摘要Objective:This study was devoted to identifying natural thrombin inhibitors from traditional Chinese medicine(TCM)and evaluating its biological activity in vitro and binding characteristics.Methods:A combination strategy containing molecular docking,thrombin inhibition assay,surface plas-mon resonance(SPR)and molecular dynamics simulation were applied to verify the study result.Results:Gallic acid was confirmed as a direct thrombin inhibitor with IC50 of 9.07 μmol/L and showed a significant inhibitory effect on thrombin induced platelet aggregation.SPR-based binding studies demon-strated that gallic acid interacted with thrombin with a KD value of 8.29 μmol/L.Molecular dynamics and binding free energy analysis revealed that thrombin-gallic acid system attained equilibrium rapidly with very low fluctuations,the calculated binding free energies was-14.61 kcal/mol.Ala230,Glu232,Ser235,Gly258 and Gly260 were the main amino acid residues responsible for thrombin inhibition by gallic acid,providing a mechanistic basis for further optimization.Conclusion:This study proved that gallic acid is a direct thrombin inhibitor with platelet aggregation inhibitory effect,which could provide a basis for the follow-up research and development for novel thrombin inhibitors.