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

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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.