Purification and Characterization of a Novel Anticoagulant Peptide from Protein Hydrolysate of Eupolyphaga sinensis Walker

Eupolyphaga sinensis Walker (ESW), an animal drug in traditional Chinese medicine, has been used clinically for thousands of years for cardiovascular disease and osteoarthritis. Many studies of ESW have reported that it had anti-inflammatory and anti-tumor activities due to the small-molecule ingredients. However, large-molecule compounds of ESW representing significant pharmacological effects such as anti-thrombotic, anti-cancer, and anti-inflammatory have not been revealed yet. Here, a novel anticoagulant peptide (P9) containing 9 amino acids was isolated from the hydrolysate of aqueous extracts of ESW. Further, P9 synthesized by solid-phase synthesis showed 70% inhibition of thrombin, prolonging thrombin time to 17 s and activated partial thromboplastin time to 33 s. Molecular docking and spectroscopy demonstrated that P9 could inhibit thrombin activity by binding to the active site of thrombin and altering the secondary structure of thrombin. In an in vivo study, P9 enhanced the intensity of Phenylhydrazine-induced cardiac staining in thrombosed zebrafish with antithrombotic activity. The results suggest that peptides originating from ESW hydrolysates could exert an anticoagulant effect, which is likely to be a potential source of bioactive peptides with anticoagulant activity.