Background: Thrombin is a central mediator of coagulation and platelet activation and a key anticoagulant target. Direct thrombin inhibitors broadly suppress thrombin proteolytic activity, potentially disrupting procoagulant and anticoagulant signaling. Exosite-targeting aptamers may enable selective modulation and programmable reversal.

Methods: We characterize AYA1809002, a 40-nt DNA aptamer targeting thrombin Exosite I.

Results: AYA1809002 bound thrombin with nanomolar affinity and inhibited fibrin formation with an IC₅₀ of 25.7 nM and K_i of 10.6 nM. Reduced 𝛾-thrombin binding supported an Exosite I-dependent mechanism. AYA1809002 preserved thrombin activity toward small peptide substrates and thrombomodulin-dependent protein C activation. It showed no measurable activity against tested coagulation, anticoagulant, fibrinolytic, and control enzymes and inhibited thrombin-mediated platelet activation while preserving responses to non-thrombin agonists. AYA1809002 showed greater functional stability than thrombin-binding aptamer NU172 following serum exposure and remained active under thermal, pH, oxidative, and photostability stress. Anticoagulant activity was rapidly and sequence-specifically reversed by a reverse-complement oligonucleotide, while lipid conjugation preserved function. AYA1809002 also retained high-affinity binding to rat thrombin and anticoagulant activity in rat plasma.

Conclusions: These findings identify AYA1809002 as a selective, stable, reversible Exosite I-targeting thrombin aptamer that suppresses key procoagulant functions while preserving thrombomodulin-dependent protein C activation.