Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Recombinant γY278H Fibrinogen Showed Normal Secretion from CHO Cells, but a Corresponding Heterozygous Patient Showed Hypofibrinogenemia

Version 1 : Received: 12 April 2021 / Approved: 13 April 2021 / Online: 13 April 2021 (11:29:08 CEST)

A peer-reviewed article of this Preprint also exists.

Kamijo, T.; Kaido, T.; Yoda, M.; Arai, S.; Yamauchi, K.; Okumura, N. Recombinant γY278H Fibrinogen Showed Normal Secretion from CHO Cells, but a Corresponding Heterozygous Patient Showed Hypofibrinogenemia. Int. J. Mol. Sci. 2021, 22, 5218. Kamijo, T.; Kaido, T.; Yoda, M.; Arai, S.; Yamauchi, K.; Okumura, N. Recombinant γY278H Fibrinogen Showed Normal Secretion from CHO Cells, but a Corresponding Heterozygous Patient Showed Hypofibrinogenemia. Int. J. Mol. Sci. 2021, 22, 5218.

Journal reference: Int. J. Mol. Sci. 2021, 22, 5218
DOI: 10.3390/ijms22105218

Abstract

We identified a novel heterozygous hypofibrinogenemia, γY278H (Hiroshima). To demonstrate the cause of reduced plasma fibrinogen levels (functional level: 1.12 g/L and antigenic level: 1.16 g/L), we established γY278H fibrinogen-producing Chinese hamster ovary (CHO) cells. An enzyme-linked immunosorbent assay demonstrated that synthesis of γY278H fibrinogen inside CHO cells and secretion into the culture media were not reduced. Then, we established an additional five variant fibrinogen-producing CHO cell lines (γL276P, γT277P, γT277R, γA279D, and γY280C) and performed further investigations. We have already established 33 γ-module variant fibrinogen-producing CHO cell lines including 6 cell lines in this study, but only the γY278H and γT277R cell lines showed disagreement, namely recombinant fibrinogen production was not reduced but the patients’ plasma fibrinogen level was reduced. Finally, we performed fibrinogen degradation assays and demonstrated that the γY278H and γT277R fibrinogens were easily cleaved by plasmin whereas their polymerization in the presence of Ca2+ and “D:D” interaction was normal. In conclusion, our investigation suggested that patient γY278H showed hypofibrinogenemia because γY278H fibrinogen is secreted normally from the patient’s hepatocytes but had accelerated degradation by plasmin in the circulation.

Subject Areas

congenital fibrinogen disorders; hypofibrinogenemia; Chinese hamster ovary cells; recombinant fibrinogen; plasmin degradation

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