Identification of serpins specific for activated protein C using a lysate-based screening assay.
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Publication Date
2018-06-08Journal Title
Sci Rep
ISSN
2045-2322
Publisher
Springer Science and Business Media LLC
Volume
8
Issue
1
Pages
8793
Language
eng
Type
Article
Physical Medium
Electronic
Metadata
Show full item recordCitation
Polderdijk, S. G., & Huntington, J. (2018). Identification of serpins specific for activated protein C using a lysate-based screening assay.. Sci Rep, 8 (1), 8793. https://doi.org/10.1038/s41598-018-27067-z
Abstract
Activated protein C (APC) is a powerful anticoagulant enzyme that proteolytically inactivates the cofactors of the Xase and prothrombinase complexes, factors VIIIa and Va. A common mutation in factor V, fVLeiden, confers resistance to APC leading to an increased risk of thrombosis in the normal population. However, when coinherited with haemophilia, fVLeiden reduces bleeding severity, suggesting that inhibition of APC may be a useful strategy for treatment of haemophilia. We previously reported on serpins that were rationally designed for improved specificity for APC over other coagulation serine proteases. Based on structural differences in the substrate binding pockets to either side of the P1 Arg, we mutated the P2 and P1' residues to Lys. Although this approach achieved APC specificity, it resulted in a reduction in the rate of APC inhibition relative to the parent containing only the P1 Arg. Here we conduct site-specific random mutagenesis at the P2 and P1' positions to determine if improvements could be made in the rate of APC inhibition. In addition to our original Lys mutations, we found that Arg and Gln also confer specificity for APC. However, in all cases specificity for APC resulted in a reduction in inhibition rate.
Keywords
Humans, Protein C, alpha 1-Antitrypsin, Serpins, Enzyme Inhibitors, Mutagenesis, Site-Directed, Binding Sites, Protein Conformation, Blood Coagulation, Models, Molecular
Identifiers
External DOI: https://doi.org/10.1038/s41598-018-27067-z
This record's URL: https://www.repository.cam.ac.uk/handle/1810/283031
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