Project 3-Abstract The overall objectives of this project are A.) to generate and employ RNA aptamers as probes to develop a more thorough understanding of the specific macromolecular interactions that control blood coagulation and B.) to utilize this new knowledge to generate potent, yet rapidly reversible, agents to regulate this process for research and clinical applications. Using high resolution X-ray crystal structure data to guide us, we will rationally generate and evaluate bifunctional EXosite-ACTive site (EXACT) inhibitors based upon aptamers and small molecule active site inhibitors. We hypothesize that such bifunctional agents will be particularly potent inhibitors of coagulation factor activity as blood feeding animals have employed this approach to create/evolve extremely potent inhibitors, such as hirudin and tick anticoagulant peptide (TAP), in nature. For such bioinspired, X-ray structure guided studies, we will covalently link an aptamer that specifically binds a specific clotting factor to an active site proteinase inhibitor to achieve potent and specific inhibition of proteinase function. Moreover, we hypothesize that an antidote oligonucleotide can made against the aptamer portion of the conjugate to rapidly and fully reverse such potent EXACT inhibitors. Our three specific aims are: Aim 1: Conduct proof-of-concept studies with thrombin to optimize an approach for potent and bivalent specific inhibition wherein specificity for the proteinase is provided by an aptamer and inhibition of active site function of thrombin arises from a small molecule ligand coupled through a linker to the aptamer. We have termed such bifunctional agents “EXACT” inhibitors as they target both an EXosite and the ACTive site on the same proteinase. Aim 2: Evaluate the generalizability of the studies on thrombin by producing and characterizing bivalent EXACT inhibitors for factor Xa (FXa), factor XIa (FXIa), activated protein C (APC) as well as a prothrombinase-specific inhibitor which does not inhibit free factor Xa. Aim 3: Characterize EXACT anticoagulant function in ex-vivo models of ECMO (extracorporeal membrane oxygenation) and in vivo models of ECMO and CPB (cardiopulmonary bypass) that lead to fulminant activation of coagulation.