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.

Figure 1. The murine model of thrombosis.

The dynamics of thrombus formation at various doses of bivalirudin (A) and RA-36 aptamer (B, C).

Figure 2. Inhibition of human and murine thrombins in fibrinogen hydrolysis with bivalirudin (A) and RA-36 aptamer (B).

Figure 3. Inhibition of human and murine plasma coagulation with bivalirudin (A) and RA-36 aptamer (B).

The coagulation test of prothrombin time was used.

Figure 4. The comparison of the antithrombotic activity of five-fold (A) and ten-fold (B) doses of bivalirudin and RA-36 aptamer.