Purification and characterization of two forms of soluble thrombomodulin from human urine.
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.
Jackson
D E
DE
Department of Medicine, Monash Medical School, Box Hill Hospital, Melbourne, Australia.
Tetaz
T J
TJ
Salem
H H
HH
Mitchell
C A
CA
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*
D018180:Thrombomodulin