-transparent.png){kind=logo}
A new class of thrombin inhibitors based on sequence-specific single-stranded DNA oligonucleotides (thrombin aptamer) has recently been identified. The aptamer-binding site on thrombin was examined by a solid-phase plate binding assay and by chemical modification. Binding assay results demonstrated that the thrombin aptamer bound specifically to alpha-thrombin but not to gamma-thrombin and that hirudin competed with aptamer binding, suggesting that thrombin's anion-binding exosite was important for aptamer-thrombin interactions. To identify lysine residues of thrombin that participated in the binding of the thrombin aptamer, thrombin was modified with fluorescein 5'-isothiocyanate in the presence or absence of the thrombin aptamer, reduced, carboxymethylated, and digested with endoproteinase Arg-C. The digestion products were analyzed by reversed-phase high performance liquid chromatography and the peptide maps compared. Four peptides with significantly decreased modification in the presence of the aptamer were identified and subjected to N-terminal sequence analysis. Results indicated that B chain Lys-21 and Lys-65, both located within the anion-binding exosite, are situated within or in close proximity to the aptamer-binding site of human alpha-thrombin. The thrombin aptamer binds to the anion-binding exosite and inhibits thrombin's function by competing with exosite binding substrates fibrinogen and the platelet thrombin receptor.
L R Paborsky, and
S N McCurdy, and
L C Griffin, and
J J Toole, and
L L Leung
December 1992,
The Journal of biological chemistry,
L R Paborsky, and
S N McCurdy, and
L C Griffin, and
J J Toole, and
L L Leung
August 1993,
The Journal of biological chemistry,
L R Paborsky, and
S N McCurdy, and
L C Griffin, and
J J Toole, and
L L Leung
January 1998,
Molecular pharmacology,
L R Paborsky, and
S N McCurdy, and
L C Griffin, and
J J Toole, and
L L Leung
March 2012,
The Analyst,
L R Paborsky, and
S N McCurdy, and
L C Griffin, and
J J Toole, and
L L Leung
September 2010,
Biochemistry,
L R Paborsky, and
S N McCurdy, and
L C Griffin, and
J J Toole, and
L L Leung
March 2001,
The Journal of biological chemistry,
L R Paborsky, and
S N McCurdy, and
L C Griffin, and
J J Toole, and
L L Leung
August 2011,
Biochimie,
L R Paborsky, and
S N McCurdy, and
L C Griffin, and
J J Toole, and
L L Leung
February 1992,
Nature,
L R Paborsky, and
S N McCurdy, and
L C Griffin, and
J J Toole, and
L L Leung
January 2009,
Methods in molecular biology (Clifton, N.J.),
L R Paborsky, and
S N McCurdy, and
L C Griffin, and
J J Toole, and
L L Leung
January 2014,
Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis,
