Analysis of ADAMTS13 Cys5 and ADAMTS13 Gly3 variants function. (A) FRETS-VWF73 was used to quantify the activity of ADAMTS13 variants Gly3 and Cys5 and compare it to the residual activity of spacer domain mutant RRYY. A total of 0.6 nM WT ADAMTS13 or ADAMTS13 variant in conditioned medium was incubated with 2 µM FRETS-VWF73 and fluorescence measured over time. A representative graph is shown (n = 5). (B) Proteolysis of recombinant full-length VWF (N1602A/C1669G/C1670G) by 8 nM ADAMTS13 or ADAMTS13 variants. Subsamples were stopped with EDTA at designated time points and analyzed by western blotting under reducing conditions using an anti-VWF monoclonal Ab that detects both full-length VWF and the 176-kDa cleavage product. (C) Binding of WT ADAMTS13 and ADAMTS13 Cys-rich Cys5, Cys6, and Gly3 variants to VWF115. VWF115 (50 nM) was immobilized on microtiter wells and incubated with increasing concentrations of ADAMTS13 or variant. Wells were washed and ADAMTS13 binding detected using a biotinylated anti-ADAMTS13 polyclonal antibody followed by streptavidin/horseradish peroxidase and orthophenylenediamine. Absorbance at 492 nm was used to quantify binding. Mean values and error bars are shown (n = 3) except for variant RRYY (n = 1).