Evidence for the interaction between ADAMTS13 TSP5-CUBs domains and VWF C-terminal region. (A) VWFA1CK and VWFA1A2A3 (depicted in Figure 1A) and the C-terminal VWF fragments (depicted in Figure 1B; 30 nM of each) were immobilized on a microtiter plate. Increasing concentrations of ADAMTS13 (0-400 nM) were applied and incubated for 2 hours at 37°C. Bound ADAMTS13 was detected by an HRP-conjugated anti-myc antibody 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) VWFA1CK, VWFA1A2A3, and VWFD4CK (30 nM of each) were immobilized on a microtiter plate and incubated with increasing concentrations of MDTCS (0-400 nM). Bound MDTCS was detected as in panel A and the K_D(app) values are listed in Table 1. (C) VWFA1CK, VWFA1A2A3, and VWFD4CK (30 nM of each) were immobilized on a microtiter plate and incubated with increasing concentrations of del(TSP5-CUB) (0-400 nM). Bound del(TSP5-CUB) was detected as in panel A and the K_D(app) values are listed in Table 1.