The single-stranded DNA aptamer-binding site of human thrombin.
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.
Paborsky
L R
LR
Gilead Sciences, Inc., Foster City, California 94404.
McCurdy
S N
SN
Griffin
L C
LC
Toole
J J
JJ
Leung
L L
LL
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D006629:Hirudins / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000032:analysis; D008969:Molecular Sequence Data; D013917:Thrombin / Q000378:metabolism*
D004277:DNA, Single-Stranded; D006629:Hirudins; D013917:Thrombin; D016650:Fluorescein-5-isothiocyanate; D008239:Lysine