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

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 conducted 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 was secreted normally from the patient's hepatocytes but then underwent accelerated degradation by plasmin in the circulation.

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

Figure 1

Immunoblotting analysis of plasma fibrinogen. One thousand-fold diluted plasma was separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) in non-reducing conditions in a 7% polyacrylamide gel (A) or reducing conditions in a 10% polyacrylamide gel (B) and plasma fibrinogen was detected by Western blotting using an anti-human fibrinogen antibody. Fibrinogen (Fbg), γ-γ dimer and Aα, Bβ, and γ chains are indicated on the right side of each panels. Lane 1: purified normal plasma fibrinogen, 2: normal plasma, 3: patient’s plasma.

Figure 2

Secretion and synthesis of fibrinogen in Chinese hamster ovary cells. Fibrinogen concentrations in the culture media (A) and cell lysates (B) were measured using an enzyme-linked immunosorbent assay. Panel (C) shows the M/C ratio of the culture media to the cell lysate. Box plots and central bars show the interquartile range and median, respectively. The significance of differences between wild-type and variant fibrinogen-producing cells is shown (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

Immunoblotting analysis of recombinant fibrinogen. Fibrinogen in Chinese hamster ovary cell lysates were determined by Western blotting using anti-human fibrinogen antibodies in non-reducing conditions in a 8% polyacrylamide gel (A) or reducing conditions in an 10% polyacrylamide gel (B) and anti-human fibrinogen γ-chain antibodies in reducing conditions in an 10% polyacrylamide gel (C). Fibrinogen (Fbg), γ-γ dimer and Aα, Bβ, and γ chain are indicated on the right side of each panel. Lane 1: purified fibrinogen, 2: wild-type, 3: γL276P, 4: γT277R, 5: γT277P, 6: γY278H, 7: γA279D, and 8: γY280C cell lines.

Figure 4

Fibrinogen degradation assay with plasmin. Recombinant fibrinogens (0.30 mg/mL) in HBS buffer containing 5 mM EDTA were incubated with 0.18 U/mL plasmin for 0–4 h at 37 °C. The fragments were analyzed by 10% SDS-PAGE and with Coomassie brilliant blue (CBB) staining. Fibrinogen (Fbg), fragments D1, D2, D3, and D4, and lower molecular weight fragment (◀) are indicated on the right side of each panel. (A) wild type, (B) γR275C, (C) γT277R, (D) γY278H.

Figure 5

Thrombin catalyzed fibrin polymerization. The polymerization of 0.18 mg/mL recombinant fibrinogens was initiated with 0.05 U/mL thrombin in the presence of 1 mM Ca²⁺ (A) or absence of Ca²⁺ (B). ●: Wild-type, ♦: γR275C, ▲: γT277R, ■: γY278H.

Figure 6

Protection assay for plasmin degradation of fibrinogen. Recombinant fibrinogen (0.30 mg/mL) in HBS buffer containing 5 mM EDTA or 1 or 5 mM Ca²⁺ was incubated with 0.18 U/mL plasmin at 37 °C for 2 h. The fragments were analyzed using 10% SDS-PAGE with CBB staining. Fragments D1, D2, D3, and D4 are indicated on the right side of panel D and lower molecular weight fragments are indicated (►). (A) wild type, (B) γR275C, (C) γT277R, (D) γY278H.

Figure 7

FXIIIa-catalyzed cross-linking of fibrinogen. Recombinant fibrinogens were cross-linked using FXIIIa for 0.5–24 h and examined in 8% SDS-PAGE gels in reducing conditions with CBB staining. Reduced fibrinogen chains (Aα, Bβ, γ chains, α-polymer, γ-γ dimer) are indicated on the right side of each panel. (A) wild type, (B) γR275C, (C) γT277R, (D) γY278H.