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The single-stranded DNA aptamer-binding site of human thrombin
L R Paborsky1, S N McCurdy, L C Griffin
1Gilead Sciences, Inc., Foster City, California 94404.
The Journal of Biological Chemistry
|October 5, 1993
Summary
New DNA aptamers specifically target alpha-thrombin, inhibiting its function. These thrombin aptamers bind to the anion-binding exosite, blocking interactions with fibrinogen and platelet receptors.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Thrombin is a key enzyme in hemostasis.
- Aptamers are short DNA or RNA molecules that bind to specific targets.
- Thrombin inhibitors are crucial for treating thrombotic disorders.
Purpose of the Study:
- To characterize the binding site of a novel thrombin aptamer on alpha-thrombin.
- To elucidate the mechanism by which thrombin aptamers inhibit thrombin activity.
Main Methods:
- Solid-phase plate binding assays to determine binding specificity.
- Chemical modification of thrombin with fluorescein isothiocyanate.
- Enzymatic digestion and peptide mapping using reversed-phase high-performance liquid chromatography.
- N-terminal sequence analysis of modified peptides.
Main Results:
- The thrombin aptamer specifically binds to alpha-thrombin, not gamma-thrombin.
- Hirudin competed with aptamer binding, indicating the involvement of the anion-binding exosite.
- Lysine residues Lys-21 and Lys-65 in the B chain were identified as being near the aptamer-binding site.
- The aptamer inhibits thrombin by competing with fibrinogen and the platelet thrombin receptor for the anion-binding exosite.
Conclusions:
- Thrombin aptamers bind to the anion-binding exosite of alpha-thrombin.
- This binding interaction inhibits thrombin's enzymatic activity.
- Thrombin aptamers represent a promising new class of anticoagulants.