Functional characterization of ADAMTS13 VR1 (E184-R193) MP domain mutants. (A-B) Proteolysis of VWF115 over time by ADAMTS13/VR1 swap variants (A) or VR1 point mutants (B) was visualized by SDS-PAGE and Coomassie staining. For more sensitive detection of the 7-kDa VWF115 cleavage product (to detect trace amounts of proteolysis), a Western blot is included below Figure 4A. (C) Proteolysis of VWF115 was quantified over time by HPLC for those variants with reduced proteolytic function. See Table 1 for the derived k_cat/K_m values. (D-E) Multimeric, plasma-derived full-length VWF was proteolyzed by 2.8nM VR1 swap variants (D) or 2.1nM point mutants (E) and analyzed by agarose gel electrophoresis and Western blotting for VWF. HMW, high molecular weight; WT, wild type.