Centre for Innovation, Canadian Blood Services, Hamilton, ON, Canada.
Department of Pathology and Molecular Medicine, McMaster University, HSC 4N66, 1280 Main Street West, Hamilton, ON, L8S 4L8, Canada.
Sci Rep. 2021 Mar 10;11(1):5565. doi: 10.1038/s41598-021-84618-7.
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.
凝血因子 XIa（FXIa）是抗血栓药物开发的新兴靶点。丝氨酸蛋白酶抑制剂 α-1 抗胰蛋白酶（AAT）的 M358R 变体可抑制 FXIa 和其他蛋白酶。我们的目的是增强 AAT M358R 对 FXIa 的特异性。我们在活性中心环位置 P13-P8 和 P7-P3 对两个 AAT M358R 噬菌体展示文库进行了随机化，并使用 FXIa 对其进行了生物淘选。还对 P2'-P3' 位置随机化的细菌表达文库进行了探测。由此产生的新型变体在大肠杆菌中表达为重组蛋白，并确定其对 FXIa 的抑制动力学。最有效的 FXIa 抑制基序为：P13-P8，HASTGQ；P7-P3，CLEVE；和 P2-P3'，PRSTE（分别加粗表示新的残基）。与 AAT M358R 相比，这些单基序变体对 FXIa 的选择性增加了 34 倍。在 AAT-RC 中结合 CLEVE 和 PRSTE 基序可使 FXIa 对凝血酶、因子 XIIa、Xa、活化蛋白 C 和激肽释放酶的选择性分别增加 279 倍、143 倍、63 倍、58 倍和 36 倍，而对 AAT M358R 的选择性则增加了 34 倍。AAT-RC 比 AAT M358R 更能延长人血浆凝固时间。AAT-RC 能快速且选择性地抑制 FXIa，值得在体内进行测试。通过噬菌体和细菌系统的选择与组合诱变相结合，可将 AAT 的特异性集中在一个靶标蛋白酶上。
