A Novel Amino Acid Substitution, Fibrinogen Bβp.Pro234Leu, Associated with Hypofibrinogenemia Causing Impairment of Fibrinogen Assembly and Secretion.

Takahiro Kaido,Yumiko Higuchi,Masahiro Yoda,Shinpei Arai,Chiaki Taira,Tomu Kamijo,Nobuo Okumura

doi:10.3390/ijms21249422

Takahiro Kaido, Yumiko Higuchi + Show 5 more

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https://doi.org/10.3390/ijms21249422

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Abstract

We identified a novel heterozygous variant, Bβp.Pro234Leu (fibrinogen Tokorozawa), which was suspected to be associated with hypofibrinogenemia. Therefore, we analyzed the assembly and secretion of this fibrinogen using Chinese hamster ovary (CHO) cells. To determine the impact on the synthesis and secretion of fibrinogen of the Bβp.P234L and γp.G242E substitutions, we established recombinant variant fibrinogen-producing CHO cell lines. Synthesis and secretion analyses were performed using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting analysis with the established cell lines. In addition, we performed fibrin polymerization using purified plasma fibrinogen and in-silico analysis. Both Bβp.P234L and γp.G242E impaired the secretion and synthesis of fibrinogen. Moreover, immunoblotting analysis elucidated the mobility migration of the Bβγ complex in Bβp.P234L. On the other hand, the fibrin polymerization of fibrinogen Tokorozawa was similar to that of normal fibrinogen. In-silico analysis revealed that the Bβp.P234 residue is located in the contact region between the Bβ and γ chains and contacts γp.G242 residue. The present study demonstrated that the Bβp.P234L substitution resulted in hypofibrinogenemia by decreasing the assembly and secretion of fibrinogen. Therefore, there is a possibility that substitutions in the contact region between the Bβ and γ chains impact the assembly and secretion of fibrinogen.

Highlights

Fibrinogen is a 340 kDa plasma glycoprotein involved in hemostasis by forming fibrin [1]
The laboratory data before the operation showed that her functional fibrinogen level was 1.36 g/L, and her prothrombin time-international normalized ratio was 1.13
Our findings showed that the secretion from the Bβp.P234L fibrinogen-producing Chinese hamster ovary (CHO) cells was impaired, similar to that from the γp.G242E fibrinogen-producing CHO cells, we speculated that the Bβp.P234L fibrinogen was absent in the Tokorozawa patient plasma, since the fibrinogen concentration in the culture media of variant fibrinogen-producing CHO cells was markedly reduced

Summary

Introduction

Fibrinogen is a 340 kDa plasma glycoprotein involved in hemostasis by forming fibrin [1]. A three-chain monomer (Aα-Bβ-γ) forms by combining the Aα chain and Bβγ complex or Bβ chain and Aαγ complex, and this monomer is held together by each N-terminal portion into a six-chain dimer (Aα-Bβ-γ), which is secreted into the circulation [1,3]. CFDs are classified according to functional and immunological fibrinogen levels as quantitative or qualitative disorders [5]. Quantitative disorders are afibrinogenemia and hypofibrinogenemia, which correspond to the complete absence of fibrinogen or decreased immunological fibrinogen levels, respectively [6]. Quantitative disorders due to mutations lead to decreased amounts of fibrinogen by causing defects in the synthesis of the constituent chains or in fibrinogen assembly, stability, or secretion [7]. Dysfibrinogenemia is mostly caused by mutations at important sites for the coagulation function [8]

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