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Recombinant neorudin (EPR-hirudin, EH), a low-bleeding anticoagulant fusion protein, is an inactive prodrug designed to be converted to the active metabolite, hirudin variant 2-Lys47 (HV2), locally at the thrombus site by FXa and/or FXIa, following activation of the coagulation system. Our aim was to evaluate the prodrug characteristics of EH by comparing the biotransformation of EH and HV2 in biological matrices, including rat blood, liver, and kidney homogenates, demonstrating the cleavage of EH to HV2 by FXa and FXIa, and comparing the conversion of EH to HV2 between fresh whole blood and whole-blood clot homogenate, using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS). Both EH and HV2 were stable in blood and unstable in the liver and kidney homogenates. Eight EH metabolites and eight HV2 metabolites identified as N-terminal fragments were found in the liver and kidney. C-terminal proteolysis is therefore the major metabolic pathway, with serine/cysteine carboxypeptidases and metallocarboxypeptidases being responsible for the degradation of EH and HV2 in the liver and kidney, respectively. EH was cleaved to release HV2 by FXIa. Higher levels of HV2 were produced from EH in the whole-blood clot homogenate, in which the coagulation system was activated compared with those in fresh whole blood. In conclusion, the metabolism of EH and HV2 shares the same cleavage pattern, and EH is transformed into HV2 when the coagulation system is activated, where FXIa is a specific enzyme. Our in vitro study revealed the anticipated prodrug characteristics of EH newly designed as an inactive prodrug of hirudin.