Disulfide Bond Heterogeneity in Factor VIII: Implications for Protein Function and Haemophilia a Therapy

Haemophilia A is a chronic, life-threatening condition caused by the deficiency or dysfunction of plasma coagulation factor VIII (FVIII). Prophylactic treatments include regular intravenous infusion of exogenously derived FVIII concentrates or FVIII gene therapy. However, both of these therapies are limited by the inherent cytotoxicity of the FVIII protein and the generation of FVIII neutralizing antibodies (inhibitors). Protein engineering approaches have aimed to reduce FVIII cytotoxicity and immunogenicity to improve the durability of replacement and gene therapy. FVIII contains eight disulphide bonds that are covalent links between pairs of cysteine residues. Using differential cysteine alkylation and mass spectrometry, we observed that all eight disulfide bonds exist in unformed states in different proportions of plasma FVIII molecules, as has been observed in other blood proteins including fibrinogen, von Willebrand factor, and platelet αIIbβ3 integrin. The FVIII bonds were unformed in 1 in 10 (C172-C198 in the A1 domain) to 1 in 2 (C1918-C1922 in the A3 domain) molecules of the FVIII populations. The different disulfide-bonded states of FVIII were predetermined and not a result of post-secretion redox events, as recombinant FVIII exhibited the same disulfide status as the plasma protein. Using targeted cysteine mutations, we found that three disulfide bonds were dispensable for sustained FVIII expression and activity, and that a FVIII with an unformed bond in the A3 domain (C1918-1922) had increased plasma stability. Additionally, von Willebrand Factor (VWF) and a panel of 4 patient-derived FVIII-neutralizing antibodies showed binding selectivity to subsets of FVIII disulfide-bonded states. These findings indicate that FVIII is constitutively produced as multiple partially disulfide-bonded states that have different properties and may aid the engineering of a FVIII with improved therapeutic properties.

No relevant conflicts of interest to declare.