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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    28 Citations (Scopus)

    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

    Cite this

    @article{dff27505ed2a4d6c9f147a7e633e0905,
    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 34-1 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.",
    author = "Jackson, {D. E.} and Tetaz, {T. J.} and Salem, {H. H.} and Mitchell, {C. A.}",
    year = "1994",
    language = "English",
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    pages = "1079--1087",
    journal = "European Journal of Biochemistry",
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    Purification and characterization of two forms of soluble thrombomodulin from human urine. / Jackson, D. E.; Tetaz, T. J.; Salem, H. H.; Mitchell, C. A.

    In: European Journal of Biochemistry, Vol. 221, No. 3, 1994, p. 1079-1087.

    Research output: Contribution to journalArticleResearchpeer-review

    TY - JOUR

    T1 - Purification and characterization of two forms of soluble thrombomodulin from human urine

    AU - Jackson, D. E.

    AU - Tetaz, T. J.

    AU - Salem, H. H.

    AU - Mitchell, C. A.

    PY - 1994

    Y1 - 1994

    N2 - 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.

    AB - 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.

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    JO - European Journal of Biochemistry

    JF - European Journal of Biochemistry

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