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A Critical Role of Carboxyl-Terminal Alpha-Helix in the Secretion of Coagulation Factor XI

1Nagoya University Graduate School of Medicine, Pathophysiological Laboratory Sciences, Nagoya, Japan, 2Nagoya University Hospital, Transfusion Medicine, Nagoya, Japan, 3Nagoya University Hospital, Clinical Laboratory, Nagoya, Japan, 4Nagoya University Hospital, Hematology and Oncology, Nagoya, Japan, 5Aichi Health Promotion Foundation, Nagoya, Japan

Abstract Number: PB0248

Meeting: ISTH 2020 Congress

Theme: Coagulation and Natural Anticoagulants » Contact Pathway

Background: Coagulation factor XI (FXI) is a plasma serine protease zymogen that belongs to the intrinsic pathway of blood coagulation. However, a molecular machinery of FXI secretion has remained unclear.

Aims: To disclose a molecular basis of FXI secretion by characterizing a novel FXI mutant identified in a Japanese FXI deficiency patient.

Methods: The patient is a 75-year-old Japanese male with FXI:C < 3%. Genetic mutation of FXI gene (F11) was analyzed with direct sequencing. Mutant FXI corresponding to the identified F11 mutation was overexpressed in HEK293 cells and examined the extracellular secretion with western blotting. Subcellular localization of the mutant FXI was investigated with immunocytochemistry.

Results: We identified a novel homozygous frameshift mutation in F11 (c.1788dupC: p.Glu597Argfs*65), resulting in a C-terminal extension. The C-terminal extended FXI was intracellularly synthesized, but represented an extracellular secretion defect. Subcellular localization analysis showed that the extended mutant FXI was not detected at the Golgi apparatus, indicating an abnormal retention within the endoplasmic reticulum. To further investigate the roles of C-terminal region in the FXI secretion, we generated a series of recombinant FXIs gradually truncated at the C-terminus. The truncated FXIs revealed that a preservation of threonine at position 622, a fourth residue from the C-terminus, was essential for the FXI secretion. In silico structure simulation predicted that the residue at position 622 engaged to constitute an alpha-helix, suggesting an importance of the C-terminal alpha-helix motif for the FXI intracellular behavior and secretion.

Conclusions: Given the observation of the secretory defect in the C-terminal extended FXI mutant (p.Glu597Argfs*65), we demonstrated the critical role of C-terminal region, especially alpha-helix motif, in the FXI extracellular secretion.

To cite this abstract in AMA style:

Hayakawa Y, Tamura S, Suzuki N, Odaira K, Tokoro M, Kawashima F, Hayakawa F, Takagi A, Suzuki A, Okamoto S, Kanematsu T, Matsushita T, Kojima T. A Critical Role of Carboxyl-Terminal Alpha-Helix in the Secretion of Coagulation Factor XI [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/a-critical-role-of-carboxyl-terminal-alpha-helix-in-the-secretion-of-coagulation-factor-xi/. Accessed March 3, 2021.

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