Mutagenesis of Site 1 abolishes the Ca²⁺-induced conformational change in ADAMTS13 observed under low-ionic-strength conditions. Analysis of putative Site 1 residue mutants. (A) 1 nM ADAMTS13 wild-type, E83A, or D173A in 150 mM NaCl and 20 mM Tris-HCl (pH 7.8) was preincubated with 0 to 5 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 derived. (B) The Abs₂₈₀ (± SD) of 1 μM purified ADAMTS13 (■; WT), E83A (▩), or D173A (□) in 20 mM Tris-HCl (pH 7.8) plus or minus 10 mM CaCl₂ was measured at 0 minutes and 60 minutes after the addition of CaCl₂. (C) Change in Abs₂₈₀ of 1 μM ADAMTS13 (WT), E83A, and D173A in 20 mM Tris-HCl (pH 7.8) and 10 mM CaCl₂ over 60 minutes.