Cardiovascular disease is the leading cause of death among urban and rural residents worldwide, and has become a major public health problem [1]. Thrombosis is the most feared complication of cardiovascular diseases [2]. Antithrombotic therapy and prevention of thrombosis remains unmet medical need [3]. However, adverse effects occur in some patients receiving anticoagulants, representative by bleeding risk, thereby delaying the patients’ recovery and, in severe cases developing long-lasting disability or mortality [4], [5], [6].

Heparin-induced thrombocytopenia (HIT) is an immune-mediated complication of heparin (or its derivatives) exposure [7]. Although rare, it is life-threatening. Warfarin needs to be routinely monitored via prothrombin time-international normalized ratio (PT-INR) and activated partial thromboplastin time (aPTT) [8]. Patients with renal failure may be at increased risk of adverse events due to excessive accumulation of fondaparinux, and the fondaparinux dose needs to be adjusted in patients with renal impairment [9], [10]. The guidance suggests that patients with moderate renal insufficiency be monitored closely for bleeding during longer durations of therapy due to potential accumulation [11]. Non–vitamin K oral anticoagulants (NOACs) (apixaban, dabigatran, rivaroxaban, bivalirudin, etc.) are associated with the risk of bleeding and angioedema [12] which can result in edema, dyspnea or even suffocation. Bivalirudin reduces the risk of bleeding compared with heparin and glycoprotein IIb/IIIa inhibition but is associated with higher rates of acute stent thrombosis and trends toward more frequent periprocedural myocardial infarction [13].

Patients receiving anticoagulants are more frequently present for emergency department care and bleeding represents approximately 80 % of these visits [14]. To improve patient safety and treatment outcome, and rapidly control the anticoagulant activity of therapeutic agents in the patients by physicians, antidote control is the safe way.

Aptamers can form specific three-dimensional conformations, allowed to specifically interact with target molecules with high binding affinity. Generally, aptamers are selected from systematic evolution of ligands by exponential enrichment (SELEX) [15], [16]. Aptamers are a promising family of molecules that can serve as therapeutics in various diseases [17], [18], be utilized in diagnostics [19], [20], [21], e.g in cardiovascular diseases (Table S1) [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], and used as specific drug delivery more than just a replacement for antibodies [40], [41].

Due to the potential as effective anticoagulant molecules to treat cardiovascular diseases and blood disorders, several aptamers have been evaluated in clinical trials and many more have demonstrated encouraging preclinical results. These aptamers target the factors in both extrinsic and intrinsic blood coagulation pathway, including human thrombin (HFIIa), HFXa, HFVIIa, HFVII, FXII (HFXIIa), HFXIa, HFIXa, HFVIII, as well as tissue factor pathway inhibitor (TFPI), Von Willebrand factor (vWF), and activated protein C (APC) [42].

Theoretical attributes of an ideal antithrombotic agent include rapid onset of action, and reversal to anticoagulation from post-procedural bleeding risk. REG1 was a novel antithrombotic system consisting of an active anticoagulant (pegnivacogin), an RNA aptamer targeting HFIXa, and a complementary oligonucleotide antidote (anivamersen) that neutralized the anticoagulant effect. However, the clinical trial of REG1 system was terminated due to the severe allergic reactions responses to the PEG group reported in the phase III study [43], [44], [45], [46], [47].

In comparison with RNA aptamers, DNAs were advantageous in easier synthesis and modification, large-scale production, lower cost, and higher stability. Herein, we screened novel DNA aptamers through SELEX in vitro to selectively recognize HFIXa and inhibit its activity. We further optimized the aptamers by truncation and site-directed simutation. We rationally designed antidotes and demonstrated the antidotes can directly bind aptamers and neutralize the anticoagulant function in vitro. Our results suggest the DNA aptamer-antidote pairs are capable of reversely controlling regulation of HFIXa activity, and are potent antidote-controlled anticoagulant agents.