title: 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.affiliation: Center for Theoretical Problems of Physicochemical Pharmacology of Russian Academy of Sciences, Moscow, Russian Federation.
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Kolyadko
authors.affiliation: Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Moscow, Russian Federation.
authors.forename: Sofya V
authors.initials: SV
authors.lastname: Lushchekina
authors.affiliation: Federal Research and Clinical Center of Pediatric Hematology, Oncology, and Immunology named after Dmitry Rogachev of Ministry of Health, Moscow, Russian Federation.
authors.forename: Tatiana A
authors.initials: TA
authors.lastname: Vuimo
authors.affiliation: Center for Theoretical Problems of Physicochemical Pharmacology of Russian Academy of Sciences, Moscow, Russian Federation.
authors.forename: Stepan S
authors.initials: SS
authors.lastname: Surov
authors.affiliation: Federal Research and Clinical Center of Pediatric Hematology, Oncology, and Immunology named after Dmitry Rogachev of Ministry of Health, Moscow, Russian Federation.
authors.forename: Ruzanna A
authors.initials: RA
authors.lastname: Ovsepyan
authors.affiliation: Center for Theoretical Problems of Physicochemical Pharmacology of Russian Academy of Sciences, Moscow, Russian Federation.
authors.forename: Vera A
authors.initials: VA
authors.lastname: Korneeva
authors.affiliation: Center "Bioengineering" of Russian Academy of Sciences, Moscow, Russian Federation.
authors.forename: Ivan I
authors.initials: II
authors.lastname: Vorobiev
authors.affiliation: Center "Bioengineering" of Russian Academy of Sciences, Moscow, Russian Federation.
authors.forename: Nadezhda A
authors.initials: NA
authors.lastname: Orlova
authors.affiliation: Waksman Institute of Microbiology, Rutgers The State University of New Jersey, Piscataway, New Jersey, United States of America.
authors.forename: Leonid
authors.initials: L
authors.lastname: Minakhin
authors.affiliation: Waksman Institute of Microbiology, Rutgers The State University of New Jersey, Piscataway, New Jersey, United States of America.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Kuznedelov
authors.affiliation: Waksman Institute of Microbiology, Rutgers The State University of New Jersey, Piscataway, New Jersey, United States of America.
authors.forename: Konstantin V
authors.initials: KV
authors.lastname: Severinov
authors.affiliation: Center for Theoretical Problems of Physicochemical Pharmacology of Russian Academy of Sciences, Moscow, Russian Federation.|Federal Research and Clinical Center of Pediatric Hematology, Oncology, and Immunology named after Dmitry Rogachev of Ministry of Health, Moscow, Russian Federation.|Department of Physics, Lomonosov Moscow State University, Moscow, Russian Federation.|Faculty of Biological and Medical Physics, Moscow Institute of Physics and Technology, Dolgoprudny, Russian Federation.|HemaCore LLC., Moscow, Russian Federation.
authors.forename: Fazoil I
authors.initials: FI
authors.lastname: Ataullakhanov
authors.affiliation: Center for Theoretical Problems of Physicochemical Pharmacology of Russian Academy of Sciences, Moscow, Russian Federation.|Federal Research and Clinical Center of Pediatric Hematology, Oncology, and Immunology named after Dmitry Rogachev of Ministry of Health, Moscow, Russian Federation.|Department of Physics, Lomonosov Moscow State University, Moscow, Russian Federation.|Faculty of Biological and Medical Physics, Moscow Institute of Physics and Technology, Dolgoprudny, Russian Federation.|HemaCore LLC., Moscow, Russian Federation.
authors.forename: Mikhail A
authors.initials: MA
authors.lastname: Panteleev
mesh_terms: D000595:Amino Acid Sequence; D001777:Blood Coagulation / Q000187:drug effects; D015195:Drug Design; D015956:Factor XIIa / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000494:pharmacology*; D010940:Plant Proteins / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D013379:Substrate Specificity; D013879:Thioredoxins / Q000378:metabolism
chemical_list: D019476:Insect Proteins; D050505:Mutant Proteins; D010940:Plant Proteins; D011480:Protease Inhibitors; C467902:infestin protein, Triatoma infestans; C052160:trypsin inhibitor, Zea mays; D013879:Thioredoxins; D015956:Factor XIIa; D015951:Factor Xa