## 4 Discussion  
The p.A354V-p.P464 Hs variant represents a paradigmatic model mutation in FVII deficiency, both on studies of bleeding phenotype and on the molecular mechanisms underlying FVII deficiency. Among the large number of coagulation protein variants, only protein C Nagoya, a thrombophilic mutation, can be compared with _F7_ p.A354V-p.P464 Hs, both in terms of molecular mechanisms (translation frameshift and elongated protein) and frequency [32]. In addition, a novel homozygous _F11_ frameshift mutation resulting in an extended FXI carboxyl-terminus, ER retention and reduced secretion was recently identified in a Japanese patient [33]. Figure 6: FXa generation in medium from cells treated with 4-PBA. FXa generation activity in FVII-deficient plasma added with medium from 4-PBA treated cells (continuous black line) and untreated (continuous gray line) cells, or in the presence of anti-TFPI aptamer (+Apt) in medium from 4-PBA treated (dotted black line) and untreated (dotted gray line) cells. RFU/s, relative fluorescence units/second. Inset: curves obtained with diluted (0.1%) medium containing rFVIIwt (black line), vector alone (dotted line) or FVII-deficient plasma alone (gray line). The analysis of clinical phenotypes in 37 homozygotes for the p.A354V-p.P464 Hfs variant significantly extends previous information about variability in bleeding severity in FVII-deficient patients. The small range of FVII:C in a group of patients with the same \(F7\) genotype supports the notion that a large variation in clinical phenotypes is detectable with relatively small variations in FVII activity, a major open question in FVII deficiency [3]. The investigation of FVII:Ag in plasma revealed that the antigen levels were clearly higher than the activity levels, suggesting a Type-II defect. The difference concerning the assay methods is relevant as the ELISA is indeed more sensitive and precise than the coagulation assay. However, both measurements display the same variability, suggesting a true variation between the studied individuals. Overall, findings in patients indicated that the elongated p.A354V-p.P464 Hfs variant circulated in plasma at low but detectable concentrations. Its activity in plasma was reduced but detectable. Our study clearly suggests that yet unknown genetic and acquired components beyond \(F7\) mutations play a role in producing variability in clinical manifestations in this deficiency. The recombinant expression of the p.A354V-p.P464 Hfs variant was aimed at providing the molecular basis underlying these observations. The Western blot analysis of patient plasma demonstrated a band with a migration pattern that was slower than that of normal plasma FVII. This pattern, indicating a protein larger in size, is in agreement with the base-pair deletion at position 464, producing a frameshift mutation that results in additional 28 residues at the protein carboxyl-terminus [16]. In fact, the FVII in plasma from patients homozygous for the p.A354V-p.P464 Hfs variant migrated in a comparable manner to that of rFVII-354V-464 Hfs, suggesting a similar processing of this protein variant in the CHO-K1 cells. When FVII is overexpressed in CHO cells, it undergoes similar post-translational modifications as plasma-derived FVII, which makes these cells the best option for the production of rFVII [34]. Chemical chaperones are known to prevent misfolded protein aggregation and alleviate ER stress, thereby improving the biosynthesis of misfolded proteins [35, 36]. We explored the possibility that the chaperone-based approach could potentiate folding and secretion of p.A354V-p.P464 Hfs variant. This would unravel biological pathways that could participate in the secretion of small amounts of the variant, which is of utmost importance to modulate bleeding phenotypes in patients. Treatment with the chemical chaperones 4-PBA and BIX increased the levels of rFVII-354V-464 Hfs in cell lysates and in the medium. However, a net effect on secretion could only be observed in cells treated with 4-PBA.