When the cofactor, factor (f) Va, protease, fXa, and substrate, prothrombin, are all attached to the same membrane surface, the prothrombinase complex converts prothrombin to thrombin by sequential cleavage at Arg320 followed by Arg271. Cleavage happens in the reverse sequence in the absence of the membrane or cofactor. Prothrombin docks on fVa in a way that displays Arg320 while hiding Arg271 from the active site of fXa, allowing the less favorable cleavage site at Arg320 to be cleaved first.
For a study, researchers developed an early prothrombin docking mode including interaction with distinct sections of the A1 and A2 domains of fV and the a1-loop linking the two domains based on the crystal structure of the prothrombinase complex from the venom of the Australian eastern brown snake, pseutarin C. They tested the hypothesized interface using site-directed PEGylation and by replacing the a1-loop in pseutarin C with those of human fV and fVIII and assessing the effect on thrombin production rate and route.
PEGylation of residues inside the suggested binding site significantly lowered the rate of thrombin formation without changing the route, but it had no effect on residues beyond the proposed interface. PEGylation of a1-loop residues also decreased the rate of thrombin production. The a1-loop sequence was discovered to be crucial in prothrombin binding and the presentation of Arg320 for early cleavage.
