Session: 321. Blood Coagulation and Fibrinolytic Factors: Poster III
Hematology Disease Topics & Pathways:
Bleeding Disorders, Diseases, Bleeding and clotting
Chinese hamster ovary (CHO-K1) cells stably expressing rFVII-160R or rFVII-354V-464Hfs were treated with the chemical chaperones 4-phenylbutyrate (4-PBA), betaine, taurine, taurourosdeoxycholic acid (TUDCA), trimethylamine N-oxide (TMAO) or lumacaftor (VX-809). The intracellular and secreted levels of FVII antigen were measured by ELISA and F7 mRNA levels were assessed by quantitative RT-PCR. Treatment of cells with 4-PBA for 48 hours increased F7 mRNA levels and increased the ratio of secreted/intracellular of both FVII mutants by ~2-2.5-fold, indicating an effect of 4-PBA FVII expression and secretion.
4-PBA is known to inhibit the activity of histone deacetylases, thereby inducing hyperacetylation of histones and increasing gene transcription. To measure histone acetylation, we next performed Western blot analysis of acetylated histone H3 (AcH3). We found that the levels of AcH3 were significantly increased after treatment with 4-PBA, indicating that induced transcription of F7 by 4-PBA could be a result of increased global histone acetylation. Since 4-PBA treatment increased the secretion of the FVII mutants, we questioned whether this also would alleviate ER stress. Using an ER stress luciferase assay, we observed that the ER stress levels in 4-PBA treated cells were significantly lower than in non-treated cells.
To determine whether 4-PBA treatment could restore the intracellular trafficking of the FVII mutants from the ER to Golgi, confocal immunofluorescence microscopy was utilized The studies revealed that upon treatment with 4-PBA, the p.Q160R mutant had a strong co-localization with the Golgi marker GM130 and with the Golgi stacking protein GRASP55, known to be associated with unconventional protein secretion. A vesicular pattern of staining could be observed in cells expressing the p.A354V-p.P464Hfs. However, these structures did not overlap with endosomal markers or markers of the ER-Golgi transport remaining the secretory route of this FVII mutant elusive.
Finally, to assess the activity of FVII mutants, we measured the generation of activated (a) factor X (FXaG) in FVII deficient plasma supplemented with conditioned medium from cells stably expressing rFVII mutants before and after 4-PBA treatment. To increase the assay sensitivity to very low FVII activity levels we also boosted the coagulation initiation phase by inhibiting the tissue factor pathway inhibitor (TFPI) through an anti-TFPI RNA aptamer. We found that treatment of cells with 4-PBA gave rise to a modest, but measurable FXaG activity in conditioned medium for both FVII mutants. Furthermore, inhibition of TFPI proportionally potentiated FXaG activity of both 4-PBA-induced FVII variants.
The present study demonstrates that chemical chaperones, such as 4-PBA, can increase intracellular trafficking through the secretory pathway of misfolded FVII mutants. Importantly, the secreted mutant proteins were found to be biologically regulated and active in the coagulation pathway. Thus, 4-PBA could have therapeutic effect in FVII deficiency caused by misfolded FVII mutants.
Disclosures: No relevant conflicts of interest to declare.
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