Factor IX mutants with enhanced catalytic activity.

Abstract

Activated coagulation factor IX (FIXa) has low catalytic activity towards its physiologic substrate FX when activated FVIII (FVIIIa) is absent. One reason for this is that the FIX surface loop 99 stabilizes FIXa in a conformation that limits access of FX to the active site. To investigate the effect of mutations in loop 99 and in the active site on FIXa activity with and without FVIIIa. Five full-length FIX mutants with amino acid exchanges in the catalytic domain of FIX were constructed and characterized by measuring their activity in FX activation in model systems and in plasma. The mutants showed no or marginally improved catalytic properties in FX activation by the intrinsic tenase complex (FIXa-FVIIIa-Ca(2+)-phospholipid). The combination of mutations Y94F and K98T hardly affected FX activation in the presence of FVIIIa, but yielded a FIX molecule that, in FIX-depleted plasma, had approximately 2.5-fold higher clotting activity and approximately 3.5-fold higher activity in a thrombin generation assay than plasma-derived FIX (pdFIX). Two FIXa mutants had considerably increased activities towards FX in the absence of FVIIIa. FIXa-Y94F/K98T/Y177F/I213V/E219G (FIXa-L) and FIXa-Y94F/A95aK/K98T/Y177F/I213V/E219G (FIXa-M) activated FX with catalytic efficiencies (k(cat)/K(m)) that, as compared with activated pdFIX, were increased 17-fold and six-fold, respectively. However, in plasma, their zymogen forms performed similarly to pdFIX. This indicates that the introduced mutations not only affected the activity of FIXa but may have also influenced the lifetime of the activated mutant molecules in plasma by modifying their activation and/or inhibition rates.