{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,3]],"date-time":"2026-03-03T15:29:42Z","timestamp":1772551782213,"version":"3.50.1"},"reference-count":54,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2024,11,21]],"date-time":"2024-11-21T00:00:00Z","timestamp":1732147200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Pharmacol."],"abstract":"<jats:sec><jats:title>Purpose<\/jats:title><jats:p>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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>We evaluated the <jats:italic>in vitro<\/jats:italic> 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 <jats:italic>in silico<\/jats:italic> analysis. Next, we employed the thrombin inhibition activity IC<jats:sub>50<\/jats:sub> to investigate the interaction between TIP and thrombin, and conducted <jats:italic>in vitro<\/jats:italic> 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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>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 <jats:italic>de novo<\/jats:italic> 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 <jats:italic>in silico<\/jats:italic> screening (molecular docking and ADMET). Then, the <jats:italic>in vitro<\/jats:italic> study revealed that P1 had a high inhibitory effect on thrombin (IC<jats:sub>50<\/jats:sub>: 2,425.5 \u00b1 109.7\u00a0\u03bcM). 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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>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.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fphar.2024.1460053","type":"journal-article","created":{"date-parts":[[2024,11,21]],"date-time":"2024-11-21T06:41:32Z","timestamp":1732171292000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":3,"title":["Identification, screening, and comprehensive evaluation of novel thrombin inhibitory peptides from the hirudo produced using pepsin"],"prefix":"10.3389","volume":"15","author":[{"given":"Xiaoyu","family":"Chai","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fulu","family":"Pan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qianqian","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xinyu","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xueyan","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dongying","family":"Qi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zirong","family":"Yi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Huan","family":"Liu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jing","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yiming","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yanli","family":"Pan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yang","family":"Liu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guopeng","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2024,11,21]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"19","DOI":"10.1016\/j.softx.2015.06.001","article-title":"GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers","author":"Abraham","year":"2015","journal-title":"SoftwareX"},{"key":"B2","doi-asserted-by":"publisher","first-page":"200","DOI":"10.1038\/s41573-020-00114-z","article-title":"Natural products in drug discovery: advances and opportunities","volume":"20","author":"Atanasov","year":"2021","journal-title":"Nat. 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