Evaluation of Antithrombotic Activity of Thrombin DNA Aptamers by a Murine Thrombosis Model

Abstract

Aptamers are nucleic acid based molecular recognition elements with a high potential for the theranostics. Some of the aptamers are under development for therapeutic applications as promising antithrombotic agents; and G-quadruplex DNA aptamers, which directly inhibit the thrombin activity, are among them. RA-36, the 31-meric DNA aptamer, consists of two thrombin binding pharmacophores joined with the thymine linker. It has been shown earlier that RA-36 directly inhibits thrombin in the reaction of fibrinogen hydrolysis, and also it inhibits plasma and blood coagulation. Studies of both inhibitory and anticoagulation effects had indicated rather high species specificity of the aptamer. Further R&D of RA-36 requires exploring its efficiency in vivo. Therefore the development of a robust and adequate animal model for effective physiological studies of aptamers is in high current demand. This work is devoted to in vivo study of the antithrombotic effect of RA-36 aptamer. A murine model of thrombosis has been applied to reveal a lag and even prevention of thrombus formation when RA-36 was intravenous bolus injected in high doses of 1.4–7.1 µmol/kg (14–70 mg/kg). A comparative study of RA-36 aptamer and bivalirudin reveals that both direct thrombin inhibitors have similar antithrombotic effects for the murine model of thrombosis; though in vitro bivalirudin has anticoagulation activity several times higher compared to RA-36. The results indicate that both RA-36 aptamer and bivalirudin are direct thrombin inhibitors of different potency, but possible interactions of the thrombin-inhibitor complex with other components of blood coagulation cascade level the physiological effects for both inhibitors.

Investigating the properties of TBA variants with twin thrombin binding domains

“…In details, at the lowest concentration used i.e 2 µM, the PT detected for TT , AglyA and AA has been 29.6 ± 0.18, 30.5 ± 1.2 and 32.6 ± 0.32 sec, respectively. These PT values are significantly higher when compared to their natural counterpart TBA (25.7 ± 0.10 sec) and to RA-36 for which a PT value of about 23 sec has been reported 12 . Otherwise, evaluation of PT in plasma incubated with Gly reveals an increased PT value superimposable to that observed for TBA.…”

“…Furthermore, animal tests have suggested a species-specificity of RA-36 9,10 . In a further study the antithrombotic activity of RA-36 has been evaluated by a murine thrombosis model 12 . Moreover, its pharmacodynamic and pharmacokinetic properties have been widely evaluated 13 .…”

Site-specific replacement of the thymine methyl group by fluorine in thrombin binding aptamer significantly improves structural stability and anticoagulant activity

“…Recently several ‘second generation’ anticoagulant aptamers have been described in literature. Among these, the duplex-quadruplex modular aptamer ( 42 , 43 , 44 ) dimeric DNA aptamers ( 45 ) and the bivalent aptamer ( 46 ) are particularly noteworthy. It should be noted that the biological activity of all these aptamers could benefit from the site specific replacement here described, since they preserve the peculiar sequence of TBA (Table 1 ) folding in the antiparallel G-quadruplex, as the structural component interacting with the thrombin.…”

The Evaluation of Pharmacodynamics and Pharmacokinetics of Anti-thrombin DNA Aptamer RA-36

“…The murine model of thrombosis has been described previously ( Zavyalova et al, 2014 ). Briefly, mice were anaesthetized with a ketamine-xylazine mixture (80 + 20 mg/kg); lidocaine was used for a local anesthesia at the site of the surgery.…”