Predicting the effects of single pathological mutations in hemophilia A and type 2N von Willebrand diseases using...

Most factor VIII (FVIII) in circulation exists in a complex with von Willebrand factor (vWF). The interaction between FVIII and vWF is vital for normal hemostatic function, and disruptions in this interaction can lead to bleeding disorders such as von Willebrand disease or hemophilia. However, the impact of pathological mutations on the binding between FVIII and vWF remains largely uncharacterized. In the current study, we used AlphaFold2-multimer and AlphaFold3 to predict the complex involving FVIII and vWF. Additionally, we explored how known mutations in FVIII or vWF, which can result in mild to severe forms of hemophilia and type 2N von Willebrand disease, affect this complex. Our predictions confirm that AlphaFold2 and AlphaFold3 can accurately model the FVIII/vWF complex in a manner consistent with existing cryogenic electron microscopy structures. However, the single pathological mutations can generally disrupt the complex interface predicted by AlphaFold2-multimer but not AlphaFold3. Molecular dynamic simulations showed that the flexibility of several common regions was affected by single pathological mutations. We further designed a new FVIII construct using AlphaFold2, which holds promise as a more effective therapeutic agent with reduced autoimmune responses. In summary, our findings suggest that in combination with molecular dynamics, AlphaFold2 is a valuable tool for swiftly assessing the impact of both known and novel mutations on hemophilia, with potential applications in precision medicine and the development of novel therapeutic interventions. SIGNIFICANCE STATEMENT: This study provides novel insights into the protein structure of the factor VIII and von Willebrand factor complex. This research demonstrates that AlphaFold2-multimer rather than AlphaFold3 can better predict the variations in the complex corresponding to clinical observations of disease severity. These findings not only deepen our comprehension of hemostatic mechanisms but also establish AlphaFold2 in combination with molecular dynamics as a useful tool for hemophilia research, with potential applications in precision medicine and the development of novel therapeutic interventions.