Abstract
The homologous coagulation factor X (FX), VII (FVII), IX (FIX) and protein C (PC) display striking differences in the carboxyl-terminus, with that of FX being the most extended. This region is essential for FVII, FIX and PC secretion. To provide experimental evidence for the role of the FX carboxyl-terminus. Recombinant FX (rFX) variants were expressed in multiple eukaryotic cell systems. Protein and activity levels were evaluated by ELISA, coagulant and amidolytic assays. Expression of a panel of progressively truncated rFX variants in HEK293 cells revealed that the deletion of up to 21 residues in the carboxyl-terminus did not significantly affect secreted protein levels, as confirmed in HepG2 and BHK21 cells. In contrast, chimeric rFX-FVII variants with swapped terminal residues showed severely reduced levels. The truncated rFX variants revealed normal amidolytic activity, suggesting an intact active site. Intriguingly, these variants, which included that resembling the activated FXβ form once cleaved, also displayed remarkable or normal pro-coagulant capacity in PT- and aPTT-based assays. This supports the hypothesis that subjects with nonsense mutations in the FX carboxyl-terminus, so far never identified, would be asymptomatic. For the first time we demonstrate that the FX carboxyl-terminal region downstream of residue K467 is not essential for secretion and provides a modest contribution to pro-coagulant properties. These findings, which might suggest an involvement of the carboxyl-terminal region in the divergence of the homologous FX, FVII, FIX and PC, help to interpret the mutational pattern of FX deficiency.
Highlights
The unique specificity and regulation of serine proteases acting in the coagulation pathway, which share high structure and sequence similarities both at the gene and protein level, stem from the evolution and divergence of specific protein regions [1]
The identification of determinants underlying the specific features of the homologous pro-coagulant factors VII (FVII), IX (FIX) and X (FX) and the anti-coagulant protein C (PC) [2,3,4] contributes to understanding the evolutionary pathways that led to their divergence and specialization within the well-orchestrated coagulation cascade [5]
In this study we addressed this issue by evaluating the secreted protein and pro-coagulant activity levels of an ample panel of recombinant factor X (FX) variants progressively truncated at the carboxyl terminus
Summary
The unique specificity and regulation of serine proteases acting in the coagulation pathway, which share high structure and sequence similarities both at the gene and protein level, stem from the evolution and divergence of specific protein regions [1]. The identification of determinants underlying the specific features of the homologous pro-coagulant factors VII (FVII), IX (FIX) and X (FX) and the anti-coagulant protein C (PC) [2,3,4] contributes to understanding the evolutionary pathways that led to their divergence and specialization within the well-orchestrated coagulation cascade [5]. Previous studies conducted by us [6,7,8] and others [9,10,11,12], through the characterization of nonsense and missense as well as frame-shifted variants of FVII, FIX and PC, have demonstrated that this region represents a key determinant for efficient secretion. In this study we addressed this issue by evaluating the secreted protein and pro-coagulant activity levels of an ample panel of recombinant FX (rFX) variants progressively truncated at the carboxyl terminus. The appreciable secretion as well as pro-coagulant and amidolytic activity levels of these FX variants help to interpret the mutational pattern of FX deficiency
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