Site 3 is a high-affinity functional Ca²⁺-binding site and is critical for ADAMTS13 activity. (A) Analysis of putative Site 3 mutants. 1 nM transiently expressed ADAMTS13 (WT), E184A, D187A, or E212A Site 3 mutants in 150 mM NaCl and 20 mM Tris-HCl (pH 7.8) were 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 D187A mutant in 20 mM Tris-HCl (pH 7.8) in the absence (□) or presence (■) of 10 mM CaCl₂ was measured at 0 minutes and 60 minutes after the addition of CaCl₂. (C) Change in Abs₂₈₀ of 1 μM purified ADAMTS13 (WT) or D187A mutant in 20 mM Tris-HCl (pH 7.8) and 10 mM CaCl₂ over 60 minutes. (D,E) Plasma-derived purified full-length VWF was pretreated with 1.5 M guanidine-HCl to denature the protein and then added to a preincubating mixture of 5 nM wild-type or D187A mutant, 150 mM NaCl, 20 mM Tris-HCl (pH 7.8), and varying CaCl₂ concentrations between 0 and 5 mM. The reaction was incubated at 37°C for 60 minutes before quenching with EDTA. Samples were analyzed by collagen-binding assay to assess the ability of the remaining VWF in the sample to bind human collagen type III (D). Samples were also run on a 1.4% agarose gel with subsequent Western blotting to detect VWF multimeric composition (E).