Summary 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 A. As factor VIII has been repeatedly described as highly immunogenic, understanding the structural nature of this immune response, how activated factor VIII (fVIIIa) binds activated platelet surfaces, and how fVIII forms a membrane-bound procoagulant complex with fIXa, 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 through multiple assays, determined the first high resolution structure of B domain-deleted factor VIII, and structurally characterized antibody inhibitors 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 and thermodynamic basis of activated platelet binding by: determining the X-ray crystal structure of an activated form of bioengineered fVIIIa; measuring the membrane binding thermodynamics of isolated C1 and C2 domains, as well as a tandem C1-C2 domain construct with isothermal titration calorimetry (ITC); and characterizing the structural and dynamic nature of C2 domain membrane binding in residue-level detail with small lipid nanodiscs with 2D 15N/1H NMR spectroscopy (Specific Aim 1). Second, we will elucidate the molecular basis for the lipid membrane-associated intrinsic tenase complex between fVIIIa and fIXa bound stably to small lipid nanodiscs (Specific Aim 2). Third, we will continue to characterize the immune response to factor VIII replacement therapy by determining high resolution cryoEM and/or X-ray 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 next generation therape...