The development of transformative therapeutics, including the possibility of a cure though gene therapy, continues to be the major research and development activity in the hemophilia space. However, progress in the field has been limited by significant hurdles including the size, complexity, immunogenicity, instability and biosynthetic inefficiency of coagulation factor VIII (FVIII). Through the study of existing vertebrate species, we have discovered that each FVIII ortholog, i.e. FVIII proteins (or genes) identified from existing species that arose from a common ancestral protein (or gene), possesses interspecies differentials in molecular, cellular and biochemical properties that affect hemostasis. Based on these data, as well as existing knowledge of FVIII biosynthesis and mechanism of action, we predict that FVIII can be bioengineered for improved effectiveness in both protein infusion and gene-based therapies. Towards this goal, we are employing ancestral protein reconstruction as a novel approach to better define these interspecies differentials and possibly identify new ones. This 'evolutionary design'-based engineering approach has certain advantages over traditional 'rational design' approaches including foremost, a priori knowledge that each FVIII ortholog is hemostatically functional. Furthermore, ancestral protein reconstruction significantly reduces the complexity associated with the identification of functional determinants within FVIII. In the current proposal, we seek to integrate ancestral sequence reconstruction, protein biochemistry, structural biology and cell biology to 1) obtain new insights into FVIII biology that will provide framework for the future development of improved FVIII therapeutics and 2) extend this approach to the hemophilia 'related' coagulation proteins, factor IX, factor VII and von Willebrand factor. The studies proposed represent a new approach towards improving our basic and translational understanding of vertebrate hemostasis and thrombosis.