PB0773 - The RNA aptamer, APTA-1 targeting evolutionarily conserved motif on thrombin cures endotoxemia and reduces thrombosis in animal models by inhibiting aggregation and secretion of platelets.

Background: Sepsis, a leading cause of critical illness, involves dysregulated host reactions to pathogen-driven infections, causing systemic inflammatory response (cytokine storm) and derangement of haemostasis resulting in excessive bleeding and clotting (coagulopathy). Developing novel drugs that effectively target inflammation and coagulopathy in sepsis is urgently needed.

Aims: APTA-1 is a 90-mer synthetic RNA aptamer designed for therapeutic intervention in sepsis. This research aimed to evaluate the therapeutic potential of APTA-1 in thrombosis and sepsis animal models and to elucidate the underlying mechanism of action.

Methods: The therapeutic potential of APTA-1 was evaluated in carotid artery thrombosis (ferric chloride) and arteriovenous shunt rat thrombosis models. To determine whether APTA-1 can improve sepsis outcomes, we induced lethal (mice) or sub-lethal (Cynomolgus monkeys) endotoxemia by administration of lipopolysaccharide. Various commercial ELISA assays were utilized to elucidate the pharmacological properties of APTA-1. Mechanistic studies using human platelets comprised aggregometry, Western blotting and fura-2-dependent Ca2+ measurements.

Results: APTA-1 inhibited thrombus formation in vivo without increasing bleeding time; in vitro analyses confirmed that APTA-1 did not affect clotting time and thrombin generation. Thus, APTA-1 has antithrombotic but not anticoagulant properties. Furthermore, APTA-1 improved survival in lethal and alleviated clinical symptoms in sub-lethal endotoxemia (Fig. 1). The pharmacological activity of APTA-1 in vivo was associated with transient enhancement of fibrinolysis and plasma pentraxin-3, whereas complement activation (C3a and C4a) was abolished. We identified the heparin-binding motif exosite II on thrombin as a crucial molecular target for APTA-1. Specifically, APTA-1 binding to thrombin inhibited platelet activation by significantly reducing protease-activated receptor (PAR)-1 and PAR-4 activation and signalling (Fig.2).

Conclusion(s): APTA-1 targets thrombin, antagonizes activation of PARs and exerts significant anti-inflammatory and antithrombotic activities. Importantly, APTA-1 has no anticoagulant activity, implying a favourable safety profile. We suggest that APTA-1 can be a promising future drug candidate for sepsis therapy.