Discovery of a Thrombin-Targeting Anticoagulant Peptide From Whitmania pigra via a "Computation-Guided Experimentation" Strategy.

Targeting thrombin to screen safe thrombin inhibitors from natural plants and animals is a critical direction in anticoagulant drug development. This study aimed to screen thrombin inhibitors from the nonbloodsucking leech Whitmania pigra (WP) and elucidate the mechanism of anticoagulation through a "computation-guided experimentation" strategy. A peptide library was constructed from WP hydrolysates, and virtual screening was performed using molecular docking and dynamics simulations. A novel thrombin-targeting anticoagulant peptide PEPWP (LRELEDALEQER) was screened out from the peptide library and validated through in vitro/in vivo experiments. PEPWP significantly prolonged thrombin time (TT) and prothrombin time (PT) in a dose-dependent manner in vitro, indicating its role in the common and extrinsic coagulation pathways. Surface plasmon resonance (SPR) analysis then confirmed strong thrombin binding (Kd = 7.242 × 10-6 mol/L). Furthermore, PEPWP prolonged TT while reducing blood viscosity in acute blood stasis rats. Finally, structural analysis revealed that PEPWP bound to Exosite II of thrombin. Arg233 and Arg101 were the key residues for the binding. In conclusion, PEPWP exhibited good anticoagulant activity and significant application potential.