Partial rescue of naturally occurring active site factor X variants through decreased inhibition by tissue factor pathway inhibitor and antithrombin.

Abstract

BACKGROUND: Activated coagulation factor X (FXa) is the serine protease component of prothrombinase, the physiological activator of prothrombin. FX Nottingham (A404T) and Taunton (R405G) are two naturally occurring mutations, identified in families with a bleeding phenotype. OBJECTIVE: To functionally characterise these FX variants. METHODS: The activity and inhibition of recombinant FX variants was quantified in plasma based and pure component assays. RESULTS: The prothrombin times in FX-depleted plasma supplemented with FX Nottingham and Taunton were greatly increased compared to wild-type (WT) FX. Kinetic investigations of activated variants in the prothrombinase complex showed kcat /Km , reduced ~50-fold and ~5-fold, respectively, explaining the prolonged PT times. The substituted residues are located in the protease domain Na+ -binding loop, important for the activity of FXa, as well as its inhibition. Both FXa Nottingham and Taunton showed reduced affinity for Na+ . Plasma-based thrombin generation assays triggered with 1pM tissue factor (TF) demonstrated only small differences in activities compared to WT FX, but large reductions at 10pM TF. Severely reduced inhibition of both FXa Nottingham and Taunton by tissue factor pathway inhibitor (TFPI) and antithrombin (AT), was shown in pure-component FXa inhibition assays. FXa Nottingham and Taunton produced higher amounts of thrombin than WT FXa in pure-component prothrombinase assays in the presence of TFPI and AT, explaining the results from the plasma-based assay. CONCLUSIONS: FX Nottingham and Taunton both display decreased proteolytic activity. However, their reduced activity in plasma triggered by low TF can be rescued by decreased inhibition by the natural FXa inhibitors, TFPI and AT.