Abstract The proposed research project continues and expands our investigation of the interaction of thrombin with the anticoagulant protein C responsible for a key feedback regulation of the coagulation response. The project addresses unresolved issues in the field using an innovative structural approach and plans to fill existing gaps in basic knowledge about protein C as a substrate of the thrombin-thrombomodulin complex and activated protein C as an enzyme that inactivates factor Va. Unraveling the architecture of multidomain factors involved in blood coagulation, complement and fibrinolysis remains a challenging task because of the difficulty of obtaining high resolution structures. This limitation is even more acute when considering complexes involving these factors and their macromolecular substrates or activators. Our approach addresses this challenge directly with cryo-EM, the new gold standard for the structural investigation of biological macromolecules. Building on our recent success in solving the structures of human coagulation factors V and Va, the proposed research project plans to revolutionize the structural enzymology of protein C in a way that is relevant to other multidomain proteins and their complexes in the blood coagulation cascade. Toward this end, we have obtained preliminary cryo-EM structures of protein C free and bound to the thrombin-thrombomodulin complex. Once fully refined as planned under aim 1, these unprecedented structures will unravel the mechanism of protein C activation and test the hypothesis that thrombomodulin promotes the interaction of thrombin with protein C by offering a scaffold that changes their conformation and alleviates electrostatic clash. In addition, we have obtained a preliminary cryo- EM structure of activated protein C free and prepared stable particles of activated protein C bound to factor Va and protein S for cryo-EM data acquisition. Progress from these studies will elucidate how the structure of activated protein C compares to that of its zymogen form and will further refine the mechanism of protein C activation. Furthermore, direct information on the epitopes of recognition of factor Va will enable a structure- based engineering of variants of activated protein C with altered specificity for potential therapeutic applications.