Localization of the single-stranded DNA binding site in the thrombin anion-binding exosite.

Q Wu,M Tsiang,J.E Sadler

doi:10.1016/s0021-9258(18)35781-8

Q Wu, M Tsiang + Show 1 more

Open Access

https://doi.org/10.1016/s0021-9258(18)35781-8

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Abstract

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.

Highlights

One of the unique features of thrombin structure is the presence of many basic amino acid residues within surface loops (12), especially ina region called the anion-binding exosite (1).These positively charged residues contribute significantly to the specificity of thrombin activity
To determine whether the DNA molecules bind to the thrombin anion-binding exosite, the effects were examined of the consensus 15-mer oligonucleotide on wild type thrombin and on mutant thrombinswith single amino acid substitutions in or near the anion-binding exosite
The results suggest that the single-stranded DNA binding site is located in the thrombin anion-binding exosite and overlaps with the binding sites for fibrinogen, the platelet thrombin receptor, and thrombomodulin

Summary

1To whom correspondence should be addressed Howard Hughes

Materials-Recombinant human thrombomodulin was expressed on the surfaceof stably transfected CV-l(l8A) or CV-l(TMncc)ells, as described previously (24, 27). Human thrombin was from Haematologic Technologies Inc., Essex Junction, VT. Recombinant human protein C was kindlyprovided by Dr S. T h e sequences of two oligonucleotides used in this study were identical to those described by Bock et al (11): consensus 15-mer (C15-mer),’ GGTTGGTGTGGTTGG; scrambled 15-mer (S15-mer), GGTGGTGGTTGTGGT. ’ The abbreviations used are: C15-mer, consensus 15-meroligonucleotide; cC15-mer, oligonucleotide with sequence complementary to the C15-mer; S15-mer, scrambled 15-meroligonucleotide; DIP, diisopropyl phosphoryl; HPLC, high-performancleiquid chromatography. Chromatography methodsa, s describedpreviously (17)P. urified plasma and recombinant prothrombinswere activated to the thrombin form by E. carinatusvenom (1pg of venom/lO pg ofprothrombin)

RESULTS

Thrombin Thrombin

Thrombh Thornbln