Blood coagulation factor VIII is a protein cofactor that is essential for the proper regulation of the clotting cascade. Deficiencies in factor VIII cause hemophilia A, the most common severe genetic bleeding disorder, affecting 1 in 5,000 males worldwide. Treatment for hemophilia A consists of therapeutic infusions of a functional form of factor VIII, termed ?replacement therapy.? Complications to factor VIII replacement therapy consist of an inhibitory immune response to the infusion in 25-30% of hemophilia patients receiving treatment. Moreover, autoimmune disorders arise in healthy individuals against native factor VIII, causing acquired hemophilia. As factor VIII has been repeatedly described as highly immunogenic, understanding the structural nature of this immune response, how factor VIII forms complexes in circulation with von Willebrand factor (VWF), and how activated factor VIII (fVIIIa) binds activated platelet surfaces, may lead to more effective therapies for hemophilia A patients. In our previous studies, we have: developed and tested a working model of membrane binding by the C- terminal (C2) domain of factor VIII, determined the first high resolution structure of B domain-deleted factor VIII, and structurally characterized inhibitory antibodies that target the factor VIII C1 and C2 domains, which have resulted in further understanding of the pathogenic anti-factor VIII immune response. To further examine the structural nature of factor VIII protein complexes that exist in circulation, this proposal will accomplish three specific aims. First, we will examine the structural basis of activated platelet binding by: determining the X-ray crystal structure of an activated form of bioengineered fVIIIa; characterizing the relative membrane binding affinity of isolated C1 and C2 domains, a tandem C1-C2 domain construct, and fVIIIa; generating a series of hemophilia A-associated point mutants in the C1 and C2 domains that we will subject to protein stability studies and membrane binding assays; and developing a quantitative assay to measure the binding of the fVIIIa/factor IXa tenase complex to lipid nanodiscs (Specific Aim 1). Second, we will elucidate the molecular basis for the factor VIII/VWF circulatory complex by determining the X-ray crystal structure of B domain-deleted factor VIII bound to the VWF D? and/or D?D3 domains (Specific Aim 2). Third, we will continue to characterize the immune response to factor VIII replacement therapy by determining the X-ray crystal structures of B domain-deleted factor VIII in complex with (1) anti-A2 domain inhibitory antibodies, and (2) hemophilia A patient-derived inhibitory antibodies (Specific Aim 3). By examining the factor VIII immune response at atomic resolution, paired with structural characterization of factor VIII circulatory complexes, our results will illustrate in detail the life cycle of factor VIII and its procoagulant activity. The structural data that result from this study will assist in the engineering and development of the next generation therapeutics to better treat hemophilia A patients worldwide.
Hemophilia A is caused by the disruption of the gene encoding blood coagulation factor VIII and is the most common form of hemophilia, affecting 1 in 5,000 males worldwide. The most significant complication in the management of hemophilia A is the development of an inhibitory immune response to therapeutically infused factor VIII. Studying the life cycle and immune response to factor VIII with a combination of structural and biochemical techniques can lead to improved therapies for hemophilia A patients.