Functional characterization of ADAMTS13 VR3 (G236-A261) MP domain mutants. (A-B) Proteolysis of VWF115 (A) or VWF115(M1606A) (B) over time by wild-type (WT) ADAMTS13 or VR3B swap variants was visualized by SDS-PAGE and Coomassie staining. (C-D) Proteolysis of VWF115 (C) or VWF115(M1606A) (D) by ADAMTS13 and VR3B swap variants was quantified over time by HPLC and represented graphically. See Table 2 for the derived k_cat/K_m values. (E) To establish the individual kinetic constants K_m and k_cat for VWF115(M1606A) proteolysis, the initial rate of proteolysis (< 15% cleavage) was analyzed by HPLC as a function of substrate concentration. The derived individual parameters are presented in Table 3. (F) 0.5nM wild-type ADAMTS13 or VR3B swap variant was used to proteolyze multimeric, recombinant VWF(M1606A) under denaturing conditions. From 0 to 60 minutes, reactions were stopped with EDTA, and changes in VWF multimeric composition were monitored by SDS agarose gel electrophoresis and Western blotting for VWF.