New Infestin-4 Mutants with Increased Selectivity against Factor XIIa.

Abstract

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.

• Authors: Vladimir N Kolyadko, Sofya V Lushchekina, Tatiana A Vuimo, Stepan S Surov, Ruzanna A Ovsepyan, Vera A Korneeva, Ivan I Vorobiev, Nadezhda A Orlova, Leonid Minakhin, Konstantin Kuznedelov, Konstantin V Severinov, Fazoil I Ataullakhanov, Mikhail A Panteleev
• Journal: PloS one (PLoS One) Vol. 10 Issue 12 Pg. e0144940 ( 2015) ISSN: 1932-6203 [Electronic] United States
• PMID: 26670620 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

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

Topics

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