Soluble VWF interaction with ADAMTS13 distal domains. (A) VWF (30 nM) was immobilized on a 96-well plate and incubated with increasing concentrations of ADAMTS13 (0-400 nM) for 2 hours at 37°C. Bound ADAMTS13 was detected by biotinylated anti–TSP2-4 polyclonal antibody, followed by streptavidin-HRP and OPD-H2O2. (B) ADAMTS13, MDTCS, and del (TSP5-CUB) (30 nM of each) were coated into corresponding wells in a microtiter plate. Soluble VWF (0-400 nM) was then added and incubated for 2 hours at 37°C. Bound VWF was detected using HRP-conjugated anti-VWF IgG. (C) VWFA2 or VWFD4CK (30 nM of either) was immobilized on a plate. Increasing concentrations of soluble VWF (0-200 nM) were preincubated for 60 minutes with 12 and 50 nM ADAMTS13, respectively. Bound ADAMTS13 was detected as in panel A. The result was plotted as relative inhibition against VWF concentration, with the binding in the absence of inhibitor taken as 100%.
