The intrinsic and extrinsic routes of the coagulation cascade converge on a single phase in which the prothrombinase complex, which includes the enzyme factor Xa (fXa), the cofactor fVa, Ca2+, and phospholipids, converts the zymogen prothrombin to the protease thrombin. 

The process involves cleavage at two sites, R271 and R320, resulting in the intermediates prethrombin 2 and meizothrombin. The molecular basis of these critical hemostasis interactions remains unknown. For a study, researchers solved two cryo-EM structures of the fVa-fXa complex, one free on nanodiscs at 5.3-Åresolution and the other bound to prothrombin at near-atomic 4.1-Å resolution. The Gla domains of fXa and prothrombin align on a plane with the C1 and C2 domains of fVa to interact with membranes in the prothrombin-fVa–fXa complex. Prothrombin and fXa emerge from this plane in curved conformations that bring their protease domains into contact with fVa’s A2 domain. The A2 domain segment 672ESTVMATRKMHDRLEPEDEE691  shuts like a lid on the fXa protease domain to set the active site orientation. 

The 696YDYQNRL702 segment binds to prothrombin and initiates the activation pathway by binding R271 to D697 and guiding R320 to the active site of fXa. The cryo-EM structure depicts prothrombin activation throughout the meizothrombin route and offers a mechanism for cleavage at the alternate R271 location. The discoveries increased the basic understanding of a critical phase in coagulation and have broad implications for other blood interactions.