OC 07.2 - Engineering a membrane-independent human prothrombinase through parsimonious mutation of factor Xa

Background: Thrombin is generated through sequential cleavage of Arg320 and Arg271 by the enzyme complex prothrombinase, composed of factor (f) Xa and fVa on membranes. The affinity of human fXa for fVa is low in the absence of membranes, however, prothrombinase from type C or D snake venoms do not require membranes. Type C delivers both components as a high-affinity complex, and type D delivers a fXa that binds to host fVa with high affinity. Both cleave prothrombin in the same order as human prothrombinase. We previously reported the crystal structure of the type C venom prothrombinase Pseutarin C.

Aims: We set out to design a membrane-independent human prothrombinase by making mutations to human fXa, guided by the structure Pseutarin C and sequence differences between human fXa and HopD, a type D venom fXa.

Methods: We made chimeras of human fXa with three loops from the catalytic domain swapped out for those of HopD at the predicted interface with fVa. The individual loop swaps had little effect on affinity, but the combined chimera bound about 2-fold more tightly. The additional swap of the EGF2 domain reduced the Kd to 50nM, and resulted in processing down the Arg320 route. Examination of model of the fXa chimera bound to fVa allowed the contribution of individual residues to be predicted. We selected 10 residues from the three loops and 7 in EGF2 for mutation to the HopD sequence.

Results: The resulting human fXa mutant bound to fVa with a Kd of 20nM, identical to HopD, and resulted in processing of prothrombin via Arg320 in the absence of membranes. Intriguingly, although HopD binds to fV with similar affinity as to fVa, the mutated human fXa is unable to bind to fV.

Conclusion(s): We created a fully functional membrane-independent human prothrombinase with only 17 mutations in fXa.