Project 4—Abstract The intrinsic Xase enzyme complex, composed of the substrate factor (F) X, cofactor FVIIIa, and protease FIXa, catalyzes the rate-limiting step of sustained coagulation. Deficiencies or dysfunction in these clotting factors leads to the bleeding disorders FX-deficiency, hemophilia A (HA), and hemophilia B (HB), respectively. The molecular details of the structure and function of FVIIIa, FIXa, and FX within the intrinsic Xase are incompletely understood. Current treatments for these diseases have limited worldwide availability and are highly burdensome. Our objectives in this proposal are to develop new therapeutic approaches for these bleeding disorders and clarify the mechanisms that govern the assembly and function of the intrinsic Xase. To this end, we have preliminary data indicating that FVIII-mimetics (a class of drugs approved for HA treatment) can be repurposed for select HB genotypes. We have identified >40 HB-causing loss-of-function FIX variants whose procoagulant activity is enhanced by FVIII-mimetics. We hypothesize that their loss-of-function is due to disrupted interactions with FVIIIa or FX; these variants also provide a new experimental system where the role of intrinsic Xase assembly of specific regions of FIXa can be biochemically and structurally interrogated. We further hypothesize that there are disease-causing loss-of-function FX variants with disrupted FVIIIa interactions whose activation could be rescued by the alternative FVIIImimetic/FIXa Xase complex. Conversely, we have also identified a class of gain-of-function FVIII variants with high specific activity, which we hypothesize is due to enhanced FVIIIa/FIXa interactions. Our experimental approach aims to 1) determine the translation potential of FVIII-mimetics for select HB and FX-deficiency genotypes as well as the potential for high-specific activity FVIII variants to address current limitations of HA gene therapy and 2) to clarify the molecular assembly and function of the intrinsic Xase complex utilizing these clinically-relevant loss-of-function and gain-of-function variants. In aim 1, using biochemical and structural approaches, we will determine the mechanism of loss-of-function of FVIII-mimetic rescuable HB-causing FIX variants with amino acid substitutions throughout the protein. In aim 2, we will develop a FVIII-mimetic adopted HB and FX-deficient mouse model and determine the in vivo hemostatic potency of FVIII-mimetics for these disorders. In aim 3, using biochemical and structural approaches, we will determine the mechanism of the high-specific activity of our new gain-of-function FVIII variants. Further, we will determine their hemostatic potency over a range of injury models in HA mice as well as their ability to enhanced gene therapy for HA. Together, these studies will provide mechanistic insights into the regulation and activity of the intrinsic Xase as well as developing alternative therapeutic approaches for these bleeding disord...