Cleavage of GST-VWF73 and rF-VWF73 by wild-type ADAMTS13 and various mutants. Purified GST-VWF73 (50nM) was incubated with the wild-type ADAMTS13 or mutants (2.5nM) in the absence (−) or presence (+) of EDTA (20mM) at 37°C for 1 hour (A) and 3 hours (B). The proteolytic cleavage product (open arrow) was determined by Western blotting. *The preexisting nonspecific bands in the purified GST-VWF73 substrate. (C) Purified recombinant rF-VWF73 at various concentrations (0-12μM) was incubated at 37°C with wild-type (WT) ADAMTS13 or mutants (2.5nM). The rate of fluorescent generation was determined at the ex 485 nm and em 535 nm. The maximal rate of fluorescent generation (V_max) with D[b]-Phe[b]-Pro[b]-Arg[b]-chloromethylketone-treated chymotrypsin (20nM) that cleaves specifically at the methionyl bond was used for a calibration. The relative mean product formation (nanomoles per second), compared with chymotrypsin at the same concentration, is expressed as a function of the substrate concentrations (micromole). The data represent the means ± SD (n = 3).