Effect of mutations at the Ca²⁺-binding site on A2 cleavage by ADAMTS13. (A) Examination of the effect of the D1596A mutation on Ca²⁺ protection of A2. In step I, the D1596A mutant A2 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 proteins were detected with Western blotting using the specific antibodies. (B) Examination of the effect of the D1498A, R1597W, and N1602A mutations on Ca²⁺ protection of A2. In step I, 2.56M urea was added. (C) Examination of the effect of the D1498A, R1597W, and N1602A mutations on the proteolysis of GST-VWF73 by ADAMTS13. No urea was added in step I and the Ca²⁺ concentration was 2.5mM in both steps.