Interaction of prothrombin with prothrombinase. Molecular surface of interaction between the A2 domain of fVa (pale green surface), fXa (deep salmon surface), and prothrombin (yellow sticks) that details how fVa orchestrates preferential binding of R320 to the active site of fXa. Shown is the segment ²⁶⁹EGRTAT²⁷⁴ comprising the site of cleavage at R271 and the longer segment ³⁰⁷KTERELLESYIDGRIVEGSD³²⁶ comprising the site of cleavage at R320. Relevant epitopes of fVa and fXa for interaction with prothrombin are colored in cyan. The interaction between E686 of fVa (orange) with K276 of fXa (white) is also labeled, along with R506 and R709 of fVa. The segment ⁶⁹⁶YDYQNRL⁷⁰² separates the 2 prothrombin segments and sequesters R271 with a strong ionic interaction with D697 fixed by a nearby interaction between E269 and R505 of fVa. The segment then organizes the proximal portion of the R320 site for interaction with the active site of fXa. Flanking D697 are Y696 in hydrophobic interaction with F535 and Y698 coupled to R310 through a cation-π interaction and in hydrophobic contact with L312. The fragment then turns toward fXa where strong electrostatic interactions involve D318 at P3, E323 at P3′ and D326 at P6’ with residues R405, K370, and K242 of fXa, respectively. An additional ionic interaction is established between K307 of prothrombin and Q240 of fXa. As a result of these interactions, R320 penetrates the active site of fXa to initiate activation along the meizothrombin pathway.