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

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

Author information

1
Center for Theoretical Problems of Physicochemical Pharmacology of Russian Academy of Sciences, Moscow, Russian Federation.
2
Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Moscow, Russian Federation.
3
Federal Research and Clinical Center of Pediatric Hematology, Oncology, and Immunology named after Dmitry Rogachev of Ministry of Health, Moscow, Russian Federation.
4
Center "Bioengineering" of Russian Academy of Sciences, Moscow, Russian Federation.
5
Waksman Institute of Microbiology, Rutgers The State University of New Jersey, Piscataway, New Jersey, United States of America.
6
Department of Physics, Lomonosov Moscow State University, Moscow, Russian Federation.
7
Faculty of Biological and Medical Physics, Moscow Institute of Physics and Technology, Dolgoprudny, Russian Federation.
8
HemaCore LLC., Moscow, Russian Federation.

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.

PMID:
26670620
PMCID:
PMC4684401
DOI:
10.1371/journal.pone.0144940
[Indexed for MEDLINE]
Free PMC Article

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