Recombinant γY278H Fibrinogen Showed Normal Secretion from CHO Cells, but a Corresponding Heterozygous Patient Showed Hypofibrinogenemia
Abstract
:1. Introduction
2. Results
2.1. Patient’s Coagulation Tests, DNA Sequence Analysis and Immunoblotting Analysis
2.2. Secretion and Synthesis of Variant Fibrinogens in CHO Cells
2.3. Fibrinogen Degradation Assay Using Plasmin
2.4. Thrombin-Catalyzed Fibrin Polymerization
2.5. Protection Assay for the Plasmin Digestion of Fibrinogen
2.6. Factor (F) XIIIa-Catalyzed Cross-Linking of Fibrinogen
3. Discussion
4. Materials and Methods
4.1. Patient and Coagulation Tests
4.2. DNA Sequence Analysis
4.3. Immunoblotting Analysis of Plasma Fibrinogen
4.4. Preparation and Production of Recombinant Fibrinogen Variants
4.5. ELISA
4.6. Immunoblotting Analysis of Recombinant Fibrinogen
4.7. Fibrinogen Degradation Assays Using Plasmin
4.8. Thrombin-Catalyzed Fibrin Polymerization
4.9. Protection Assay for the Plasmin Digestion of Fibrinogen
4.10. FXIIIa-Catalyzed Cross-Linking of Fibrinogen
4.11. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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Variant | Recombinant Fibrinogen Production | Patients’ Plasma Fibrinogen Level | Disagreement | ||
---|---|---|---|---|---|
M/C (M/C of Wild-Type) | Reference | Antigenic (g/L) | Reference | ||
γC153R | NA (2.09 ± 1.38) | [23] | 0.87 | [23] | ◯ |
γR275C | Not reduced | [15] | 2.91 | [17] and 26 cases | ◯ |
γR275H | Not reduced | [24] | 2.02 | [17] and 31 cases | ◯ |
γT277P | 0.03 ± 0.02 (1.24 ± 0.31) | This manuscript | 1.18 | [21] | ◯ |
γT277R | 2.03 ± 0.50 (1.24 ± 0.31) | 0.79 | [22] | ✕ | |
γY278H | 0.72 ± 0.17 (1.24 ± 0.31) | 1.16 | This manuscript | ✕ | |
γA279D | 1.62 ± 0.48 (1.24 ± 0.31) | 2.7 | [25] | ◯ | |
γY280C | 0.82 ± 0.20 (1.24 ± 0.31) | NA * | [20] and 1 case | ◯ | |
γG284R | 0.05 ± 0.01 (0.77 ± 0.22) | [24] | 1.00 | [19] | ◯ |
γA289V | 0.07 ± 0.02 (0.88 ± 0.17) | [26] | 0.50 | [26] and 1 case | ◯ |
γG292V | 0.35 ± 0.12 (0.88 ± 0.17) | [26] | 1.20 | [27] | ◯ |
γT305A | 1.12 ± 0.14 (1.66 ± 0.40) | [28] | 1.12 | [28] | ✕ |
γH307Y | 0.31 ± 0.09 (1.66 ± 0.40) | [28] | 0.86 | [29] | ◯ |
γN308K | 1.74 ± 0.62 (1.66 ± 0.40) | [28] | 2.46 | [30] and 4 cases | ◯ |
γN308I | Not reduced | [31] | NA * | [32] | ◯ |
γS313N | 0.06 ± 0.01 (1.05 ± 0.17) | [18] | 0.58 | [33] | ◯ |
γT314P | 0.08 ± 0.04 (0.77 ± 0.22) | [24] | 0.47 | [34] | ◯ |
γD316N | 0.26 ± 0.05 (0.77 ± 0.22) | [24] | 1.36 | [21] and 1 case | ◯ |
γD318Y | 0.70 ± 0.17 (1.30 ± 0.19) | [35] | 2.9 | [36] | ◯ |
γΔ319 | 0.03 ± 0.01 (1.30 ± 0.19) | [35] | NA * | Czwalinna A, 2004 [12] (on-line submission) | ◯ |
γN319K | 0.05 ± 0.01 (1.30 ± 0.19) | [35] | NA | Meyer M, 2000 [12] (congress abstract) | NA |
γΔ320 | 0.09 ± 0.03 (1.30 ± 0.19) | [35] | 0.80 | [37] and 1 case | ◯ |
γD320E | 0.06 ± 0.03 (1.30 ± 0.19) | [35] | 1.30 | [38] | ◯ |
γD320G | 0.11 ± 0.02 (1.30 ± 0.19) | [35] | 0.85 | [35] and 1 case | ◯ |
γC326S | 0.03 ± 0.02 (2.17 ± 0.57) | [39] | 0.81 | [40] and 3 cases | ◯ |
γC326Y | 0.02 ± 0.03 (2.17 ± 0.57) | [39] | 1.50 | [41] and 1 case | ◯ |
γM336I | 0.05 ± 0.01 (1.05 ± 0.17) | [18] | NA * | [41] and 1 case | ◯ |
γA341D | 0.15 ± 0.06 (1.05 ± 0.17) | [18] | 1.37 | [42] | ◯ |
γN345D | 0.09 ± 0.02 (1.05 ± 0.17) | [18] | <0.5 | [41] | ◯ |
γD364H | Not reduced | [43] | 3.40 | [22] | ◯ |
γG366S | 0.03 ± 0.02 (0.77 ± 0.22) | [24] | 0.95 | [21] | ◯ |
γR375G | 1.10 ± 0.25 (0.94 ± 0.10) | [44] | 2.50 | [45] | ◯ |
γR375W | 0.09 ± 0.10 (0.94 ± 0.10) | [44] | 1.10 | 13 cases | ◯ |
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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. https://doi.org/10.3390/ijms22105218
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. International Journal of Molecular Sciences. 2021; 22(10):5218. https://doi.org/10.3390/ijms22105218
Chicago/Turabian StyleKamijo, Tomu, Takahiro Kaido, Masahiro Yoda, Shinpei Arai, Kazuyoshi Yamauchi, and Nobuo Okumura. 2021. "Recombinant γY278H Fibrinogen Showed Normal Secretion from CHO Cells, but a Corresponding Heterozygous Patient Showed Hypofibrinogenemia" International Journal of Molecular Sciences 22, no. 10: 5218. https://doi.org/10.3390/ijms22105218
APA StyleKamijo, T., Kaido, T., Yoda, M., Arai, S., Yamauchi, K., & Okumura, N. (2021). Recombinant γY278H Fibrinogen Showed Normal Secretion from CHO Cells, but a Corresponding Heterozygous Patient Showed Hypofibrinogenemia. International Journal of Molecular Sciences, 22(10), 5218. https://doi.org/10.3390/ijms22105218