Binding of ADAMTS13 (or variants) to VWF immobilized on solid surfaces. (A) Binding of ADAMTS13 and variants to VWF immobilized on a microtiter plate. FL-A13, delCUB, and MDTCS (0 to 200 nM) were incubated without (control) or with VWF (10 μg/mL, 100 μL per well) immobilized on a microtiter plate for 1 hour. The bound ADAMTS13 and variants were determined by mouse anti-V5 IgG, followed by rabbit antimouse IgG, peroxidase-conjugated and OPD-H₂O₂. The K_D (S) was determined by fitting the data into nonlinear regression. (B) Binding of ADAMTS13 and variants to immobilized VWF on Affi-gel 10. FL-A13, delCUB, MDTCS, and metalloprotease domain (M) (about 50 nM) were incubated at 37°C for 1 hour without (−) or with (+) VWF covalently immobilized onto the Affi-gel 10. After extensive washing with TBS and 20 mM Tris-HCl, pH 7.5, 500 nM NaCl, the bound ADAMTS13 and variants were eluted from the beads with SDS-gel sample buffer and determined by Western blot with anti-V5. The amount of input FL-A13, delCUB, and MDTCS is the same with the signal of only FL-A13 shown in lane 1 (IN).