Localization of the single-stranded DNA binding site in the thrombin anion-binding exosite.
Single-stranded DNA molecules containing a 15-nucleotide consensus sequence have been reported to inhibit thrombin activity. The mechanism of the inhibition was studied using a consensus 15-mer oligonucleotide and two recombinant mutant thrombins: the anion-binding exosite mutant thrombin R70E, and thrombin K154A, in which the mutation was located in a surface loop outside of the exosite. The consensus 15-mer oligonucleotide inhibited both fibrinogen-clotting and platelet-activation activities of plasma-derived thrombin, recombinant wild type thrombin, and mutant thrombin K154A in a sequence-specific and dose-dependent manner, whereas it did not inhibit either activity of mutant thrombin R70E. The 15-mer oligonucleotide also inhibited thrombomodulin-dependent protein C activation by plasma-derived thrombin. In competition equilibrium binding experiments, binding of 125I-labeled diisopropyl phosphoryl-thrombin to thrombomodulin was completely inhibited by the consensus 15-mer oligonucleotide with a Kd value of 2.68 +/- 0.16 nM. These results suggest that Arg-70 in the anion-binding exosite of thrombin is a key determinant for interaction with specific single-stranded DNA molecules, and that binding of single-stranded DNA molecules to the exosite prevents the interaction of thrombin with fibrinogen, the platelet thrombin receptor, and thrombomodulin.
Wu
Q
Q
Howard Hughes Medical Institute, Jewish Hospital of St. Louis, Washington University School of Medicine, Missouri 63110.
Tsiang
M
M
Sadler
J E
JE
D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism* / Q000494:pharmacology; D015539:Platelet Activation / Q000187:drug effects; D011486:Protein C / Q000378:metabolism; D011516:Prothrombin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D018179:Receptors, Thrombin; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013917:Thrombin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D011516:Prothrombin; D005340:Fibrinogen; D013917:Thrombin