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

Abstract

BackgroundThrombin-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. ObjectivesTo establish a reliable assay that can monitor FXI-thrombin binding and is suitable for high throughput screening. MethodsA 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. ResultsThe 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. ConclusionsWe 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.