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

Summary. 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.