Functional and binding studies of gallic acid showing platelet aggregation inhibitory effect as a thrombin inhibitor

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 plasmon 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 lmol/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 lmol/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. (C) 2021 Tianjin Press of Chinese Herbal Medicines. Published by ELSEVIER B.V.