| First Author | Nielsen AL | Year | 2017 |
| Journal | Proc Natl Acad Sci U S A | Volume | 114 |
| Issue | 47 | Pages | 12454-12459 |
| PubMed ID | 29109275 | Mgi Jnum | J:254329 |
| Mgi Id | MGI:6101374 | Doi | 10.1073/pnas.1618713114 |
| Citation | Nielsen AL, et al. (2017) Engineering of a membrane-triggered activity switch in coagulation factor VIIa. Proc Natl Acad Sci U S A 114(47):12454-12459 |
| abstractText | 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. |
