## 1 Introduction  
Factor (F) VII is a vitamin K-dependent glycoprotein synthesized in the liver that undergoes extensive post-translational modifications prior to secretion [1, 2, 3]. It is secreted into the blood where it circulates at a concentration of roughly 0.5 \(\upmu\)g/mL (10 nM) [4]. The FVII protein contains an amino-terminal \(\gamma\)-carboxoglutamic acid domain followed by two epidermal growth factor (EGF)-like domains and a carboxy-terminal protease domain [5]. The single-chain native FVII is a zymogen that, upon binding to its cofactor,tissue factor (TF), becomes activated, resulting in a two-chained activated molecule (FVIIa). The TF/FVIIa catalytic complex, which is considered the key initiator of blood coagulation, activates downstream clotting factors leading to the production of thrombin, the key effector for the final formation of the clot [6,7]. Congenital FVII deficiency (OMIM #227500) is a rare, autosomal recessive bleeding disorder caused by mutations in the \(F7\) gene resulting in a concomitant deficiency of plasma FVII protein (FVII antigen, FVII:Ag) and FVII procoagulant activity (FVIIC) (Type I deficiency) or in low FVII:C levels with normal, near-normal, or reduced FVII:Ag (Type II deficiency) [8]. In FVII deficiency, the spectrum of bleeding symptoms is widely variable [9,10]. Most patients exhibit mild symptoms such as mucocutaneous bleeds, whereas 10-15% of patients may experience potentially life-threatening hemorrhages such as central nervous system (CNS) or gastrointestinal (GI) bleeds [11, 12, 13]. The \(F7\) p.A354V-p.P464 Hs compound variant is caused by the p.A354V missense change, with pleiotropic effects in the FVII catalytic domain [14], and a cytosine deletion causing a frameshift at the C-terminal codon 464, which produces an elongated protein [15]. This variant represents the most frequent \(F7\) alteration in Central Europe [13,16, 17, 18] reported in the FVII variant database ([[https://f7-db.eahad.org/](https://f7-db.eahad.org/)]([https://f7-db.eahad.org/](https://f7-db.eahad.org/)), accessed on 1 May 2021) [19] and is associated with a bleeding phenotype due to severely reduced FVII:C and FVII:Ag in plasma [16,20]. We previously reported minimal FVII secretion in cells expressing the recombinant (r) FVII p.A354V-p.P464 Hs (rFVII-354V-464 Hs) variant [21,22], resembling the patient phenotype. Our data suggested that the combined protein alterations were associated with a defective folding and that the misfolded state led to impaired trafficking, endoplasmic reticulum (ER) retention and ER stress. Very few studies on the clinical manifestations of the p.A354V-p.P464 Hfs variant have been performed with a small number of patients [13,16], and detailed studies on the residual activity of this variant are hampered by the low levels of FVII in plasma and very reduced secretion after recombinant expression [13]. The relatively high frequency for a symptomatic mutation in a rare bleeding disorder, as well as the presence of small amounts of residual activity of this variant, provides a unique opportunity to investigate, in a constant \(F7\) mutation background, the poor consistency between FVII levels and the clinical manifestations, a key issue in FVII deficiency [23]  
In the present study, we collected and analyzed data on FVII plasma activity and bleeding phenotype in 37 p.A354V-p.P464 Hs homozygotes as a means to deepen our knowledge on the genotype-plasma and clinical phenotype relationships. The rFVII-354V-464 Hs was produced in Chinese hamster ovary (CHO) K1 cells and compared to patient phenotypes and plasma FVII from a subset of patients. We recently demonstrated that the chemical chaperone 4-phenylbutyrate (4-PBA) could improve the secretion and biological activity of another recombinant FVII variant characterized by impaired secretion and triggering of ER stress [24].