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

Blood. 2001 Sep 15;98(6):1662-6. doi: 10.1182/blood.v98.6.1662.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • ADAM Proteins
  • ADAMTS13 Protein
  • Amino Acid Sequence
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Humans
  • Metalloendopeptidases / chemistry*
  • Metalloendopeptidases / isolation & purification*
  • Molecular Sequence Data

Substances

  • ADAM Proteins
  • Metalloendopeptidases
  • ADAMTS13 Protein
  • ADAMTS13 protein, human