Paborsky L R, McCurdy S N, Griffin L C, Toole J J, Leung L L
Gilead Sciences, Inc., Foster City, California 94404.
J Biol Chem. 1993 Oct 5;268(28):20808-11.
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.
最近发现了一类基于序列特异性单链DNA寡核苷酸（凝血酶适体）的新型凝血酶抑制剂。通过固相板结合试验和化学修饰研究了凝血酶上的适体结合位点。结合试验结果表明，凝血酶适体特异性结合α-凝血酶，而不结合γ-凝血酶，水蛭素与适体结合竞争，这表明凝血酶的阴离子结合外位点对适体-凝血酶相互作用很重要。为了鉴定参与凝血酶适体结合的凝血酶赖氨酸残基，在有或没有凝血酶适体的情况下，用5'-异硫氰酸荧光素修饰凝血酶，还原、羧甲基化，并用内肽酶Arg-C消化。通过反相高效液相色谱分析消化产物，并比较肽图。鉴定出在适体存在下修饰显著减少的四种肽，并进行N端序列分析。结果表明，位于阴离子结合外位点内的B链Lys-21和Lys-65位于人α-凝血酶的适体结合位点内或紧邻该位点。凝血酶适体与阴离子结合外位点结合，并通过与外位点结合底物纤维蛋白原和血小板凝血酶受体竞争来抑制凝血酶的功能。
