing conditions as determined by SDS/PAGE. Amino acid analysis of the 63/57-kDa soluble thrombomodulin confirmed sequence identity with human thrombomodulin and demonstrated N-terminal heterogeneity. Compared to membrane-type thrombomodulin, the purified 63/57-kDa soluble thrombomodulin was more active as a cofactor for protein-C activation. The second major thrombomodulin fragment urine is an inactive 35-kDa thrombomodulin polypeptide derived from the N-terminal extracellular region of thrombomodulin.
authors.affiliation: Department of Medicine, Monash Medical School, Box Hill Hospital, Melbourne, Australia.
authors.forename: D E
authors.initials: DE
authors.lastname: Jackson
authors.forename: T J
authors.initials: TJ
authors.lastname: Tetaz
authors.forename: H H
authors.initials: HH
authors.lastname: Salem
authors.forename: C A
authors.initials: CA
authors.lastname: Mitchell
mesh_terms: D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010084:Oxidation-Reduction; D012995:Solubility; D018180:Thrombomodulin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014462:Ultrafiltration; D014556:Urine / Q000737:chemistry*
chemical_list: D018180:Thrombomodulin