title: WEDGE: an anticoagulant thrombin mutant produced by autoactivation.
abstract: BACKGROUND
  The production of therapeutically relevant proteases typically involves activation of a zymogen precursor by external enzymes, which may raise regulatory issues about availability and purity. Recent studies of thrombin precursors have shown how to engineer constructs that spontaneously convert to the mature protease by autoactivation, without the need for external enzymes.
  
  
  OBJECTIVE
  Autoactivation is an innovative strategy that promises to simplify the production of proteases of therapeutic relevance, but has not been tested in practical applications. The aim of this study was to provide a direct test of this strategy.
  
  
  METHODS
  An autoactivating version of the thrombin mutant W215A/E217A (WE), which is currently in preclinical development as an anticoagulant, was engineered.
  
  
  CONCLUSIONS
  The autoactivating version of WE can be produced in large quantities, like WE made in BHK cells or Escherichia coli, and retains all significant functional properties in vitro and in vivo. The results serve as proof of principle that autoactivation is an innovative and effective strategy for the production of trypsin-like proteases of therapeutic relevance.
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO, USA.
authors.forename: D C
authors.initials: DC
authors.lastname: Wood
authors.forename: L A
authors.initials: LA
authors.lastname: Pelc
authors.forename: N
authors.initials: N
authors.lastname: Pozzi
authors.forename: M
authors.initials: M
authors.lastname: Wallisch
authors.forename: N G
authors.initials: NG
authors.lastname: Verbout
authors.forename: E I
authors.initials: EI
authors.lastname: Tucker
authors.forename: A
authors.initials: A
authors.lastname: Gruber
authors.forename: E
authors.initials: E
authors.lastname: Di Cera
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000925:Anticoagulants / Q000008:administration & dosage / Q000378:metabolism*; D001777:Blood Coagulation / Q000187:drug effects; D002384:Catalysis; D004789:Enzyme Activation; D007275:Injections, Intravenous; D009154:Mutation; D010215:Papio; D010314:Partial Thromboplastin Time; D015202:Protein Engineering / Q000379:methods*; D011516:Prothrombin / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D013917:Thrombin / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000925:Anticoagulants; D011994:Recombinant Proteins; D011516:Prothrombin; C012007:prethrombins; D013917:Thrombin
keywords: anticoagulants; blood coagulation factors; protein engineering; thrombin; zymogens