New Infestin-4 Mutants with Increased Selectivity against Factor XIIa

PLoS One. 2015 Dec 15;10(12):e0144940. doi: 10.1371/journal.pone.0144940. eCollection 2015.


Factor XIIa (fXIIa) is a serine protease that triggers the coagulation contact pathway and plays a role in thrombosis. Because it interferes with coagulation testing, the need to inhibit fXIIa exists in many cases. Infestin-4 (Inf4) is a Kazal-type inhibitor of fXIIa. Its specificity for fXIIa can be enhanced by point mutations in the protease-binding loop. We attempted to adapt Inf4 for the selective repression of the contact pathway under various in vitro conditions, e.g., during blood collection and in 'global' assays of tissue factor (TF)-dependent coagulation. First, we designed a set of new Inf4 mutants that, in contrast to wt-Inf4, had stabilized canonical conformations during molecular dynamics simulation. Off-target activities against factor Xa (fXa), plasmin, and other coagulation proteases were either reduced or eliminated in these recombinant mutants, as demonstrated by chromogenic assays. Interactions with fXIIa and fXa were also analyzed using protein-protein docking. Next, Mutant B, one of the most potent mutants (its Ki for fXIIa is 0.7 nM) was tested in plasma. At concentrations 5-20 μM, this mutant delayed the contact-activated generation of thrombin, as well as clotting in thromboelastography and thrombodynamics assays. In these assays, Mutant B did not affect coagulation initiated by TF, thus demonstrating sufficient selectivity and its potential practical significance as a reagent for coagulation diagnostics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Blood Coagulation / drug effects
  • Drug Design
  • Factor XIIa / antagonists & inhibitors*
  • Factor XIIa / metabolism
  • Factor Xa / metabolism
  • Humans
  • Insect Proteins / chemistry
  • Insect Proteins / genetics
  • Insect Proteins / pharmacology*
  • Kinetics
  • Molecular Sequence Data
  • Mutant Proteins / pharmacology*
  • Plant Proteins / pharmacology
  • Protease Inhibitors / pharmacology
  • Protein Binding / drug effects
  • Substrate Specificity
  • Thioredoxins / metabolism


  • Insect Proteins
  • Mutant Proteins
  • Plant Proteins
  • Protease Inhibitors
  • infestin protein, Triatoma infestans
  • trypsin inhibitor, Zea mays
  • Thioredoxins
  • Factor XIIa
  • Factor Xa

Grant support

The project was supported by Russian Science Foundation grant 14-14-00195 to MAP, with the exception of all molecular dynamics and docking studies that were supported by Dynasty Foundation Fellowship to SVL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. HemaCore LLC provided support in the form of salaries for authors VNK, TAV, SSS, RAO, VAK, FIA, MAP, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.