Structural Studies of Blood Coagulation Factor VIII in Protein Complexes

A deficiency in blood coagulation factor VIII (fVIII) is responsible for the inherited bleeding disorder hemophilia A, which affects approximately 1 in 5000 males. The development of inhibitory antibodies is a significant issue faced by hemophilia A patients receiving therapeutic infusions of fVIII. The C-terminal C2 domain of fVIII has been shown to be highly immunogenic and the site of binding for numerous antibodies of both the classical and non-classical classifications. A detailed understanding of the structural components involved in C2-antibody binding interactions is vital for the development of improved therapeutics for hemophilia patients. Here we present the structure of the classical antibody 3E6 bound to human C2 at 2.6 Å resolution. Previous studies have suggested that pairs of classical and non-classical antibodies may exhibit binding cooperativity. Comparison of the C2:3E6 structure with a previously determined ternary structure of C2 bound simultaneously to both 3E6 and the non-classical antibody G99 reveals that cooperativity is not the result of dramatic conformational changes at the binding interface. Rather, changes in B-factor ratios between the C2 complexes and the isolated C2 domain suggest that dynamic changes upon 3E6 binding may lead to more favorable binding of a second antibody on the protein's opposite face. Studies of the fVIII C2 domain were then extended to attempts to understand the structural basis for the interactions between fVIII and its circulatory partner von Willebrand factor (vWF), which is crucial for maintaining fVIII levels in plasma. The inability for vWF to bind fVIII is the basis for another blood coagulation disorder, von Willebrand's disease type 2N, which can lead to hemophilia-type levels of fVIII in the blood and subsequent bleeding episodes. Previous publications have suggested a role for C2 in the binding of vWF. Attempts were made to formulate protein complexes between two vWF domain mutants, TIL'E' and D'D3, and the human C2 domain, but were ultimately unsuccessful. This outcome corroborates recent publications suggesting that the fVIII C1 domain may play a more active role in binding vWF than the C2 domain. In order to further study this possibility, complexes were formed between recombinant B-domain deleted fVIII and TIL'E' or D'D3 and X-ray crystal trials initiated. Hopefully, greater understanding of the structural interactions between fVIII and other proteins will lead to improved therapies for patients suffering from fVIII-related diseases.