Monitoring complex formation in the blood-coagulation cascade using aptamer-coated SAW sensors.
Specific binding of the anticoagulants heparin and antithrombin III to the blood clotting cascade factor human thrombin was recorded as a function of time with a Love-wave biosensor array consisting of five sensor elements. Two of the sensor elements were used as references. Three sensor elements were coated with RNA or DNA aptamers for specific binding of human thrombin. The affinity between the aptamers and thrombin, measured using the biosensor, was within the same range as the value of K(D) measured by filter binding experiments. Consecutive binding of the thrombin inhibitors heparin, antithrombin III or the heparin-antithrombin III complex to the immobilized thrombin molecules, and binding of a ternary complex of heparin, anithrombin III, and thrombin to aptamers was evaluated. The experiments showed attenuation of binding to thrombin due to heparin-antithrombin III complex formation. Binding of heparin activated the formation of the inhibitory complex of antithrombin III with thrombin about 2.7-fold. Binding of the DNA aptamer to exosite II appeared to inhibit heparin binding to exosite I.
Gronewold
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TM
Center of Advanced European Studies and Research, Aptamer Biosensors, Ludwig-Erhard-Allee 2, 53175 Bonn, Germany.
Glass
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Quandt
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Famulok
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D000162:Acoustics / Q000295:instrumentation*; D000990:Antithrombin III / Q000737:chemistry; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D001777:Blood Coagulation / Q000502:physiology*; D001780:Blood Coagulation Tests / Q000295:instrumentation / Q000379:methods; D003199:Computer Systems; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006493:Heparin / Q000737:chemistry; D006801:Humans; D009841:Oligonucleotides / Q000737:chemistry; D025941:Protein Interaction Mapping / Q000295:instrumentation* / Q000379:methods; D013917:Thrombin / Q000032:analysis* / Q000737:chemistry*; D013918:Thrombin Time / Q000295:instrumentation* / Q000379:methods
D009841:Oligonucleotides; D000990:Antithrombin III; D006493:Heparin; D013917:Thrombin