Thrombin inhibitors based on single-stranded DNA aptamers

Abstract

Investigation of inhibitory effect of two single-stranded DNA thrombin-inhibiting aptamers (15TBA and 31TBA) on fibrin polymerization in fibrinogen solutions and comparison of anticoagulant properties of these aptamers by a new global coagulation test of thrombodynamics. Measurement of aptamers' functional stability in human plasma and blood in vitro in order to investigate the involvement of 3'-exonuclease in fast decrease of aptamers' functional activity in vivo. Thrombin inhibition activity was measured in a buffer system in vitro as effects of aptamers on fibrin polymerization. Anticoagulant activity was investigated by measuring the spatial clot growth rate in the presence of aptamers. The stability of aptamers during incubation in human plasma was investigated in vitro by measuring activated partial thromboplastin time. Both aptamers dose-dependently inhibit fibrin polymerization in a buffer solution (IC50=10 nm for 15TBA and 3 nm for 31TBA) and are effective anticoagulants in human plasma (IC50 for spatial clot growth rate decreasing are 9.5 μmol/l and 4.0 μmol/l for 15TBA and 31TBA, correspondingly). Both aptamers remain stable in plasma or whole blood in vitro for at least 4 h. It was shown that 31TBA was 2-3 times more effective than 15TBA. Both aptamers were stable in human plasma and whole blood in vitro. So, the 3'-exonuclease could not be the reason for fast decrease of aptamers' functional activity in vivo. The main role in the removal of oligonucleotides from the circulation is played obviously by the liver.

The Risk Factors of Thrombogenic, Thrombophilia, and the Principle for Heparin Prophylaxis in Personalized Medicine

“…This method was widely used to study hemophilia, mechanisms of action of antihemophilic drugs, and the development of new ones [71,72]. There are some data about the use of this method in pharmacology, such as the development of thrombin inhibitors [73], a study of their antidotes [74], or study of the procoagulant activity of microparticles [75]. Clinical studies of the capacity of thrombodynamics to identify the development of procoagulant states are presented by the studies of patients with sepsis [76].…”

Highly specific detection of thrombin using an aptamer-based suspension array and the interaction analysis via microscale thermophoresis

“…Two DNA aptamers with 15-mer (denoted as TBA1 in this article) and 29-mer (denoted as TBA2 in this article) have been specifically selected against thrombin corresponding to its two electropositive exosites, namely fibrinogen and heparin sites, situated on the sides of the active site. [20][21][22][23][24][25] Recently, based on the molecular interaction, many selective and sensitive aptamer-based methods have been developed, such as electrochemical and electrochemiluminescence assays, 26 which require complicated electrode modification and multistep washing processes, and gold nanoparticle-based assays 27 which may not be suitable for high-throughput screening and large-scale clinical examination.…”

Graphene oxide modified with aptamer-conjugated gold nanoparticles and heparin: a potent targeted anticoagulant

“…Moreover, oligonucleotide aptamers are easily degraded by blood nucleases and have a short lifetime (∼2 min) in blood, limiting their potential use. 11 Conjugation of aptamers through phosphorothioate linkages and by the use of lockednucleic acids has been shown to be efficient in improving the half-life and binding affinity of aptamers. 12 However, toxicity of the expensive non-natural modified aptamers is a concern.…”