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A FRET-based assay for the quantitation of the thrombin-factor XI interaction
Awital Bar Barroeta1, J Arnoud Marquart1, Joost C M Meijers2
1Department of Molecular Hematology, Sanquin Research, Amsterdam, the Netherlands.
Thrombosis Research
|April 22, 2022
Summary
Researchers developed a new assay to measure how thrombin and Factor XI (FXI) bind. This assay can be used for high-throughput screening to find drugs that may help with bleeding disorders.
Area of Science:
- Biochemistry
- Biophysics
- Pharmacology
Background:
- Thrombin activates Factor XI (FXI), which is crucial for blood clot stability and resistance to fibrinolysis.
- Enhancing this FXI activation could benefit patients with bleeding disorders, such as those with FXI deficiency.
Purpose of the Study:
- To develop a robust, high-throughput screening assay for monitoring thrombin-FXI binding.
Main Methods:
- A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was established to quantify thrombin-FXI interaction.
- Assay robustness was tested by varying NaCl, MgCl2, and DMSO concentrations.
- Signal stability was assessed through multiple freeze-thaw cycles.
Main Results:
- The TR-FRET assay demonstrated a stable signal suitable for high-throughput screening.
- The assay showed sensitivity and robustness.
- Modulation of the thrombin-FXI interaction by non-labeled FXI was successfully measured.
Conclusions:
- A validated TR-FRET assay for quantifying the thrombin-FXI interaction has been developed.
- The assay is suitable for high-throughput screening.
- This assay can be utilized to identify small molecules that modulate the thrombin-FXI interaction.
