Abstract
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.
Highlights
Coagulation Factor XIa (FXIa) is an emerging target for antithrombotic agent development
We previously showed that the lists of the most abundant sequences identified via sequencing of selected clones or deep sequencing exhibited considerable overlap, in that case for the alpha-1 antitrypsin (AAT) P7-P3 library probed with thrombin[39]
This study was prompted by the unmet clinical needs for FXIa inhibition and by our previous demonstrations that: novel variants of AAT M358R could be selected using phage d isplay[39] or lysate s creening[41]; and that AAT M358R-expressing T7 phages could be selected by FXIa-mediated biopanning from a population of phages not expressing this serpin[38]
Summary
Coagulation Factor XIa (FXIa) is an emerging target for antithrombotic agent development. 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. Circulating levels of FXI can be reduced by an antisense oligonucleotide agent specific for FXI mRNA; a phase 2 trial demonstrated that this approach was superior to low molecular weight heparin in preventing thrombosis and did not increase bleeding in patients undergoing knee r eplacements[17]. It takes several weeks for the antisense approach to reduce FXI levels in the circulation.
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