PURIFICATION AND CHARACTERIZATION OF 2 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 ultrafiltra tion, chromatography on DEAE-Sepharose, affinity chromatography on dii sopropyl-phosphate - thrombin and/or monoclonal anti-thrombomodulin Ig G affigel followed by reverse-phase HPLC. An active soluble form of th rombomodulin was purified 1600-fold from 34-1 urine. The purified prot ein migrated as a doublet, with molecular mass 76/72 kDa under reducin g conditions and 63/57 kDa under non-reducing conditions as determined by SDS/PAGE. Amino acid analysis of the 63/57-kDa soluble thrombomodu lin confirmed sequence identity with human thrombomodulin and demonstr ated N-terminal heterogeneity. Compared to membrane-type thrombomoduli n, the purified 63/57-kDa soluble thrombomodulin was more active as a cofactor for protein-C activation. The second major thrombomodulin fra gment in urine is an inactive 35-kDa thrombomodulin polypeptide derive d from the N-terminal extracellular region of thrombomodulin.