A FRET-based assay for the quantitation of the thrombin-factor XI interaction

Thrombin-mediated activation of FXI supports clot stability and protects the clot from fibrinolysis. This generally poor activation could be enhanced to stimulate coagulation, which might serve patients experiencing bleeding, for example due to FXI deficiency. To establish a reliable assay that can...

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Bibliographic Details
Published in:Thrombosis research 2022-06, Vol.214, p.23-28
Main Authors: Bar Barroeta, Awital, Marquart, J. Arnoud, Meijers, Joost C.M.
Format: Article
Language:English
Subjects:
Bleeding disorder
Blood Coagulation Tests
Factor XI
Factor XI - metabolism
Factor XI Deficiency
Fluorescence Resonance Energy Transfer
High-throughput screening
Humans
Thrombin
Thrombin - metabolism
TR-FRET
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Summary:Thrombin-mediated activation of FXI supports clot stability and protects the clot from fibrinolysis. This generally poor activation could be enhanced to stimulate coagulation, which might serve patients experiencing bleeding, for example due to FXI deficiency. To establish a reliable assay that can monitor FXI-thrombin binding and is suitable for high throughput screening. A time-resolved fluorescence resonance energy transfer assay was set up to measure binding between FXI and thrombin in a dose-dependent manner. This assay was subjected to varying concentration of NaCl, MgCl2, and DMSO to test the robustness of the output signal. Moreover, the stability of the signal was tested after going through various freeze-thaw cycles. The assay produces a stable signal that meets the sensitivity and robustness criteria for application in high-throughput screening. Moreover, it was possible to measure modulation of the interaction with non-labelled FXI. We have established and validated a time-resolved fluorescence resonance energy transfer assay that can quantify the thrombin-FXI interaction. We propose that the assay is compatible with high-throughput screening. Thus, the assay could be used to screen for small molecules that interfere with the interaction on a high-throughput scale.
ISSN:0049-3848
1879-2472
DOI:10.1016/j.thromres.2022.04.006