KB-VWF-D3.1 binds to the VWF A3 domain. (A) Binding of KB-VWF-D3.1 (1 μg/mL) to various concentrations of VWF domain-Fc fusion proteins (0-10 nM) that were captured onto anti-human Fc antibodies. Bound KB-VWF-D3.1 was probed using peroxidase-labeled polyclonal rabbit anti-cMyc antibodies and detected following hydrolysis of 3,3',5,5'-tetramethylbenzidine. Blue squares, A1-Fc; red triangles, A2-Fc; red circles, A3-Fc: green circles, D4-Fc. Data represent mean ± SD of 3 experiments. (B) In silico simulation of KB-VWF-D3.1 (colored structures) docking on the VWF A3 domain (grey structure). Shown are the top 30–ranked structures of KB-VWF-D3.1, which all bind in a similar fashion to the A3 domain. (C) Single-structure representation of KB-VWF-D3.1 binding to the A3 domain. (D) The VWF A3 domain, with residues in red representing amino acids predicted to be in the epitope of KB-VWF-D3.1. Residues known to affect collagen binding are indicated in yellow. (E) Amino acid sequence of the VWF A3 domain, with the residues predicted to harbor the epitope for KB-VWF-D3.1 in red. Residues previously reported to be involved in collagen binding²² are boxed. (F) Inhibition of pdVWF binding to collagen-type III by KB-VWF-D3.1 (red circles), monoclonal antibody Mab505 (blue squares) and nanobody C37h (green circles). Presented is residual pdVWF binding vs nanobody/antibody concentration. Data represent mean ± SD of 3 experiments. (G) Binding of pdVWF (red symbols) or degraded-VWF (green symbols) to immobilized Mab505 (5 μg/mL; circles) or C37h (5 μg/mL; squares). Bound pdVWF was probed using peroxidase-labeled polyclonal anti-VWF antibodies and detected via hydrolysis of 3,3',5,5'-tetramethylbenzidine. Data represent mean ± SD of 3 experiments. OD, optical density.