Ca²⁺ dependence of the VWF115 proteolysis by ADAMTS13. (A) 20 nM purified ADAMTS13 in 150 mM NaCl, 20 mM Tris-HCl (pH 7.8) was preincubated with 5 mM CaCl₂ for 60 minutes (+ Preinc), 0 minutes (−Preinc), or in the absence of CaCl₂ (−Ca²⁺) before addition of 1.6 μM VWF115 substrate. Reactions were incubated at 37°C and stopped with EDTA, and VWF115 cleavage was quantified by HPLC. (B) The reaction conditions were as in panel A, but initial rates of substrate proteolysis by fully purified ADAMTS13 were determined as a function of preincubation time with 5 mM CaCl₂. (C) Reactions were set up again as in panel A, except that the concentration of CaCl₂ was varied and the K_D(app) (± SD; n = 4) was derived for purified ADAMTS13. (D) 1 nM of ADAMTS13 in dialyzed conditioned medium was used under identical conditions to that used in panel C, and the K_D(app) (± SD; n = 4) was again derived. (E) 1 nM of ADAMTS13 in dialyzed conditioned medium was studied as in panel A, except that preincubation was performed in the presence of 10 mM (final concentration) of CaCl₂, BaCl₂, MgCl₂, NiSO₄, MnCl₂, or CuSO₄. (F) 1 nM ADAMTS13 was pretreated with 15 mM EDTA to remove the Zn²⁺ ions, dialyzed and equilibrated in 150 mM NaCl, 20 mM Tris-HCl (pH 7.8), 5 mM CaCl₂, and varying concentrations of ZnCl₂ between 0 and 100 μM for 60 minutes at 37°C before addition of 1.6 μM VWF115 substrate. Reactions were stopped with EDTA after 10 minutes and proteolysis analyzed by HPLC, from which a K_D(app) for Zn²⁺ of 1.5 μM (± 0.6 μM) was derived.