# In-Silico Characterization of von Willebrand Factor Bound to FVIII
## 3 Results  
### Protein Docking  
Protein-protein docking studies were carried out with PyDock on the model of full-length secreted form of FVIII, to predict VWF/FL-FVIII complex (Figure 2). To validate the protein-protein docking and the computational structure interface analysis approaches, we also docked the BDD-FVIII (PDB: 2R7E) with the fragment of von Willebrand factor (PDB: 6N29) (Figure 3). [PERSON] predicted that von Willebrand factor interacts with the B domain of secreted full-length FVIII with slightly more negative predicted binding free energy, compared to the BDD-FVIII/VWF complex (Table 1).  
Figure 2: **VWF/FL-FVIII complex.** (**A**) FL-FVIII is represented in cyan (light-chain) and green cartoons (heavy-chain, including domain (**B**)), VWF is represented with magenta cartoon. (**B**) Surfaces representation as mesh. Capital letters refer to protein domains of FVIII.  
[PERSON] et al. in 2014 identified the D'D3 (PDB: 6N29) as the domain of von Willebrand factor (VWF) that interacts with FVIII [46]. [PERSON] et al. in 2015 found that the VWF interacts with the A3 and C1 domain in light chain of the B domain-deleted FVIII [47]. The [PERSON] output for VWF/BDD-FVIII complex is in accordance with the study results from [PERSON] et al. 2015 and [PERSON] et al. 2021 (Figures 3 and 4) [13; 47].  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Complex** & **FVIII-Interacting** & **Electrostatic** & **Desolvation** & **VDW** & \(\Delta\mathbf{G_{binding}}\) \\  & **Domain** & **Component** & **Component** & **Component** & **Component** & \(\Delta\mathbf{G_{binding}}\) \\ \hline B domain-deleted FVIII & A3-C1-light chain & \(-44.864\) & \(-8.012\) & \(71.540\) & \(-45.722\) \\ \hline Secreted full-length FVIII & B domain & \(-37.144\) & \(-10.343\) & \(-6.838\) & \(-48.172\) \\ \hline \hline \end{tabular}
\end{table}
Table 1: **Pydock output.** Predicted interactions between FVIII structure models and von Willebrand factor fragment (PyDock).  
Figure 3: **VWF/BDD-FVIII complex.** (**A**) BDD-FVIII is represented in cyan (light-chain) and green cartoon, VWF is represented with magenta cartoon. (**B**) Surfaces representation as mesh. Capital letters refer to protein domains of FVIII.  
[PERSON] et al., 2021, have solved the structure of the bioengineered clinical-stage FVIII substitute, BIVV001 or \(\rm{efanesococog\ alfa}\) (PDB: 7 KWO). In \(\rm{efanesococog\ alfa}\), the VWF-D\({}^{\prime}\)D3 is covalently linked to an Fc domain of a BDD-FVIII, resulting in a stabilized VWF-D\({}^{\prime}\)D3/BDD-FVIII complex (Figure 4).  
The two structures of predicted VWF/BDD-FVIII and of \(\rm{efanesococog\ alfa}\) have been superimposed, and RMSD of alignment was 1.4 A. PRODIGY rescoring (Table 2) provided binding energy and dissociation constants for analyzed complexes. The binding energy (\(-14.1\) kcal/mol, pK4 10.4) of the predicted complex VWF/FL-FVIII, characterized by the binding of VWF with B domain, was higher (less favorable), although comparable to the binding energy of VWF bound to the A3-C1 domains of BDD-FVIII (\(-15.3\) kcal/mol, pK4 11.3), resembling the slight differences predicted by PyDock. We included the prediction of binding free energy of VWF in \(\rm{efanesococog\ alfa}\). In the \(\rm{efanesococog\ alfa}\) atomic model, the predicted binding free energy of protein-protein interactions between VWF and FVIII was more negative (\(\sim\)4 logs), compared to binding free energy of the other complexes predicted through protein-protein docking (VWF/FL-FVIII and VWF/BDD-FVIII) (Table 2). Therefore, our computational approach is in accordance with the experimental findings reporting highly stable VWF and FVIII interactions in \(\rm{efanesococog\ alfa}\) or BIVV001.  
Figure 4: **Efanesococog alfa** (**A**) FVIII is represented in green cartoon (heavy chain) and cyan cartoon (light chain). VWF is represented with magenta cartoon. (**B**) Surfaces representation as mesh. Capital letters refer to protein domains of FVIII.