## 4 Discussion
We previously reported an effect of 4-PBA on the secretion of the FVII p.Q160R variant [24] and on a protein C variant [37], and similar results were reported for FIX in a cellular model of hemophilia B [38]. In the present study, we observed an increased colocalization with the _trans_-Golgi network marker TGN46 and with the vesicular marker COPII. The export of cargo out of the ER is mediated by COPII-coated vesicles that bud from the ER at ER-exit sites (reviewed in [39] and [40]), and the _trans_-Golgi network is a distinct membranous compartment at the _trans_ face of the Golgi apparatus thought to be involved in the sorting of secretory proteins [41]. This could indicate that treatment with 4-PBA might facilitate transport out of the ER and into the secretory pathway, as reported for other FVII variants [24]. Our previous studies demonstrated increased ER stress in cells expressing rFVII-354 VHfs and upregulation of the unfolded protein response [22]. In our model, 4-PBA and BIX increased the expression levels of the ER chaperone BiP, which can bind to exposed hydrophobic residues of unfolded proteins and plays a key role in protein folding and quality control in the ER [42]. In a recent study by [PERSON] et al., it was shown that inadequate BiP expression upon bulk expression of secretory immunoglobulin M heavy chain caused proteotoxicity [43]. The authors argued that pharmacological intervention against proteotoxicity should primarily be aimed at promoting a favorable level of BiP versus the client protein. For 4-PBA, the increase in BiP was accompanied by a reduced ratio of BAX/BCL-2, suggesting an antiapoptotic state in the cells supporting this hypothesis. In addition to increasing the levels of BiP, 4-PBA also increased the expression of the ER chaperone GRP94 and both chaperones increased the levels of the transcription factor sXBP1, which is known to increase the transcription of key ER chaperones. Based on these observations, we might hypothesize that the enhanced folding capacity of the ER following chaperone treatment contributes to the increased levels of the FVII variant in the cell lysates. Even small changes of folding capacity in the patients' hepatocytes would be candidates to produce the small differences observed in plasma p.A354V-p.P464 Hs levels among patients, which in turn could potentially contribute to producing differences in bleeding symptoms. Both 4-PBA and BIX are reported to have antiapoptotic effects [44; 45; 46]. We observed a decreased rate of apoptosis upon treatment with 4-PBA, whereas BIX treatment resulted in a slightly increased rate of apoptosis. A possible explanation for the increased rate of apoptosis seen in BIX-treated cells could be the intracellular accumulation of rFVII-354V-464 Hs causing proteotoxicity. This is supported by the net negative effect on secretion following BIX treatment and the lack of GRP94 induction and relatively low BiP induction compared to 4-PBA. Of note, the only known effects of BIX are as a molecular chaperone inducer, whereas 4-PBA is reported to have many effects, including working both as a chemical chaperone and reducing ER stress [35]. Despite the similarities that we report between the plasma and the recombinant protein, the latter was characterized by very low levels of FXa generation activity. Even the residual function of 4-PBA-induced rFVII-354V-464 Hs was very reduced (0.1% of rFVIIwt), compared to in vivo, where the residual FVII coagulant activity has a median value of approximately 1%. In the stable cell lines used in this study, the FVII:Ag in the conditioned medium was also severely reduced, and no FXaG could be measured in the conditioned medium unless concentrated several folds. The low secretion and clotting activity of this variant in vitro, observed in independent experiments and in different laboratories [13], and even after chaperone treatment, appears to be a feature of the p.A354V-p.P464 Hs variant.