title: Fasxiator, a novel factor XIa inhibitor from snake venom, and its site-specific mutagenesis to improve potency and selectivity.
abstract: 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.
  
  
  OBJECTIVE
  Goals were to (i) identify novel anticoagulants selectively targeting intrinsic coagulation pathway and (ii) characterize and further improve the properties of the identified anticoagulants.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pharmacy, National University of Singapore, Singapore City, Singapore.
authors.forename: W
authors.initials: W
authors.lastname: Chen
authors.forename: L P D
authors.initials: LP
authors.lastname: Carvalho
authors.forename: M Y
authors.initials: MY
authors.lastname: Chan
authors.forename: R M
authors.initials: RM
authors.lastname: Kini
authors.forename: T S
authors.initials: TS
authors.lastname: Kang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D001777:Blood Coagulation / Q000187:drug effects*; D002038:Bungarotoxins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D017838:Bungarus / Q000378:metabolism*; D016893:Carotid Stenosis / Q000097:blood / Q000139:chemically induced / Q000517:prevention & control*; D002712:Chlorides; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D015945:Factor XIa / Q000378:metabolism; D065427:Factor Xa Inhibitors / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D005290:Ferric Compounds; D006801:Humans; D007700:Kinetics; D008297:Male; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010314:Partial Thromboplastin Time; D011487:Protein Conformation; D011517:Prothrombin Time; D011994:Recombinant Proteins / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013927:Thrombosis / Q000097:blood / Q000139:chemically induced / Q000517:prevention & control*
chemical_list: D002038:Bungarotoxins; D002712:Chlorides; D065427:Factor Xa Inhibitors; D005290:Ferric Compounds; D011994:Recombinant Proteins; C000602467:fasxiator, Bungarus fasciatus; D015945:Factor XIa; C024555:ferric chloride
keywords: anticoagulants; carotid artery thrombosis; factor XIa; protease inhibitors; snake venom