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 Blood, 29 January 2009, Vol. 113, No. 5, pp. 1149-1157.
Prepublished online as a Blood First Edition Paper on December 1, 2008; DOI 10.1182/blood-2008-03-144683.

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THROMBOSIS AND HEMOSTASIS

A functional calcium-binding site in the metalloprotease domain of ADAMTS13

Michelle D. Gardner1, Chan K. N. K. Chion1, Rens de Groot1, Anuja Shah1, James T. B. Crawley1, and David A. Lane1

1 Department of Haematology, Imperial College London, London, United Kingdom

ADAMTS13 regulates the multimeric size of von Willebrand factor (VWF). Its function is highly dependent upon Ca2+ ions. Using the initial rates of substrate (VWF115, VWF residues 1554-1668) proteolysis by ADAMTS13 preincubated with varying Ca2+ concentrations, a high-affinity functional ADAMTS13 Ca2+-binding site was suggested with KD(app) of 80 µM (± 15 µM) corroborating a previously reported study. When Glu83 or Asp173 (residues involved in a predicted Ca2+-binding site in the ADAMTS13 metalloprotease domain) were mutated to alanine, Ca2+ dependence of proteolysis of the substrate was unaffected. Consequently, we sought and identified a candidate Ca2+-binding site in proximity to the ADAMTS13 active site, potentially comprising Glu184, Asp187, and Glu212. Mutagenesis of these residues within this site to alanine dramatically attenuated the KD(app) for Ca2+ of ADAMTS13, and for D187A and E212A also reduced the Vmax to approximately 25% of normal. Kinetic analysis of the Asp187 mutant in the presence of excess Ca2+ revealed an approximately 13-fold reduction in specificity constant, kcat/Km, contributed by changes in both Km and kcat. These results were corroborated using plasma-purified VWF as a substrate. Together, our results demonstrate that a major influence of Ca2+ upon ADAMTS13 function is mediated through binding to a high-affinity site adjacent to its active site cleft.


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R. de Groot, A. Bardhan, N. Ramroop, D. A. Lane, and J. T. B. Crawley
Essential role of the disintegrin-like domain in ADAMTS13 function
Blood, May 28, 2009; 113(22): 5609 - 5616.
[Abstract] [Full Text] [PDF]


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