Evidence for an ADAMTS13 binding site on VWF distal to its A2 domain. (A) Of each VWF fragment (depicted in Figure 1A), 30 nM was immobilized on a 96-well microtiter plate and incubated with increasing concentrations of recombinant ADAMTS13 (0-300 nM) in the presence of 10 mM EDTA, for 2 hours at 37°C. The bound ADAMTS13 was detected by a biotinylated anti–TSP2-4 polyclonal antibody followed by streptavidin-HRP and OPD-H₂O₂. The curves were fitted to the one-binding site model using GraphPad Prism 4. The calculated K_D(app) values are listed in Table 1. (B) VWFA2, VWFA1A2A3, and VWFA1CK (50 nM of each) were coated on a microtiter plate and soluble VWFA2 was used as a competitor for their binding to ADAMTS13 (A2/A2, A1A2A3/A2, and A1CK/A2, respectively). In separate experiments, soluble VWFA1A2A3 was tested as an inhibitor for the binding between immobilized VWFA2 and ADAMTS13 (A2/A1A2A3). Different concentrations (range 0-600 nM) of the competitor (VWFA2 or VWFA1A2A3) were preincubated with 12 to 25 nM ADAMTS13, at 37°C for 40 to 60 minutes, and subsequently added to the wells. The detection of the bound ADAMTS13 was performed as described in panel A. The binding in the absence of soluble inhibitor was taken as 100% and the result was plotted as a percentage of this maximal binding against the inhibitor concentration. The curves were fitted using Graph Pad Prism 4 software to the one-site competition model and IC₅₀ values are listed in Table 2. (C) VWFA1CK (50 nM) was immobilized on a microtiter plate and preincubated for 40 to 60 minutes with increasing concentrations (0-400 μg/mL) of either a monoclonal antibody directed against the VWF D4 domain (mAb RU8) or a polyclonal antibody against the VWF CK domain (pAb C-20). ADAMTS13 (12 nM) was applied, incubated for 2 hours at 37°C, and detected as in panel A. The binding in the absence of the inhibitory antibody was taken as 100% binding and the relative binding plotted against the antibody concentration.