Engineering a membrane-independent human prothrombinase through parsimonious mutation of factor Xa

Thrombin is generated from its precursor prothrombin by sequential cleavage at Arg320 and Arg271 by the prothrombinase complex, composed of factor (F)Xa and FVa on phospholipid (PL) surfaces. The affinity of human FXa for FVa is low in the absence of PL. However, FXa orthologs from the venom of group D snakes bind to FVa with high affinity, forming an active complex without PL. We previously characterized the properties of the FXa ortholog hopsarin D (HopD) from Hoplocephalus stephensii.

In this study, we set out to create a PL-independent human prothrombinase by making mutations to FXa, guided by a model of the prothrombinase complex and the sequence differences between human FXa and HopD.

We assessed the contribution of individual domains of FXa to its binding to FVa by swapping each with the corresponding domain of HopD. We then chose 3 loops in the serine protease domain predicted to be in contact with FVa to swap to those of HopD. Eventually, 10 residues from the 3 loops in the serine protease domain and 7 from the epidermal growth factor 2 domain were selected for mutation.

The resulting M17 FXa variant bound to FVa with a dissociation constant of 21 nM, similar to HopD, and together efficiently processed prothrombin through the meizothrombin intermediate in the absence of PL.

We conclude that the role of PL membranes in prothrombinase assembly and function is limited to improving the affinity of FXa for FVa and that the M17-FVa complex is likely to be structurally equivalent to human prothrombinase.