title: Purification of human von Willebrand factor-cleaving protease and its identification as a new member of the metalloproteinase family.
abstract: von Willebrand factor (vWF) is synthesized in megakaryocytes and endothelial cells as a very large multimer, but circulates in plasma as a group of multimers ranging from 500 to 10 000 kd. An important mechanism for depolymerization of the large multimers is the limited proteolysis by a vWF-cleaving protease present in plasma. The absence or inactivation of the vWF-cleaving protease results in the accumulation of large multimers, which may cause thrombotic thrombocytopenic purpura. The vWF-cleaving protease was first described as a Ca(++)-dependent proteinase with an apparent molecular weight of approximately 300 kd. Thus far, however, it has not been isolated and characterized. In this study, the purification of human vWF-cleaving protease from a commercial preparation of factor VIII/vWF concentrate by means of several column chromatographic steps, including 2 steps of heparin-Sepharose column, is reported. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the anion exchange and gel filtration column fractions showed that the vWF-cleaving protease activity corresponded to a protein band of 150 kd. After reduction, it migrated with an apparent weight of 190 kd. The amino terminal sequence of the 150-kd band was AAGGIL(H)LE(L)L(D)AXG(P)X(V)XQ (single-letter amino acid codes), with the tentative residues shown in parentheses. A search of the human genome sequence identified the vWF-cleaving protease as a new member of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin type I motif) family of metalloproteinase. An active site sequence of HEIGHSFGLEHE (single-letter amino acid codes) was located at 150 residues from the N terminus of the protein.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA 98195, USA. fujikawa@u.washington.edu
authors.forename: K
authors.initials: K
authors.lastname: Fujikawa
authors.forename: H
authors.initials: H
authors.lastname: Suzuki
authors.forename: B
authors.initials: B
authors.lastname: McMullen
authors.forename: D
authors.initials: D
authors.lastname: Chung
mesh_terms: D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006801:Humans; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000302:isolation & purification*; D008969:Molecular Sequence Data
chemical_list: D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human