4-thio-deoxyuridylate-modified thrombin aptamer and its inhibitory effect on fibrin clot formation, platelet aggregation and thrombus growth on subendothelial matrix.

Abstract

BACKGROUND:

The consensus thrombin aptamer C15-mer is a single-stranded DNA of 15 nucleotides [d(GGTTGGTGTGGTTGG)] that was identified by the selection of thrombin-binding molecules from a large combinatorial library of oligonucleotides. It is capable of inhibiting thrombin at nanomolar concentrations through binding to a specific region within thrombin exosite 1. As has been shown in our earlier studies, the 4-thio-deoxyuridylate (s4dU)-containing oligonucleotides have high affinity for a number of proteins, due to the reduced hydrophilic character of the modified oligonucleotide.

METHODS:

Three different analogs of the original thrombin-inhibiting sequence, in which some of the thymidylate residues were replaced by 4-thio-deoxyuridylates, were synthesized. The inhibitory effect of modified aptamers was tested on thrombin-catalyzed fibrin clot formation and fibrinopeptide A release from fibrinogen, thrombin-induced platelet aggregation/secretion, and the formation of thrombus on coverslips coated with human collagen type III, thrombin-treated fibrinogen or subendothelial matrix of human microvascular endothelial cells.

RESULTS:

As compared with the C15-mer, the analog with the sequence GG(s4dU)TGG(s4dU)G(s4dU)GGT(s4dU)GG (UC15-mer) showed a 2-fold increased inhibition of thrombin-catalyzed fibrin clot formation, fibrinopeptide A release, platelet aggregation and secretion in human plasma and thrombus formation on thrombin-treated fibrinogen surfaces under flow conditions. Concerning the inhibition of thrombin-induced fibrin formation from purified fibrinogen and activation of washed platelets, UC15-mer was 3-fold and twelve-fold more effective than C15-mer, respectively.

CONCLUSION:

The replacement of four thymidylate residues in C15-mer by 4-thio-deoxyuridylate resulted in a new thrombin aptamer with increased anticoagulant and antithrombotic properties.

• Authors: S Mendelboum Raviv, A Horváth, J Aradi, Z Bagoly, F Fazakas, Z Batta, L Muszbek, J Hársfalvi
• Journal: Journal of thrombosis and haemostasis : JTH (J Thromb Haemost) Vol. 6 Issue 10 Pg. 1764-71 (Oct 2008) ISSN: 1538-7836 [Electronic] England
• PMID: 18665927 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• 4-thio-2'-deoxyuridylate
• Aptamers, Nucleotide
• Deoxyuracil Nucleotides
• Thionucleotides
• thrombin aptamer
• Fibrinopeptide A

Topics

• Aptamers, Nucleotide (chemical synthesis, pharmacology)
• Base Sequence
• Blood Coagulation (drug effects)
• Deoxyuracil Nucleotides
• Drug Evaluation, Preclinical
• Endothelial Cells
• Endothelium, Vascular (cytology)
• Fibrinopeptide A (metabolism)
• Humans
• Perfusion
• Platelet Aggregation (drug effects)
• Structure-Activity Relationship
• Thionucleotides
• Thrombosis (prevention & control)