PMID- 33692375 OWN - NLM STAT- MEDLINE DCOM- 20211208 LR - 20211214 IS - 2045-2322 (Electronic) IS - 2045-2322 (Linking) VI - 11 IP - 1 DP - 2021 Mar 10 TI - Identification of an alpha-1 antitrypsin variant with enhanced specificity for factor XIa by phage display, bacterial expression, and combinatorial mutagenesis. PG - 5565 LID - 10.1038/s41598-021-84618-7 [doi] LID - 5565 AB - Coagulation Factor XIa (FXIa) is an emerging target for antithrombotic agent development. The M358R variant of the serpin alpha-1 antitrypsin (AAT) inhibits both FXIa and other proteases. Our aim was to enhance the specificity of AAT M358R for FXIa. We randomized two AAT M358R phage display libraries at reactive centre loop positions P13-P8 and P7-P3 and biopanned them with FXIa. A bacterial expression library randomized at P2'-P3' was also probed. Resulting novel variants were expressed as recombinant proteins in E. coli and their kinetics of FXIa inhibition determined. The most potent FXIa-inhibitory motifs were: P13-P8, HASTGQ; P7-P3, CLEVE; and P2-P3', PRSTE (respectively, novel residues bolded). Selectivity for FXIa over thrombin was increased up to 34-fold versus AAT M358R for these single motif variants. Combining CLEVE and PRSTE motifs in AAT-RC increased FXIa selectivity for thrombin, factors XIIa, Xa, activated protein C, and kallikrein by 279-, 143-, 63-, 58-, and 36-fold, respectively, versus AAT M358R. AAT-RC lengthened human plasma clotting times less than AAT M358R. AAT-RC rapidly and selectively inhibits FXIa and is worthy of testing in vivo. AAT specificity can be focused on one target protease by selection in phage and bacterial systems coupled with combinatorial mutagenesis. FAU - Bhakta, Varsha AU - Bhakta V AD - Centre for Innovation, Canadian Blood Services, Hamilton, ON, Canada. FAU - Hamada, Mostafa AU - Hamada M AD - Department of Pathology and Molecular Medicine, McMaster University, HSC 4N66, 1280 Main Street West, Hamilton, ON, L8S 4L8, Canada. FAU - Nouanesengsy, Amy AU - Nouanesengsy A AD - Department of Pathology and Molecular Medicine, McMaster University, HSC 4N66, 1280 Main Street West, Hamilton, ON, L8S 4L8, Canada. FAU - Lapierre, Jessica AU - Lapierre J AD - Department of Pathology and Molecular Medicine, McMaster University, HSC 4N66, 1280 Main Street West, Hamilton, ON, L8S 4L8, Canada. FAU - Perruzza, Darian L AU - Perruzza DL AD - Department of Pathology and Molecular Medicine, McMaster University, HSC 4N66, 1280 Main Street West, Hamilton, ON, L8S 4L8, Canada. FAU - Sheffield, William P AU - Sheffield WP AD - Centre for Innovation, Canadian Blood Services, Hamilton, ON, Canada. sheffiel@mcmaster.ca. AD - Department of Pathology and Molecular Medicine, McMaster University, HSC 4N66, 1280 Main Street West, Hamilton, ON, L8S 4L8, Canada. sheffiel@mcmaster.ca. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20210310 PL - England TA - Sci Rep JT - Scientific reports JID - 101563288 RN - 0 (Peptide Library) RN - 0 (Recombinant Proteins) RN - 0 (SERPINA1 protein, human) RN - 0 (alpha 1-Antitrypsin) RN - EC 3.4.21.27 (Factor XIa) SB - IM MH - Escherichia coli/chemistry/genetics/metabolism MH - Factor XIa/*chemistry MH - *Gene Expression MH - Humans MH - *Mutagenesis MH - *Peptide Library MH - Recombinant Proteins/biosynthesis/chemistry/genetics MH - alpha 1-Antitrypsin/biosynthesis/*chemistry/genetics PMC - PMC7946950 COIS- The authors declare no competing interests. EDAT- 2021/03/12 06:00 MHDA- 2021/12/15 06:00 PMCR- 2021/03/10 CRDT- 2021/03/11 06:15 PHST- 2020/11/03 00:00 [received] PHST- 2021/02/15 00:00 [accepted] PHST- 2021/03/11 06:15 [entrez] PHST- 2021/03/12 06:00 [pubmed] PHST- 2021/12/15 06:00 [medline] PHST- 2021/03/10 00:00 [pmc-release] AID - 10.1038/s41598-021-84618-7 [pii] AID - 84618 [pii] AID - 10.1038/s41598-021-84618-7 [doi] PST - epublish SO - Sci Rep. 2021 Mar 10;11(1):5565. doi: 10.1038/s41598-021-84618-7.