Western blot and proteolytic activity of ADAMTS13 and mutants. (A) Western blotting with anti-V5 IgG detects WT and single point mutants at the position 660 in the concentrated condition medium (∼ 50nM per lane). Arrowhead indicates the intact full-length ADAMTS13, ∼ 195 kDa; and double stars, degradation product. (B) Relative proteolytic activity (%) of WT and single point variants assessed by the cleavage of rF-VWF73. Data are mean ± SD (n = 3). All ADAMTS13 mutants except for R660K had relative activity less than 20% of WT. (C) Proteolytic cleavage of multimeric VWF by ADAMTS13 and single point mutants under denaturing conditions. Plasma-derived VWF (37.5 μg/mL or 150nM) was incubated at 37°C with 0.2nM of recombinant WT-ADAMTS13 and point mutants in the presence of 1.5M urea for 4 hours. The proteolytic cleavage of VWF was determined by 1% agarose gel electrophoresis and Western blotting. + indicates the presence of 10mM EDTA in the reaction; −, the absence of EDTA in the reaction; HMW, high molecular weight multimers; and P, cleavage product.