Proteolytic cleavage of VWF115 by spacer domain ADAMTS13 variants with mutations in Y658-Y665. (A) Each single alanine variant (2nM each) in region Y658-Y665 or wild-type ADAMTS13 was incubated with 6μM VWF115 substrate at 37°C. After 0, 30, and 60 minutes samples were taken, and the reaction was stopped with EDTA and analyzed on SDS-PAGE. The VWF115 cleavage products of 10 and 7 kDa are indicated. (B) Similar reactions were set up by the use of ADAMTS13-RY1, RY2, YY, and RYY. Conditioned medium derived from nontransfected cells was included as a negative control (−ve Ctrl). (C) Cleavage products generated in activity assays with 2nM wild-type ADAMTS13 or ADAMTS13-RYY and 2μM VWF115 activity at the times indicated were quantified by HPLC. VWF115 proteolysis is depicted in percentages and mean values of 3 experiments, with error bars representing SD are shown. (D) From the graph in panel C, the catalytic efficiencies (k_cat/K_m) for VWF115 proteolysis were derived and mean values ± SD are shown in the table.