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


CONCLUSIONS
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.
Mendelboum Raviv
S
S
Clinical Research Centre, Medical and Health Science Centre, University of Debrecen, Debrecen, Hungry.
Horváth
A
A
Aradi
J
J
Bagoly
Z
Z
Fazakas
F
F
Batta
Z
Z
Muszbek
L
L
Hársfalvi
J
J
D052157:Aptamers, Nucleotide / Q000138:chemical synthesis / Q000494:pharmacology*; D001483:Base Sequence; D001777:Blood Coagulation / Q000187:drug effects*; D003856:Deoxyuracil Nucleotides; D004353:Drug Evaluation, Preclinical; D042783:Endothelial Cells; D004730:Endothelium, Vascular / Q000166:cytology; D005344:Fibrinopeptide A / Q000378:metabolism; D006801:Humans; D010477:Perfusion; D010974:Platelet Aggregation / Q000187:drug effects*; D013329:Structure-Activity Relationship; D013873:Thionucleotides; D013927:Thrombosis / Q000517:prevention & control*
C009461:4-thio-2'-deoxyuridylate; D052157:Aptamers, Nucleotide; D003856:Deoxyuracil Nucleotides; D013873:Thionucleotides; C081676:thrombin aptamer; D005344:Fibrinopeptide A