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Kunitz-type proteins or peptides have been found in many blood-sucking animals, but the identity of them in leeches remained elusive. In the present study, five Kunitz-type peptides named WPK1-WPK5 were identified from the leech Whitmania pigra. Recombinant WPK1-WPK5 were expressed in Pichia pastoris GS115, and their inhibitory activity against Factor XIa (FXIa) was tested. WPK5 showed inhibitory activity against FXIa with an IC50 value of 978.20 nM. To improve its potency, the loop replacement strategy was used. The loop 1 (TGPCRSNLER) and loop 2 (QYGGC) in WPK5 were replaced by loop 1 (TGPCRAMISR) and loop 2 (FYGGC) in PN2KPI, respectively, and the resulting peptide named WPK5-Mut showed an IC50 value of 8.34 nM to FXIa, which is about 100-fold the potency of FXIa compared to that of WPK5. WPK5-Mut was further evaluated for its extensive bioactivity in vitro and in vivo. It dose-dependently prolonged APTT on both murine plasma and human plasma, and potently inhibited FeCl3-induced carotid artery thrombosis in mice at a dose of 1.5 mg/kg. Additionally, WPK5-Mut did not show significant bleeding risk at a dose of 6 mg/kg. Together, these results showed that WPK5-Mut is a promising candidate for the development of an antithrombotic drug.