# In-Silico Characterization of von Willebrand Factor Bound to FVIII
## 1 Introduction  
Hemophilic A patients are treated periodically with the coagulation factor FVIII substitutes, such as purified hemoderivatives or FVIII biological drugs, which are currently biological advanced therapies approved for treatment of hemophilia A. This class of drugs includes recombinant full-length FVIII and B domain-deleted FVIII [1]. B domain-deleted FVIII (BDD-FVIII) biologics have been developed to improve the biotechnological production of these proteins. Furthermore, BDD-FVIII conjugated with Fc immunoglobulin fragment was reported to have the highest plasma half-life, providing the opportunity ofscheduling a low number of infusions [2]. Therefore, BDD-FVIII biologics (products with extended half-life, EHL) have been claimed to decrease the number of infusions in hemophilic patients. EHL products may allow less frequent dosing; however, due to inter-patient differences in FVIII plasma stability and clearance, less frequent infusions may cause longer time periods with relatively low FVIII plasma levels, which could increase the risk of bleeding. The safety cutoff for plasma FVIII levels was set to >1%, and patients with levels >12% were subjected to less bleeding events, especially at joints [2; 3]. Specifically, clinical trials carried out so far highlighted a similar capability of different FVIII substitutes on bleeding prevention, measured as annualized bleeding rate [2]. Interestingly, a pharmacokinetic (PK) modeling study predicted that mean values of FVIII plasma levels were similar in patients treated with either BDD-FVIII or full-length recombinant FVIII [1]. Furthermore, this study predicted that patients treated with full-length FVIII (infusions every 48 h) spent more time with FVIII above the 10 IU dL-1 than patients treated with BDD-FVIII product infusions every 72 h. Therefore, full-length FVIII could be characterized by higher plasma stability than B domain-deleted FVIII substitutes compared to BDD-FVIII [1]. This data raised a controversy [4], but the high plasma stability of FL-FVIII was confirmed at pre-clinical level [5]. FVIII half-life is strongly influenced by von Willebrand Factor (VWF), which is reported to bind with FVIII [6; 7; 8]. Several studies reported that the binding sites of VWF at FVIII are in the A3 and C1 FVIII domains [9; 10; 11; 12] Therefore, with the aim to improve BDD-FVIII plasma stability, a novel FVIII substitute has been developed, \(\acute{\text{e}}\)fanesococog alfa (BIVV001). \(\acute{\text{e}}\)fanesococog alfa is an investigational biologic drug where VWF D'-D3 domain is covalently bound (through a cleavable XTEN polypeptide linker) to an engineered BDD-FVIII [13]. The atomic model of cryo-electron microscopy (Cryo-EM) of \(\acute{\text{e}}\)fanesococog alfa was built and deposited on Protein Data Bank (PDB:7 KWO); in this atomic model VWF binding to A3 and C1 domains of FVIII is confirmed. \(\acute{\text{a}}\)f\(\acute{\text{e}}\)VIII is a 2351 a.a pre-protein that, during processing in the endoplasmic reticulum, is cleaved and reassembled in a heavy and a light chain, interacting by means of Van der Waals interactions, and through a metal complex interaction with a divalent cation (Figure 1). Figure 1: **FVIII processing. ** Capital letters refer to protein domains, while lowercase letters refer to loops linking two protein domains (modified from Pipe SW. Haemophilia 2009 [14]). After secretion, the processed full-length FVIII (FL-FVIII) is cleaved mainly at a.a 1313, corresponding to the secreted inactivated form of FVIII, containing about 572 amino acids of B domain (secreted full-length FVIII) (Figure 1) [14,15]. However, several heterogenous forms of secreted FL-FVIII were found in plasma [16]. The domain B function has been linked to protection of FVIII against premature proteolysis. Moreover, B domain can inhibit FVIII binding to activated platelets, decreasing overall the inactivation rate of FVIII. B domain has also been involved in modulation of FVIII clearance through binding to the sialoglycoprotein receptor [14].