Highly selective and rapidly activatable fluorogenic Thrombin sensors and application in human lung tissue.

Area of Science:

• Biochemistry
• Enzyme kinetics
• Fluorescence spectroscopy

Background:

• Thrombin is a critical enzyme in coagulation and other physiological processes.
• Developing selective and stable probes for thrombin activity is essential for research and diagnostics.
• Existing probes may lack specificity or stability in complex biological environments.

Purpose of the Study:

• To design and synthesize Förster Resonance Energy Transfer (FRET)-based peptide substrates for thrombin.
• To identify probes with high selectivity for thrombin and resistance to other proteases.
• To develop optimized probes for detecting thrombin activity in ex vivo biological samples.

Main Methods:

• Synthesis of a library of FRET-based peptides.
• Enzyme activity assays using thrombin, Factor Xa, and matrix metalloproteases.
• Evaluation of probe stability in human inflammatory cell lysates and supernatant.
• Optimization of probe properties, including solubility and resistance to plasmin.
• Application of the optimized probe for detecting thrombin in ex vivo fibrotic human tissue.

Main Results:

• Identified FRET-based probes with selective activation by thrombin.
• Probes exhibited low fluorescent background signals and stability against Factor Xa and MMPs.
• A second generation of probes showed improved solubility and Plasmin resistance.
• The optimized probe successfully detected thrombin activity in ex vivo fibrotic human tissue.

Conclusions:

• Novel FRET-based peptide probes were successfully developed for selective thrombin detection.
• These probes offer improved stability and performance compared to existing methods.
• The optimized probe is a valuable tool for investigating thrombin activity in complex biological contexts, such as fibrotic diseases.