Purification and characterization of two forms of soluble thrombomodulin from human urine

D. E. Jackson, T. J. Tetaz, H. H. Salem, C. A. Mitchell

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    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 34-1 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 in urine is an inactive 35-kDa thrombomodulin polypeptide derived from the N-terminal extracellular region of thrombomodulin.

    Original languageEnglish
    Pages (from-to)1079-1087
    Number of pages9
    JournalEuropean Journal of Biochemistry
    Volume221
    Issue number3
    Publication statusPublished - 1994

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