Evaluation of ADAMTS-13 activity in plasma using recombinant von Willebrand Factor A2 domain polypeptide as substrate

Miguel A. Cruz; Jody Whitelock; Jing-fei Dong

doi:10.1160/TH03-06-0398

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2003; 90(06): 1204-1209
DOI: 10.1160/TH03-06-0398

New Technologies and Diagnostic Tools

Schattauer GmbH

Evaluation of ADAMTS-13 activity in plasma using recombinant von Willebrand Factor A2 domain polypeptide as substrate

Miguel A. Cruz

¹The Thrombosis Research Section, Department of Medicine, Baylor College of Medicine, Houston, Texas, USA

,

Jody Whitelock

¹The Thrombosis Research Section, Department of Medicine, Baylor College of Medicine, Houston, Texas, USA

,

Jing-fei Dong

¹The Thrombosis Research Section, Department of Medicine, Baylor College of Medicine, Houston, Texas, USA

› Author Affiliations

Abstract

Summary

The metalloprotease ADAMTS-13 cleaves von Willebrand factor (VWF), and is absent or severely reduced in the plasma of patients with thrombotic thrombocytopenia purpura (TTP). Under physiologic flowing conditions, the enzyme cleaves endothelial cell-derived ultra-large VWF multimers at the Y842/M843 peptide bond located in the A2 domain, where many mutations associated with Type 2A VWD cluster. These VWF mutants are more susceptible for cleavage activity, decreasing the large VWF multimers in the plasma. The susceptibility of a recombinant VWF-A2 domain to ADAMTS-13 and the potential application in detecting enzyme activity were investigated. In vitro, fluid phase cleavage of VWF by ADAMTS-13 requires denaturing conditions and prolonged incubation in order to estimate enzyme activity. We have measured ADAMTS-13 activity based on enzyme cleavage of a recombinant VWF-A2 domain under non-denaturing conditions. In our assay, enzyme activity was absent in plasma from congenital and acquired TTP patient, and blocked by each EDTA, monoclonal antibody VP-1 (peptide-specific antibody against residues 828-842 of VWF), and an ADAMTS-13 antibody purified from plasma of an acquired TTP patient. This novel recombinant VWF-A2 protein has potential utility as matrix for a rapid clinical measurement of plasma ADAMTS-13 activity.

Keywords

von Willebrand Factor - recombinant protein - ADAMTS-13 - proteolysis detection

Full Text

References

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