Identification of ADAMTS13 disintegrin-like domain residues important for proteolysis of VWF. (A) Activity assays containing 0.5 nM wild-type or mutant ADAMTS13 and 6 μM VWF115 were incubated at 37°C. At the times indicated, reactions were stopped with EDTA and analyzed by SDS-PAGE. Proteolysis was assessed by the generation of the 10-kDa and 7-kDa VWF115 cleavage products. (B) Multimeric VWF activity assays were set up using 1 nM wild-type ADAMTS13 or VS1 mutants (D330A and Q333A) under denaturing conditions. Reactions were stopped after 2 hours with EDTA followed by VWF multimer analysis. (C) Multimeric VWF activity assays were set up using 3.5 nM wild-type ADAMTS13 or HVR mutants under denaturing conditions. Reactions were stopped after 1 hour with EDTA followed by VWF multimer analysis.