Fasxiator, a novel factor XIa inhibitor from snake venom, and its site‐specific mutagenesis to improve potency and selectivity

• Published in: Journal of thrombosis and haemostasis 2015-02, Vol.13 (2), p.248-261
• Main Authors: Chen, W., Carvalho, L. P. D., Chan, M. Y., Kini, R. M., Kang, T. S.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; anticoagulants; Binding, Competitive; Blood Coagulation - drug effects; Bungarotoxins - genetics; Bungarotoxins - isolation & purification; Bungarotoxins - metabolism; Bungarotoxins - pharmacology; Bungarus - metabolism; carotid artery thrombosis; Carotid Stenosis - blood; Carotid Stenosis - chemically induced; Carotid Stenosis - prevention & control; Chlorides; Disease Models, Animal; Dose-Response Relationship, Drug; Factor Xa Inhibitors - isolation & purification; Factor Xa Inhibitors - metabolism; Factor Xa Inhibitors - pharmacology; factor XIa; Factor XIa - metabolism; Ferric Compounds; Humans; Kinetics; Male; Mice, Inbred C57BL; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Partial Thromboplastin Time; protease inhibitors; Protein Conformation; Prothrombin Time; Recombinant Proteins - pharmacology; snake venom; Structure-Activity Relationship; Thrombosis - blood; Thrombosis - chemically induced; Thrombosis - prevention & control
• ISSN: 1538-7933; 1538-7836; 1538-7836
• DOI: 10.1111/jth.12797

Summary Background Bleeding remains a major limitation of standard anticoagulant drugs that target the extrinsic and common coagulation pathways. Recently, intrinsic coagulation factors are increasingly being investigated as alternative targets for developing anticoagulant drugs with lower bleeding risk. Objectives Goals were to (i) identify novel anticoagulants selectively targeting intrinsic coagulation pathway and (ii) characterize and further improve the properties of the identified anticoagulants. Methods and Results We have isolated and sequenced a specific factor XIa (FXIa) inhibitor, henceforth named Fasxiator, from the venom of the banded krait snake, Bungarus fasciatus. It is a Kunitz‐type protease inhibitor that prolonged activated partial thromboplastin time without significant effects on prothrombin time. Fasxiator was recombinantly expressed (rFasxiator), purified, and characterized to be a slow‐type inhibitor of FXIa that exerts its anticoagulant activities (doubled activated partial thromboplastin time at ~ 3 μmol L–1) by selectively inhibiting human FXIa in in vitro assays. A series of mutants were subsequently generated to improve the potency and selectivity of recombinant rFasxiator. rFasxiatorN17R,L19E showed the best balance between potency (IC50 ~ 1 nmol L–1) and selectivity (> 100 times). rFasxiatorN17R,L19E is a competitive slow‐type inhibitor of FXIa (Ki = 0.86 nmol L–1), possesses anticoagulant activity that is ~ 10 times stronger in human plasma than in murine plasma, and prolonged the occlusion time of mice carotid artery in FeCl3‐induced thrombosis models. Conclusion We have isolated an exogenous FXIa specific inhibitor, engineered it to improve its potency by ~ 1000 times and demonstrated its in vitro and in vivo efficacy. These proof‐of‐principle data supported the further development of Fasxiator as a novel anticoagulant candidate.