Overall Program-Abstract Blood coagulation derives from a series of specific proteolytic activation reactions that are catalyzed with narrow and defined specificity by trypsin-like serine proteinases. In several instances, these proteinases function in membrane assembled enzyme complexes. Distinctive protein substrate specificities and the modulation of function by interactions with membranes, cofactors and ligands are hallmarks of the proteolytic reactions of blood coagulation. There are major gaps in the current understanding of the molecular bases for these unique features that underlie the function of the hemostatic reactions. This program proposes an integrated approach focused on the modulation of enzymic function and specificity that uniquely arises from macromolecular interactions that underlie the action of the hemostatic enzymes. Project 1 (Krishnaswamy) uses the constituents of the prothrombinase complex as a paradigm to investigate functional and structural mechanisms underlying regulation of zymogen, proteinase and cofactor function. Project 2 (Camire) will investigate molecular mechanisms at play in the conversion of factor V to the cofactor, factor Va and the surprising new biological insights that these mechanisms reveal. Project 3 (Sullenger) employs RNA aptamers in a unique inhibitor strategy that combines specific aptamers targeting exosites linked to active site ligands to form potent and readily reversible bivalent EXosite-ACTive site (EXACT) inhibitors to modulate coagulation reactions for therapeutic gain. Project 4 (Samelson-Jones) is an ESI-led project that investigates mechanisms underlying the structural correlates of IXa and VIIIa function to develop novel approaches for the treatment of hemophilia B or to improve approaches for hemophilia A treatment by gene therapy. The objectives of the five projects will be supported by an administrative core (Core A) and a core that provides support for molecular biology, protein expression and structural biology (Core B). Overall, this project applies the expertise of the individual investigators towards addressing major unanswered questions in hemostasis and thrombosis extending from biochemical and structural insights, to biological function and the translation of of these insights to the treatment of disease. The proposed approaches will provide new insights into the chemistry and biology of the blood coagulation reactions with implications for an understanding of normal hemostasis and thrombosis and the treatment of clotting or bleeding disorders.