Open AccessPurification and characterization of two forms of soluble thrombomodulin from human urine
Author(s) -
JACKSON Denise E.,
TETAZ Tim J.,
SALEM Hatem H.,
MITCHELL Christina A.
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.tb18827.x
Subject(s) - thrombomodulin , thrombin , affinity chromatography , chemistry , ultrafiltration (renal) , biochemistry , high performance liquid chromatography , microbiology and biotechnology , chromatography , biology , enzyme , platelet , immunology
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.