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J. Biol. Chem., Vol. 281, Issue 2, 850-857, January 13, 2006

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Zinc and Calcium Ions Cooperatively Modulate ADAMTS13 Activity^*

Patricia J. Anderson, Koichi Kokame, and J. Evan Sadler1

From the Howard Hughes Medical Institute, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110 and the National Cardiovascular Center Research Institute, Osaka 565-8565, Japan

ADAMTS13 is a metalloproteinase that cleaves von Willebrand factor (VWF) multimers. The metal ion dependence of ADAMTS13 activity was examined with multimeric VWF and a fluorescent peptide substrate based on Asp¹⁵⁹⁶–Arg¹⁶⁶⁸ of the VWF A2 domain, FRETS-VWF73. ADAMTS13 activity in citrate-anticoagulated plasma was enhanced 2-fold by zinc ions, 3-fold by calcium ions, and 6-fold by both ions, suggesting cooperative activation. Cleavage of VWF by recombinant ADAMTS13 was activated up to 200-fold by zinc ions (K_{D app} 0.5 µM), calcium ions (K_{D app} 4.8 µM), and barium ions (K_{D app} 1.7 mM). Barium ions stimulated ADAMTS13 activity in citrated plasma but not in citrate-free plasma. Therefore, the stimulation by barium ions of ADAMTS13 in citrated plasma appears to reflect the release of chelated calcium and zinc ions from complexes with citrate. At optimal zinc and calcium concentrations, ADAMTS13 cleaved VWF with a K_{m app} of 3.7 ± 1.4 µg/ml (15 nM for VWF subunits), which is comparable with the plasma VWF concentration of 5–10 µg/ml. ADAMTS13 could cleave 14% of VWF pretreated with guanidine HCl, suggesting that this substrate is heterogeneous in susceptibility to proteolysis. ADAMTS13 cleaved FRETS-VWF73 with a K_{m app} of 3.2 ± 1.1 µM, consistent with an 200-fold decrease in affinity compared with VWF. ADAMTS13 cleaved VWF and FRETS-VWF73 with roughly comparable catalytic efficiency of 55 µM^–1 min^–1 and 18 µM^–1 min^–1, respectively. The striking preference of ADAMTS13 for VWF suggests that substrate recognition depends on structural features or exosites on multimeric VWF that are missing from FRETS-VWF73.

Received for publication, April 26, 2005 , and in revised form, October 7, 2005.

^* This work was supported in part by American Heart Association National Scientist Development Award 0530110N (to P. J. A.) and by National Institutes of Health Grant HL72917. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Howard Hughes Medical Institute, WA University School of Medicine, 660 S. Euclid, Box 8022, St. Louis, MO 63110. Tel.: 314-362-9029; Fax: 314-454-3012; E-mail: esadler{at}im.wustl.edu.

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