Putative Site 2 is not a high-affinity functional binding site. Analysis of putative Site 2 mutants. 1 nM transiently expressed ADAMTS13 (WT) or E164A or D166A Site 2 mutant in 150 mM NaCl and 20 mM Tris-HCl (pH 7.8) were preincubated with 0 to 8 mM CaCl₂ for 60 minutes at 37°C before addition of 1.6 μM VWF115 substrate. After 10 minutes, reactions were stopped with EDTA, and VWF115 cleavage was quantified by HPLC, from which initial rates of substrate proteolysis were determined. Initial rates are plotted as a function of Ca²⁺ concentration, from which the K_D(app) was determined.