Naturally occurring truncated proteins: decreased protein secretion and increased activity result in asymptomatic coagulation factor deficiency

Factor VII (FVII), IX (FIX), X and protein C (PC), having distinct protein properties and specificities, belong to the coagulation serine protease family that evolved from a common ancestor. In spite of high homology at the gene and protein levels, they show remarkable differences in the carboxyl-terminal region. The C-terminus of FIX and PC has been shown to be essential for secretion but not function. To address this issue in FVII we took advantage from the identification of the homozygous R402Stop nonsense mutation in two asymptomatic FVII deficient patients. FVII protein and coagulant activity levels in patients’ plasma were 0,8% and 5% of normal, respectively, thus suggesting the presence of truncated molecules (FVII-R402Stop, natural stop codon at 407 position), poorly secreted but with improved procoagulant activity. Functional FXa and Thrombin generation assays in plasma confirmed these observations. To investigate these features, we expressed the naturally truncated FVII-402Stop and the FVII variants 403-406Stop. Similarly to the natural mutant, the rFVII-403Stop and rFVII-402Stop variants were poorly secreted (~1%). We found an inverse relationship between secreted protein levels in media and the extent of the deletion for the rFVII-406Stop (50-60% of WT), rFVII-405Stop (15-20%) and rFVII-404Stop (9-12%). FXa generation and coagulation assays revealed a normal specific activity for the rFVII-406Stop, rFVII-405Stop, rFVII-404Stop variants. Intriguingly, upon concentration of media, the specific activity of the rFVII-402Stop appeared to be 387±11% of rFVII-wt, thus recapitulating the mild coagulation phenotype of patients. Altogether these findings point toward multiple functional roles of the FVII carboxyl-terminal region, whose variation might have contributed to the divergence of this protein from the other coagulation serine proteases.