Abstract
Factor XIa (FXIa) is a serine protease that catalyzes the activation of Factor IX (FIX) in the blood coagulation cascade. FXIa and its precursor FXI are emergent therapeutic targets for the development of safer anticoagulant agents. Here, we sought a novel DNA-based agent to inhibit FXIa. Towards this goal, an 80 base, single-stranded DNA aptamer library (containing a 40 base randomized core) was screened for FXIa-binding candidates, using ten rounds of positive and negative selection. After selection, 6 of 89 different sequences inhibited FXIa-mediated chromogenic substrate S2366 cleavage. The most active anti-FXIa aptamer had a hypervariable central sequence 5′-AACCTATCGGACTATTGTTAGTGATTTTTATAGTGT-3′ and was designated Factor ELeven Inhibitory APtamer (FELIAP). FELIAP, but not a scrambled aptamer control (SCRAPT), competitively inhibited FXIa-catalyzed S2366 cleavage, FIX activation, and complex formation with antithrombin. No effect of FELIAP on FXI activation was observed. FELIAP inhibited plasma clotting and thrombin generation assays to a significantly greater extent than SCRAPT. Immobilized FELIAP bound FXIa with strong affinity and an equilibrium binding constant (KD) in the low nanomolar range determined using surface plasmon resonance. FELIAP is the first FXIa-inhibitory aptamer to be described and constitutes a lead compound to develop related aptamers for in vivo use.
Highlights
The coagulation system can function in a protective or pathological manner
After 4 and 10 rounds of selection, we noted no inhibition of Factor XIa (FXIa)-mediated amidolysis when the selected aptamer pool was introduced into the reaction
Our protocol favoured the selection of aptamers binding at or near the active site of FXIa over those binding to other portions of the enzyme
Summary
The coagulation system can function in a protective or pathological manner. Haemostatic blood clots prevent excessive blood loss at sites of vascular injury[1], whereas thrombotic clots occlude blood vessels and prevent the flow of blood to critical organs, such as the heart or brain[2, 3]. Warfarin attenuates clotting by reducing the hepatic synthesis of multiple coagulation factors[5], whereas dabigatran inhibits thrombin and rivaroxaban, apixaban and edoxaban inhibit activated factor X (FXa)[6]. Inhibition of FXI has the potential to attenuate thrombosis without impairing hemostasis. In support of this concept, knockdown of FXI in patients undergoing elective knee replacement was more effective than enoxaparin, the current standard of care, at preventing postoperative venous thromboembolism and did not increase the risk of bleeding[18]. We describe the selection and characterization of a DNA aptamer that binds the active site of FXIa and inhibits its enzymatic action on both artificial and natural substrates
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