Abstract Work under previous support has continued to advance basic knowledge in the field of hemostasis and thrombosis with significant contributions to the structural understanding of thrombin dynamics and prothrombin activation. Unraveling the architecture of coagulation factors and their complexes remains a challenging task because of the difficulty of obtaining high resolution structures for proteins containing multiple domains. We have addressed this challenge by being among the pioneers of using cryo-EM in the field of hemostasis and thrombosis. We have solved the structures of factors V, Va, V short and most notably that of the prothrombin- prothrombinase complex that has documented, for the first time, the initial step of prothrombin activation along the meizothrombin pathway. We will build upon the progress made under previous support and address the structural basis of prothrombin activation, that remains an important unresolved issue in the field. Using cryo- EM, we will dissect the entire kinetic scheme that defines how prothrombinase activates prothrombin along the meizothrombin (cleavage at R320 first) and prethrombin-2 (cleavage at R271 first) pathways. We will characterize the interaction of prothrombinase with prothrombin and meizothrombin (aim 1), and solve the structures of prothrombin, meizothrombin, and fXa in the free form (aim 2). These studies will unravel the nature of conformational changes and underlying mechanism leading to assembly of the various complexes in the kinetic scheme of activation. We will test the hypothesis that cleavage at R320 takes place with prothrombin in the collapsed closed form whilst cleavage at R271 requires meizothrombin or prothrombin to switch to the elongated open form. We also hypothesize that prothrombinase cleaves R320 and R271 in two different conformations. The proposal is supported by solid preliminary cryo-EM data of the prothrombin-prothrombinase complex on nanodiscs, the meizothrombin-prothrombinase complex, as well as of prothrombin, meizothrombin, and fXa in the free form. Success of the project will significantly advance basic knowledge on the most important step of the coagulation response and provide a strategy for the study of other interactions of biological relevance to blood physiopathology.