Targeted codelivery of nitric oxide and hydrogen sulfide for enhanced antithrombosis efficacy

Thrombosis is a leading cause of mortality worldwide. As important gaseous signaling molecules, both nitric oxide (NO) and hydrogen sulfide (H₂S) demonstrate antiplatelet and anticoagulant functions, but little attention has been given to their synergistic effect and the underlying mechanism. In the present study, we developed an NO/H₂S codelivery system based on enzyme prodrug therapy (EPT) strategy in which the prodrugs are specifically recognized by the engineered β-galactosidase. Targeted codelivery of NO and H₂S <i>in vivo</i> was demonstrated by near-infrared fluorescence imaging and confirmed by measuring plasma and tissue levels; as a result, the side effects caused by systemic delivery, such as bleeding time, were reduced. Delivery of an optimized combination of NO and H₂S with a low combination index (CI) results in a synergistic effect on the inhibition of platelet adhesion and activation. Mechanistically, NO and H₂S cooperatively enhance the cGMP level through redox-based posttranslational modifications of phosphodiesterase 5A (PDE5A), which leads to activation of the cGMP/PKG signaling pathway. Furthermore, targeted codelivery of NO and H₂S demonstrates enhanced therapeutic efficacy for thrombosis in two mouse models of FeCl₃-induced arterial thrombosis and deep vein thrombosis. Collectively, these results confirm the synergistic efficacy of NO and H₂S for antithrombotic therapy, and the codelivery system developed in this study represents a promising candidate for clinical translation.