A recombinant prodrug, single-chain urokinase-type plasminogen activator (scuPA) fused to an anti–PECAM-1 antibody single-chain variable fragment (anti–PECAM scFv/scuPA) targets endothelium and augments thrombolysis in the pulmonary vasculature. To avoid premature activation and inactivation and to limit systemic toxicity, we replaced the native plasmin activation site in scFv/low-molecular-weight (lmw)–scuPA with a thrombin activation site, generating anti–PECAM scFv/uPA-T that (1) is latent and activated by thrombin instead of plasmin; (2) binds to PECAM-1; (3) does not consume plasma fibrinogen; (4) accumulates in mouse lungs after intravenous injection; and (5) resists PA inhibitor PAI-1 until activated by thrombin. In mouse models of pulmonary thrombosis caused by thromboplastin and ischemia-reperfusion (I/R), scFv/uPA-T provided more potent thromboprophylaxis and greater lung protection than plasmin-sensitive scFv/uPA. Endothelium-targeted thromboprophylaxis triggered by a prothrombotic enzyme illustrates a novel approach to time- and site-specific regulation of proteolytic reactions that can be modulated for therapeutic benefit.