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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. This Article Full Text (PDF) All Versions of this Article: blood-2008-03-144683v1 113/5/1149    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Gardner, M. D Articles by Lane, D. A. Search for Related Content PubMed PubMed Citation Articles by Gardner, M. D Articles by Lane, D. A. Social Bookmarking                   What's this?  Previous Article  |  Next Article  Submitted March 11, 2008 Accepted October 23, 2008 A functional calcium binding site in the metalloprotease domain of ADAMTS13 Michelle D Gardner, Chan K. N. K. Chion, Rens de Groot, Anuja Shah, James T.B. Crawley*, and David A. Lane Department of Haematology, Imperial College London, London, United Kingdom * Corresponding author; email: j.crawley{at}imperial.ac.uk. 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 pre-incubated with varying Ca2+ concentrations, a high affinity functional ADAMTS13 Ca2+ binding site was suggested with KD(app) of 80 ± 15µM, 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 close 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 ~25% normal. Kinetic analysis of the Asp187 mutant in the presence of excess Ca2+ revealed a ~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. CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: 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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