# In-Silico Characterization of von Willebrand Factor Bound to FVIII
## 4 Discussion  
FVIII is a key protein involved in the coagulation cascade, and genetic defects in the FVIII gene (F8) cause hemophilia A, an x-linked recessive inherited disease. Hemophilia A is a rare, life-threatening disease affecting 1 in 6000 males, which causes spontaneous and prolonged hemorrhages due to FVIII deficiency. FVIII replacement therapy is the major therapeutic strategy for treatment of hemophilia A, and FVIII medications are listed by the World Health Organization (WHO) as essential medicines [48]. FVIII substitutes consist of full-length FVIII extracted and purified from human plasma, along with several biologic drugs, such as recombinant full-length FVIII (rFL-FVIII) and recombinant B domain-deleted FVIII (rBDD-FVIII). BDD-FVIII products have been developed to improve production yield and standardize recombinant protein production processes, but rBDD-FVIII were also found to be associated to increased FVIII plasma half-life; therefore, these products are also denominated as extended half-life (EHL) FVIII [49]. Interaction between von Willebrand Factor (VWF) and FVIII has been reported to improve FVIII plasma stability and pharmacokinetics properties, without modification of FVIII pharmacodynamics [8; 9; 50]. From this perspective, a novel FVIII investigational replacement medication has been developed, i.e., the BIVV001 or efanesoctocog alfa, which is a recombinant fusion protein in which the B domain-deleted FVIII is covalently linked to VWF, through a XTEN polypeptide linker [13]. VWF binds with most favorable energy to A3-C1 domains of FVIII light chain, however, binding of VWF to other domains of FVIII should not be excluded, since FVIII and VWF stoichiometry has not been univocally identified [21; 22]. Moreover, the VWF binding to full-length FVIII has been linked to increased FVIII stability and decreased FVIII immunogenicity (i.e., inhibitor formation). This explains the trend of FL-FVIII medication to show a longer half-life over time and a similar or lower rate of inhibitor formation in treatment-naive patients, compared to BDD-FVIII medications [5; 8; 20]. These data can also be attributed to binding of B domain to other plasma proteins, such as albumin [20]. Our in silico study could be considered as a small step to translational investigation, because it outlined how B domain in FL-FVIII/VWF would be an additional, but not the most favorable, binding site for VWF, thus putatively contributing to high plasma stability of FL-FVIII medication. Our in silico study provided a structural insight on the binding of von Willebrand Factor to FVIII, and our computational data are in accordance with the experimental findings, i.e., FVIII A3-C1 domain is the most stable binding site of VWF. Our computational approach suggested that one of the driving forces of VWF binding at this preferential binding site could be related to conformational modifications of FVIII, through modulation of allosteric residues. The PISA and PCN analysis of topological parameters suggested an unfavorable binding of VWF at B domain of FVIII, due to an increased stability of the interface between the heavy and the light chain, compared to VWF/BDD-FVIII and efanesoctocog alfa. In fact, the most stable VWF/FVIII complex (efanesoctocog alfa) was characterized by highest (less favorable) interaction energy between heavy and light chains of FVIII. Differences between the modeled VWF/BDD-FVIII complex and the atomic model of efanesoctocog alfa are not likely to be attributed to the lower resolution of BDD-FVIII X-ray structure (PDB:2R7E), compared to the atomic model of efanesoctocog alfa (PDB: 7 KWO). Furthermore, the X-ray structure of BDD-FVIII showed 4% of Phi and Psi angles in disallowed regions of the Ramachandran plot, but these residues are located in disordered loops of A1 and A2 domains of heavy chain, a region not involved in the binding with VWF [51].