Zinc and Calcium Ions Cooperatively Modulate ADAMTS13 Activity

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 (KDapp ∼0.5 μm), calcium ions (KDapp ∼4.8 μm), and barium ions (KDapp ∼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 Kmapp 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 Kmapp 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.