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DNA Aptamers to Thrombin Exosite I. Structure-Function Relationships and Antithrombotic Effects

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Abstract

DNA aptamers (oligonucleotides) interacting with thrombin exosite I contain G-quadruplex, two T-T, and one T-G-T loops in their structure. They prevent exosite I binding with fibrinogen and thrombin receptors on platelet surface, thereby suppressing thrombin-stimulated formation of fibrin from fibrinogen and platelet aggregation. Earlier, we synthe-sized original antithrombin aptamer RE31 (5′-GTGACGTAGGTTGGTGTGGTTGGGGCGTCAC-3′) that contained (in addition to G-quadruplex) a hinge region connected to six pairs of complementary bases (duplex region). In this study, we compared properties of RE31 aptamer and its analogues containing varying number of bases in the duplex region and nucleotide insertions in the hinge region. Reduction in the number of nucleotides in the duplex region by 1 to 4 pairs (in comparison with RE31 aptamer) resulted in the decrease of the structural stability of aptamers (manifested as lower melting temperatures) and their ability to inhibit thrombin-stimulated fibrin formation in human blood plasma in tests of thrombin, prothrombin, and activated partial thromboplastin times. However, an increase in the number of bases by 1 to 2 pairs did not cause significant changes in the stability and antithrombin activity of the aptamers. Insertions into the hinge region of RE31 aptamer decreased its antithrombin activity. Investigation of RE31 antithrombotic properties demonstrated that RE31 (i) slowed down thrombin formation in human blood plasma (thrombin generation test), (ii) accelerated lysis of fibrin clot by tissue plasminogen activator in in vitro model, and (iii) suppressed arterial thrombosis in in vivo model. Based on the obtained data, RE31 aptamer can be considered as a potentially effective antithrombotic compound.

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Abbreviations

APTT:

activated partial thromboplastin time

PCI:

potato carboxypeptidase inhibitor

TAFI:

thrombin-activated fibrinolysis inhibitor

TBA:

thrombin-binding aptamer

tPA:

tissue plasminogen activator

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Authors and Affiliations

  1. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992, Moscow, Russia

    V. A. Spiridonova, T. M. Novikova, V. A. Sizov & V. S. Shashkovskaya

  2. National Medical Research Center for Cardiology, Russian Ministry of Health, 121552, Moscow, Russia

    E. V. Titaeva, A. B. Dobrovolsky, E. B. Zharikova & A. V. Mazurov

Authors
  1. V. A. Spiridonova
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  2. T. M. Novikova
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  3. V. A. Sizov
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  4. V. S. Shashkovskaya
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  5. E. V. Titaeva
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  6. A. B. Dobrovolsky
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  7. E. B. Zharikova
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  8. A. V. Mazurov
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Corresponding author

Correspondence to A. V. Mazurov.

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Conflict of interest. The authors declare no conflict of interest in financial or any other sphere.

Ethical approval. This article does not contain any studies with human participants. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Russian Text © The Author(s), 2019, published in Biokhimiya, 2019, Vol. 84, No. 12, pp. 1876–1885.

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Spiridonova, V.A., Novikova, T.M., Sizov, V.A. et al. DNA Aptamers to Thrombin Exosite I. Structure-Function Relationships and Antithrombotic Effects. Biochemistry Moscow 84, 1521–1528 (2019). https://doi.org/10.1134/S0006297919120113

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  • DOI: https://doi.org/10.1134/S0006297919120113

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