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New Infestin-4 Mutants with Increased Selectivity against Factor XIIa
Vladimir N Kolyadko1, Sofya V Lushchekina2, Tatiana A Vuimo3
1Center for Theoretical Problems of Physicochemical Pharmacology of Russian Academy of Sciences, Moscow, Russian Federation.
Plos One
|December 17, 2015
Summary
Infestin-4 mutants selectively inhibit factor XIIa (fXIIa), a key player in thrombosis and coagulation testing. This engineered inhibitor shows promise for diagnostic applications by specifically targeting the contact pathway without affecting tissue factor-initiated coagulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- Factor XIIa (fXIIa) is a serine protease initiating the coagulation contact pathway, implicated in thrombosis.
- Inhibition of fXIIa is crucial for accurate coagulation testing and managing thrombotic conditions.
- Infestin-4 (Inf4) is a Kazal-type inhibitor of fXIIa, with potential for enhanced specificity through protein engineering.
Purpose of the Study:
- To engineer Infestin-4 (Inf4) mutants for selective inhibition of fXIIa.
- To assess the efficacy of these mutants in repressing the contact pathway under various in vitro conditions.
- To evaluate the potential of engineered Inf4 as a diagnostic reagent in coagulation assays.
Main Methods:
- Design and molecular dynamics simulation of novel Inf4 mutants with stabilized conformations.
- Chromogenic assays to determine off-target activities against proteases like fXa and plasmin.
- Protein-protein docking to analyze interactions with fXIIa and fXa.
- In vitro testing of potent mutants in plasma using thromboelastography and thrombodynamics assays.
Main Results:
- Engineered Inf4 mutants exhibited reduced or eliminated off-target activities against other coagulation proteases.
- Mutant B demonstrated high potency (Ki for fXIIa = 0.7 nM) and selectivity.
- Mutant B (5-20 μM) selectively delayed contact-activated thrombin generation and clotting in global assays without affecting tissue factor-initiated coagulation.
Conclusions:
- Engineered Inf4 mutants offer enhanced specificity for fXIIa inhibition.
- Mutant B shows significant potential as a selective reagent for the repression of the contact pathway.
- These findings highlight the practical significance of engineered Inf4 for coagulation diagnostics and research.
