In vitro expression of mutant factor VII proteins and characterization of their clinical significance

Mashayekhi,Amir; Shahbazi,Shirin; Omrani,Mirdavood; Mahdian,Reza

doi:10.3892/mmr.2017.8158

In vitro expression of mutant factor VII proteins and characterization of their clinical significance

Authors:
- Amir Mashayekhi
- Shirin Shahbazi
- Mirdavood Omrani
- Reza Mahdian
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Affiliations: Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 1431633443, Iran, Department of Medical Genetics, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran 1985717443, Iran, Department of Molecular Medicine, Pasteur Institute of Iran, Tehran 1316943551, Iran
Published online on: November 27, 2017 https://doi.org/10.3892/mmr.2017.8158
Pages: 2738-2742

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Abstract

Abstract. Factor VII (FVII) serves an essential role in the initiation of blood coagulation. Mutations in conserved residues within its serine protease domain may lead to dysregulated coagulation activity. The objective of the present study was to elucidate the impact of altering two conserved residues, H348R and S282R, on the functional properties of the FVII protein. The mutation‑harboring fragments were derived from genomic DNA of a FVII deficient patient. The fragments were integrated into a pcDNA vector containing FVII cDNA of HepG2 cells. The wild-type and mutated FVII constructs were transfected into CHO‑K1 cells as a mammalian cell model. The coagulation activity, antigen levels and intracellular localization of the recombinant proteins were studied in association with their pathological importance. Results indicated that FVII activity was not detectable in conditioned media of the cells transfected with the mutated constructs. The H348R mutation reduced the expression of intracellular and secreted forms of the FVII protein. Following S282R transfection, intracellular FVII expression showed no significant variation; however, extracellular protein was reduced. The pattern of intracellular localization of mutated FVII remained unaltered in comparison to the wild-type protein. In conclusion, the present study suggested that missense mutations within the serine protease domain of FVII affect extracellular levels in addition to the coagulation activity of FVII. These results may contribute to further understanding of the molecular pathogenesis of FVII deficiency and the development of pharmaceutical candidates with improved therapeutic properties.

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