Purification of human von Willebrand factor-cleaving protease and its identification as a new member of the metalloproteinase family

von Willebrand factor (vWF) is synthesized in megakaryocytes and endothelia l cells as a very large multimer, but circulates in plasma as a group of mu ltimers ranging from 500 to 10 000 kd. An important mechanism for depolymer ization of the large multimers is the limited proteolysis by a vWF-cleaving protease present in plasma. The absence or inactivation of the vWF-cleavin g protease results in the accumulation of large multimers, which may cause thrombotic thrombocytopenic purpura. The vWF-cleaving protease was first de scribed as a Ca++-dependent proteinase with an apparent molecular weight of approximately 300 kd. Thus far, however, it has not been isolated and char acterized. In this study, the purification of human vWF-cleaving protease f rom a commercial preparation of factor VIII/vWF concentrate by means of sev eral column chromatographic steps, including 2 steps of heparin-Sepharose c olumn, is reported. Sodium dodecyl sulfate-polyacrylamide gel electrophores is analysis of the anion exchange and gel filtration column fractions showe d 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. Th e 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 i n parentheses. A search of the human genome sequence identified the vWF-cle aving protease as a new member of the ADAMTS (a disintegrin and metalloprot einase with thrombospondin type I motif) family of metalloproteinase. An ac tive site sequence of HEIGHSFGLEHE (single-letter amino acid codes) was loc ated at 150 residues from the N terminus of the protein. (C) 2001 by The Am erican Society of Hematology.