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Further characterization of ADAMTS-13 inactivation by thrombin

J K Lam1, C K N K Chion, S Zanardelli

  • 1Department of Haematology, Imperial College London, London, UK.

Journal of Thrombosis and Haemostasis : JTH
|March 16, 2007
PubMed
Abstract

Insights

ADAMTS-13, an enzyme regulating von Willebrand factor, is protected from thrombin inactivation in normal plasma. This protection mechanism is crucial for maintaining its function in blood coagulation.

Area of Science:

  • Biochemistry
  • Hematology
  • Protease activity

Background:

  • The metalloprotease ADAMTS-13 regulates von Willebrand factor (VWF) multimeric size and platelet tethering.
  • In vitro studies show ADAMTS-13 is vulnerable to inactivation by thrombin.

Purpose of the Study:

  • To investigate the inactivation of ADAMTS-13 by thrombin.
  • To determine the physiological relevance of this interaction.

Main Methods:

  • N-terminal sequencing and mutagenesis to identify thrombin cleavage sites on ADAMTS-13.
  • Utilized a library of thrombin mutants to assess exosite I function.
  • Measured VWF binding affinity and VWF-cleaving function post-proteolysis.
  • Assessed ADAMTS-13 behavior during plasma coagulation.

Main Results:

  • Identified R257 and R1176 as primary thrombin cleavage sites on ADAMTS-13.
  • Thrombin proteolysis reduced ADAMTS-13's VWF affinity by 8-fold, impairing VWF cleavage.
  • Thrombin-cleaved ADAMTS-13 retained binding and proteolysis of a VWF A2 domain substrate.
  • Endogenous ADAMTS-13 resisted coagulation-induced fragmentation in normal plasma.
  • ADAMTS-13 proteolysis by thrombin had a K(m) higher than physiological ADAMTS-13 concentrations, with low affinity for thrombin (K(D) 95 nM).

Conclusions:

  • ADAMTS-13 is protected from rapid thrombin inactivation in normal plasma.
  • The protection mechanism's status in pathological conditions with sustained thrombin generation requires further investigation.

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