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dc.contributor.authorPolderdijk, Stéphanie GI
dc.contributor.authorHuntington, James A
dc.date.accessioned2018-10-03T04:43:41Z
dc.date.available2018-10-03T04:43:41Z
dc.date.issued2018-06-08
dc.identifier.issn2045-2322
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/283031
dc.description.abstractActivated 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.
dc.format.mediumElectronic
dc.languageeng
dc.publisherSpringer Science and Business Media LLC
dc.rightsAttribution 4.0 International
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subjectHumans
dc.subjectProtein C
dc.subjectalpha 1-Antitrypsin
dc.subjectSerpins
dc.subjectEnzyme Inhibitors
dc.subjectMutagenesis, Site-Directed
dc.subjectBinding Sites
dc.subjectProtein Conformation
dc.subjectBlood Coagulation
dc.subjectModels, Molecular
dc.titleIdentification of serpins specific for activated protein C using a lysate-based screening assay.
dc.typeArticle
prism.issueIdentifier1
prism.publicationDate2018
prism.publicationNameSci Rep
prism.startingPage8793
prism.volume8
dc.identifier.doi10.17863/CAM.30394
dcterms.dateAccepted2018-05-18
rioxxterms.versionofrecord10.1038/s41598-018-27067-z
rioxxterms.licenseref.urihttp://www.rioxx.net/licenses/all-rights-reserved
rioxxterms.licenseref.startdate2018-06-08
dc.contributor.orcidHuntington, Jim [0000-0003-0076-7204]
dc.identifier.eissn2045-2322
rioxxterms.typeJournal Article/Review
cam.issuedOnline2018-06-08


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Attribution 4.0 International
Except where otherwise noted, this item's licence is described as Attribution 4.0 International