Hirudin is a potent thrombin inhibitor limited by short circulation half-life and bleeding complications. We engineered an FXa-activatable hirudin thrombus-targeted prodrug, yet the N-terminal IEGR peptide introduced unwanted basal activity. After further screening, we found this residual function was fully silenced by a P-selectin-binding peptide upstream of IEGR, with no compromise to FXa-mediated cleavage. Our final construct PXHV2 incorporates both peptides, C-terminal human serum albumin to extend half-life, and an albumin-embedded cyclic RGD sequence for constitutive platelet recruitment. PXHV2 remains catalytically inert until FXa proteolysis restores thrombin-suppressive capacity. In murine electrical and laser-induced thrombosis models, PXHV2 delayed arterial and microvascular occlusion and maintained 120 min pre-injury antithrombotic protection, unlike rapidly ineffective free hirudin. PXHV2 elicited no increase in tail bleeding relative to saline controls. Our data identify PXHV2 as a long-lived, thrombus-selective prodrug with durable efficacy and minimal bleeding risk.
