Proteolytic cleavage of multimeric VWF by ADAMTS13 and variants under denaturing conditions. (A) Human plasma-derived VWF (37.5 μg/mL or 150nM) predenatured with 1.5M urea was mixed with WT, M1, M2, M3, M4, and M5 (0.04nM and 0.2nM) in the absence or in the presence of EDTA (10mM; last lane) and dialyzed against 10mM Tris-HCl, pH 8.0, containing 1.5M urea at 37° for 4 hours. The cleavage of VWF was determined by agarose (1%) gel electrophoresis and Western blotting with rabbit anti-VWF IgG (1:5000), followed by IRDye 800CW-labeled goat anti–rabbit IgG (1:10 000). (B) The relative activity was determined by ImageJ quantitation of the ratio of cleavage product (P, arrowhead) to high molecular weight (HMW) VWF multimers in each sample. The specific activity was normalized to that of WT (1 arbitrary unit). Data are mean ± SEM from 3 independent experiments (n = 3).