Binding of ADAMTS13 and mutants to immobilized VWF73 and multimeric VWF. The microtiter plate was coated without (nonspecific binding controls) or with 100 μL of purified GST-VWF73 (2.0 μg/mL; panel A) or plasma-derived VWF (12.5 μg/mL; panel B). After being blocked with 2.5% BSA in 20mM Tris-HCl, 150mM NaCl for 30 minutes, 100 μL of wild-type ADAMTS13 or mutants at the concentration indicated diluted with PBS containing 10mM EDTA, 0.5% BSA, and 0.05% Tween 20 were added for 2 hours. The plate was washed 3 times, and the bound wild-type (WT) ADAMTS13 and mutants were detected by anti-V5 IgG, peroxidase conjugated (1:1000). The data in both panels A and B represent the means of 3 independent experiments. The apparent dissociation constants (K_D; app) were determined by fitting the data into the Michaelis-Menten equation with SigmaPlot software.