A human FVIII inhibitor modulates FVIII surface electrostatics at a VWF‐binding site distant from its epitope

Abstract

Summary. Background: BO2C11 is a human monoclonal factor (F) VIII inhibitor. When bound to the C2 domain of FVIII, the Fab fragment of BO2C11 (Fab BO2C11 ) buries a surface of C2 that contains residues participating in a binding site for von Willebrand factor (VWF). BO2C11 has thus been proposed to neutralize FVIII by steric hindrance. Objectives: The BO2C11 epitope on C2 overlaps with residues located at the periphery of the putative VWF binding site; hence, most of the residues that constitute the VWF binding site on C2 and a3 remain accessible for VWF interaction following BO2C11/ FVIII complex formation. We thus investigated the contribution of alternative molecular mechanisms to FVIII inactivation by BO2C11. Methods: Continuum electrostatic calculations were applied to the crystal structure of C2, free or Fab BO2C11 -complexed. In silico predictions were confirmed by site-directed mutagenesis and VWF-binding assays of the mutated FVIII. Results: Binding of Fab BO2C11 to C2 induced perturbations in the electrostatic potential of C2 and in the local electrostatic parameters of 18 charged residues in C2, which are distant from the BO2C11 epitope. Nine of the predicted electrostatic hotspots clustered on the VWF-binding site of C2. Mutation of some of the predicted electrostatic hotspots has been associated with hemophilia A and reduced VWF binding in vitro. Conclusions: Inhibitors may neutralize FVIII by alteration of protein surface electrostatics at a long distance from their epitope. Perturbation of the electrostatic environment of C2, either upon binding by anti-FVIII antibodies or consecutive to missense mutations in the F8 gene, may lead to hampered VWF binding and reduced FVIII residence time in circulation.

The 1.7 Å X-Ray Crystal Structure of the Porcine Factor VIII C2 Domain and Binding Analysis to Anti-Human C2 Domain Antibodies and Phospholipid Surfaces

“…Furthermore, the frequency of phylogenetic differences on the non-classical face of the protein, along with the observation that some of the sequence differences are not conservative, suggests that the face of the C2 domain that harbors the non-classical epitope is largely solvent accessible in circulation. By contrast, the face containing the classical epitope displays less phylogenetic differences, suggesting this region is responsible for binding other proteins in circulation, such as VWF, which is supported by previous findings [ 11 , 29 , 54 ].…”

Structure of the factor VIII C2 domain in a ternary complex with 2 inhibitor antibodies reveals classical and nonclassical epitopes

“…38,41 The 3E6 epitope partially consists of Lys2183, Asp2187, and Arg2215, each of which is expected to be involved in VWF binding (Figure 2). 52 Moreover, Lys2183 forms a direct contact with multiple 3E6 residues and is largely buried at the binding interface (Figure 2A), which is consistent with a Lys2183Ala mutant that decreases 3E6 binding by an order of magnitude. 41 By contrast, multiple residues at the C2 domain/G99 interface are suggested to be involved in binding both factors IXa and Xa.…”

“…Visualization of the electrostatic surface potential for both the C2/3E6 and the C2/G99 binary structures supports this hypothesis (supplemental Figure 1), which is consistent with previous hypotheses regarding antibody inhibition of VWF binding. 52,56 A third hypothesis is that the binding of 3E6 affects the dynamics of the fVIII C2 domain, which could modify flexible loops that may be important to allow membrane engagement. Analysis of the crystallographic thermal B factors does not support this hypothesis, however, as the average B factors for the 2 hydrophobic loops (2199/2200 and 2251/2252) display higher than average values, which are often concomitant with dynamic flexibility.…”

In silico calculated affinity of FVIII‐derived peptides for HLA class II alleles predicts inhibitor development in haemophilia A patients with missense mutations in the F8 gene

“…Besides, multiple ‘high risk’ F8 genotypes have been located in the C2 domain of FVIII: W2229C, P2300L, and N2286K . It remains to be elucidated, however, whether the elevated hydrophobicity of the C2 domain as compared to the other domains of the molecule , or the role of the C2 domain in binding to von Willebrand factor or to phospholipids on activated membranes, account for the observed differences.…”