Xiaoyu Chai ^1* Fulu Pan ¹ Qianqian Wang ^1* Xinyu Wang ^1* Xueyan Li ¹ Dongying Qi ^1* Zirong Yi ^1* Huan Liu ^1* Jing Zhang ^2* Yiming Zhang ^2* Yanli Pan ^2* Yang Liu ^1* Guopeng Wang ^1*

• ¹ Beijing University of Chinese Medicine, Beijing, China
• ² Institute of Information on Traditional Chinese Medicine, China Academy of Chinese Medical Sciences, Beijing, China

Purpose: The inhibition of thrombin has proven to be an efficacious therapeutic approach for managing cardiovascular disease (CVD), with widespread implementation in clinical settings. Oral ingestion of peptides and protein drugs is influenced by gastrointestinal digestive enzymes. We aimed to evaluate the thrombin inhibitory properties of hirudo hydrolysates (HHS) produced by pepsin and propose a comprehensive approach to screen and evaluate thrombin inhibitors. Methods: We evaluated the in vitro inhibitory properties of the hirudo extract, both before and after hydrolysis with pepsin, toward thrombin. We screened for the most potent thrombin inhibitory peptide (TIP) using nano liquid chromatography-tandem mass spectrometry (Nano LC-MS/MS) coupled with in silico analysis. Next, we employed the thrombin inhibition activity IC50 to investigate the interaction between TIP and thrombin, and conducted in vitro evaluations of its anticoagulant effects (APTT, TT, PT), as well as its ability to inhibit platelet aggregation. Furthermore, we utilized UV-Vis spectroscopy to explore structural changes in thrombin upon binding with TIP and employed molecular dynamics simulations to delve deeper into the potential atomic-level interaction modes between thrombin and TIP. Results: The retention rate of thrombin inhibition for HHS was found to be between 60% and 75%. A total of 90 peptides from the HHS were identified using LC-MS/MS combined with de novo sequencing. Asn-Asp-Leu-Trp-Asp-Gln-Gly-Leu-Val-Ser-Gln-Asp-Leu (NDLWDQGLVSQDL, P1) was identified as the most potent thrombin inhibitory peptide after in silico screening (molecular docking and ADMET). Then, the in vitro study revealed that P1 had a high inhibitory effect on thrombin (IC50: 2425.5±109.7 μM). P1 exhibited a dose-dependent prolongation of the thrombin time (TT) and a reduction in platelet aggregation rate. Both UV-Vis spectroscopy and molecular dynamics simulations demonstrated that P1 binds effectively to thrombin. Conclusion: Overall, the results suggested that HHS provides new insights for searching and evaluating potential antithrombotic compounds. The obtained P1 can be structurally optimized for in-depth evaluation in animal and cellular experiments.