Monitoring complex formation in the blood-coagulation cascade using aptamer-coated SAW sensors

Area of Science:

• Biochemistry
• Biomolecular Engineering
• Biophysics

Background:

• Human thrombin is a key factor in the blood clotting cascade.
• Anticoagulants like heparin and antithrombin III are crucial for regulating blood coagulation.
• Understanding their specific interactions is vital for developing effective antithrombotic therapies.

Purpose of the Study:

• To quantify the binding affinity of aptamers to human thrombin using a Love-wave biosensor.
• To investigate the sequential binding interactions of anticoagulants with immobilized human thrombin.
• To evaluate the impact of anticoagulant complex formation on thrombin binding to aptamers.

Main Methods:

• Utilized a Love-wave biosensor array with aptamer-functionalized sensor elements for real-time binding analysis.
• Measured the binding kinetics and affinity of human thrombin to RNA and DNA aptamers.
• Assessed the binding of heparin, antithrombin III, and their complex to immobilized thrombin and aptamer-thrombin complexes.

Main Results:

• Aptamer affinity for human thrombin was comparable to values obtained from filter binding assays.
• Heparin-antithrombin III complex formation was observed to reduce binding to immobilized thrombin.
• Heparin binding to exosite I of thrombin was inhibited by DNA aptamer binding to exosite II.
• Heparin binding activated antithrombin III-thrombin inhibitory complex formation approximately 2.7-fold.

Conclusions:

• Love-wave biosensors provide a sensitive platform for studying anticoagulant-thrombin interactions.
• Aptamers can be designed to modulate the binding of anticoagulants to human thrombin.
• These findings offer insights into the complex regulatory mechanisms of blood coagulation and potential therapeutic strategies.