Solution competition assay of ADAMTS13 with VWF A2 domain glycan variants. WT VWF A2 domain (50 nM) was immobilized on a microtiter plate. A total of 30 nM (inactive) iADAMTS13 (E225A) was separately preincubated with varying concentration of the VWF A2 domain glycan variants (0-500 nM) in the presence of 1 mM EDTA or 5mM CaCl2 for 1 hour at 37°C: N1574Q (A), PNGase (B), GnT−/− (C), and EndoH (D). The preincubated VWF A2/ADAMTS13 was added to the microtiter plate for 1 or 2 hours, where the preincubated VWF A2 domain variants compete with the immobilized WT VWF A2 domain for binding to the iADAMTS13. After washing, iADAMTS13 bound to the immobilized VWF A2 domain was detected by a biotinylated anti–TSP2-4 polyclonal antibody followed by streptavidin–horseradish peroxidase and o-phenylenediamine hydrochloride. Preincubation controls containing 0 nM VWFA2 and 30 nM iADAMTS13 represented 0% solution binding of the VWF A2 domain to iADAMTS13, and 0 nM VWF A2 and 0 nM iADAMTS13 represented 100% solution binding of VWF A2 to ADAMTS13. Results were plotted as percent solution binding against preincubation concentration of the VWF A2 domain variant and fitted using the 1-site binding model (GraphPad). Results are means ± SEM of at least 3 independent experiments.
