Factor VIII with a 237 amino acid B‐domain has an extended half‐life in F8‐knockout mice
| Autor: | Gert Bolt, Peter B. Johansen, Helle Heibroch Petersen, Henrik Rahbek-Nielsen, D. M. Karpf, Peder Lisby Nørby, Mette Loftager, Marianne Kjalke, Lars Thim, E. Bloem, G. E. Blouse |
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| Rok vydání: | 2019 |
| Předmět: |
congenital
hereditary and neonatal diseases and abnormalities animal diseases media_common.quotation_subject Genetic enhancement 030204 cardiovascular system & hematology Hemophilia A Cell Line Gene Knockout Techniques 03 medical and health sciences 0302 clinical medicine Protein Domains Von Willebrand factor In vivo hemic and lymphatic diseases Animals Internalization media_common Mice Knockout chemistry.chemical_classification Factor VIII biology Coagulants Half-life Hematology Molecular biology Recombinant Proteins In vitro Amino acid Disease Models Animal chemistry Knockout mouse biology.protein Low Density Lipoprotein Receptor-Related Protein-1 Half-Life Protein Binding |
| Zdroj: | Journal of Thrombosis and Haemostasis. 17:350-360 |
| ISSN: | 1538-7836 |
| Popis: | Essentials Factor (F)VIII with an intermediate-length B-domain showed higher levels in murine gene therapy. FVIII with different B-domain lengths were analysed. FVIII variants with B-domains between 186 and 240 amino acids (aa) have extended half-life in mice. Reduced cell binding of FVIII with a 237aa B-domain may explain the extended half-life. SUMMARY: Background Factor VIII consists of the A1-domain, A2-domain, B-domain, A3-domain, C1-domain, and C2-domain. FVIII with an intermediate-length B-domain of 226 amino acids (aa) has previously been evaluated in murine gene therapy studies. Objective To characterize FVIII with intermediate-length B-domains in vitro and in vivo in F8-knockout (KO) mice. Methods and results FVIII molecules with B-domains of 186-240aa had longer half-lives in F8-KO mice than FVIII molecules with shorter or longer B-domains. FVIII with a B-domain containing the 225 N-terminal aa fused to the 12 C-terminal aa of the wild-type B-domain (FVIII-237) had a 1.6-fold extended half-life in F8-KO mice as compared with FVIII with a 21aa B-domain (FVIII-21). The in vitro and in vivo activity of FVIII-237 were comparable to those of FVIII-21, as was binding to von Willebrand factor. Cell binding to LDL receptor-related protein 1 (LRP-1)-expressing cells was markedly reduced for FVIII-237 as compared with FVIII-21, whereas the affinity for LRP-1 was not reduced in surface plasmon resonance (SPR) studies. FVIII-21 cell binding and internalization could be inhibited by a fragment consisting of the 226 N-terminal aa of the FVIII B-domain, and SPR analysis suggested that this B-domain fragment might bind with weak affinity to FVIII-21. Conclusion Reduced cell binding of FVIII-237 might explain the observed extended half-life in F8-KO mice. This may contribute to the increased FVIII levels measured in murine gene therapy studies using FVIII constructs with similar B-domain lengths. |
| Databáze: | OpenAIRE |
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