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

Fatma Işık Üstok, Ph.D.
University of Cambridge
Cambridge, U.K.

Thrombin is generated through sequential cleavage of Arg320 and Arg271 by the enzyme complex prothrombinase composed of factor (f) Xa and fVa on membranes. Fatma Işık Üstok, Ph.D., presents information in which the team aims to design a membrane-independent human prothrombinase by making mutations to human fXa, guided by the structure pseutarin C and sequence difference between human fXa and HopD. The authors made chimeras of human fXa with the EGF2 domain and three loops from the catalytic domain swapped out for those of HopD that shared membrane independence with HopD, which were eventually whittled down to just 17 point mutations. The resulting human fXa mutant bound to fVa with a Kd of 20nM, identical to HopD, and resulted in processing of prothrombin via the Arg320 route. In conclusion, a fully functional membrane-independent human prothrombinase with only 17 mutations in fXa were created.