Effect of Ca²⁺ binding on A2 cleavage by ADAMTS13. (A) Examination of the protective effect of Ca²⁺ with 2-step ADAMTS13 cleavage assays. In step I, the wtA2 protein was denatured under different urea concentrations and Ca²⁺ was supplemented as indicated. In step II, the Ca²⁺ concentration was adjusted to 2.5mM. The cleaved and intact A2 proteins were detected with Western blotting using the specific antibodies. (B) Examination of the effect of Ca²⁺ on the proteolysis of the GST-VWF73 proteins by ADAMTS13. No urea was added in step I. (C) Examination of the effect of Ca²⁺ on the proteolysis of the wtA2 protein by ADAMTS13 without adjustment of the Ca²⁺ concentration in step II. (D) Examination of the effect of Ca²⁺ ion on the proteolysis of wtA2 by ADAMTS13 with 1-step assays.