Abstract Number: PB0712
Meeting: ISTH 2020 Congress
Background: Factor XIII (FXIII) is activated by thrombin to catalyze covalent cross-linking formation between fibrin molecules, making clot stable and resistant to fibrinolysis. While FXIII is expected to be a novel target of therapeutic anticoagulation, FXIII reportedly prevents pulmonary emboli in mice by stabilizing deep vein thrombi. It remains to be elucidated how suppression of FXIII-mediated mechanisms influences thrombosis. Basic aspects of inhibition by direct thrombin inhibitors (DTIs) of FXIII activation are expected to provide insights into the concern.
Aims: The present study aims to characterize effects of DTIs on FXIII activation from the perspectives of enzyme kinetics.
Methods: The synthetic substrate-based assay for FXIII activity quantification was applied to analyses based on enzyme kinetics. The Lineweaver-Burk plot and the Dixon plot were used to assess inhibition types. The Hill plot using drug concentrations was subjected to calculation of Hill coefficients measuring inhibition cooperativity as well as half maximal inhibitory concentrations (IC50). The Hill plot using FXIII concentrations was subjected to calculation of Hill coefficients measuring substrate cooperativity.
Results: According to combination of observations from the Lineweaver-Buk plot with observations about how FXIII concentrations impact the IC50 of each DTI, dabigatran and argatroban exhibited non-competitive inhibition or mixed inhibition mimicking non-competitive binding while hirudin and bivalirudin exhibited mixed inhibition mimicking uncompetitive binding. Dabigatran and argatroban exhibited positive inhibition cooperativity while bivalirudin exhibited negative inhibition cooperativity. The Hill plot analysis was inapplicable to the dose-response curve of hirudin putatively because of its irreversibility. Hill coefficients calculated using FXIII concentrations were around 1 with or without DTIs, proving that DTIs do not induce positive or negative substrate cooperativity of FXIII.
Conclusions: The results revealed the properties of effects of DTIs on FXIII activation from the perspective of enzyme kinetics, providing insights into the relevance of FXIII-mediated mechanisms to thrombosis.
To cite this abstract in AMA style:Fujimori Y, Wakui M, Nakamura S, Kondo Y, Ozaki Y, Oka S, Nakagawa T, Katagiri H, Murata M. Assessment of in vitro Effects of Direct Thrombin Inhibitors on Activation of Coagulation Factor XIII through Enzyme Kinetics in the Synthetic Substrate-Based Assay [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/assessment-of-in-vitro-effects-of-direct-thrombin-inhibitors-on-activation-of-coagulation-factor-xiii-through-enzyme-kinetics-in-the-synthetic-substrate-based-assay/. Accessed May 6, 2021.
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