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Engineering of a membrane-triggered activity switch in coagulation factor VIIa.
- Source :
-
Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2017 Nov 21; Vol. 114 (47), pp. 12454-12459. Date of Electronic Publication: 2017 Nov 06. - Publication Year :
- 2017
-
Abstract
- Recombinant factor VIIa (FVIIa) variants with increased activity offer the promise to improve the treatment of bleeding episodes in patients with inhibitor-complicated hemophilia. Here, an approach was adopted to enhance the activity of FVIIa by selectively optimizing substrate turnover at the membrane surface. Under physiological conditions, endogenous FVIIa engages its cell-localized cofactor tissue factor (TF), which stimulates activity through membrane-dependent substrate recognition and allosteric effects. To exploit these properties of TF, a covalent complex between FVIIa and the soluble ectodomain of TF (sTF) was engineered by introduction of a nonperturbing cystine bridge (FVIIa Q64C-sTF G109C) in the interface. Upon coexpression, FVIIa Q64C and sTF G109C spontaneously assembled into a covalent complex with functional properties similar to the noncovalent wild-type complex. Additional introduction of a FVIIa-M306D mutation to uncouple the sTF-mediated allosteric stimulation of FVIIa provided a final complex with FVIIa-like activity in solution, while exhibiting a two to three orders-of-magnitude increase in activity relative to FVIIa upon exposure to a procoagulant membrane. In a mouse model of hemophilia A, the complex normalized hemostasis upon vascular injury at a dose of 0.3 nmol/kg compared with 300 nmol/kg for FVIIa.<br />Competing Interests: Conflict of interest statement: A.L.N., A.B.S., H.L.H., P.S.G., J.B., K.L., M.K.-H., C.D.L., B.B.S., O.H.O., and H.Ø. are employed by Novo Nordisk A/S.<br /> (Copyright © 2017 the Author(s). Published by PNAS.)
- Subjects :
- Allosteric Regulation
Animals
Blood Coagulation drug effects
Disease Models, Animal
Factor VIIa genetics
Factor VIIa pharmacology
Factor VIIa therapeutic use
Female
Hemophilia A physiopathology
Humans
Kinetics
Male
Mice
Mice, Inbred C57BL
Mice, Knockout
Molecular Dynamics Simulation
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins pharmacology
Recombinant Proteins therapeutic use
Thromboplastin genetics
Thromboplastin pharmacology
Thromboplastin therapeutic use
Biological Therapy methods
Factor VIIa chemistry
Hemophilia A therapy
Protein Engineering methods
Thromboplastin chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1091-6490
- Volume :
- 114
- Issue :
- 47
- Database :
- MEDLINE
- Journal :
- Proceedings of the National Academy of Sciences of the United States of America
- Publication Type :
- Academic Journal
- Accession number :
- 29109275
- Full Text :
- https://doi.org/10.1073/pnas.1618713114