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Naturally derived pentacyclic triterpenoids, such as betulinic acid and its acetylated derivatives, 3\u03b2-acetoxybetulinic acid, exhibit promising antiplatelet aggregation activity in validated in vitro and ex vivo assays; however, the molecular determinants underlying their direct thrombin inhibition remain unexplored. Results: Docking and MM\/GBSA analyses revealed that Baa exhibits the strongest binding affinity (\u0394G = \u221229.58 \u00b1 2.97 kcal\/mol), exceeding those of Ba (\u221220.94 \u00b1 5.81 kcal\/mol) and Asp (\u221218.87 \u00b1 4.18 kcal\/mol). Baa forms a highly persistent hydrogen bond with Trp96 (95.5% occupancy) and extensive hydrophobic contacts with Trp215, Leu99, Ile174, and Tyr60A residues defining thrombin\u2019s aryl-binding pocket. MD trajectories demonstrated that Baa binding reduced solvent-accessible surface area (SASA) and residue fluctuations, indicating enhanced structural compaction and stability. In contrast, Ba exhibited weaker, transient hydrogen bonding, while Asp bound primarily near the catalytic triad. The triterpenes exhibit limited oral bioavailability, free PAINS alerts, favourable permeability and metabolic stability. Conclusions: Acetylation at C-3 (acetoxy substitution) substantially enhances thrombin binding via cooperative hydrogen bonding and van der Waals stabilisation, explaining the superior experimental inhibitory potency of Baa. These findings provide a mechanistic framework for structure-guided optimisation of triterpenoid-based thrombin inhibitors and support their further experimental development. Methods: In this study, molecular docking, molecular dynamics (MD) simulations (400 ns), and MM\/GBSA free energy analyses were employed to elucidate the binding mechanisms of 3\u03b2-acetoxybetulinic acid (Baa), betulinic acid (Ba), and aspirin (Asp) within the thrombin receptor active site. The simulations were explicitly grounded in previously reported chromogenic antithrombin assays and platelet aggregation studies and were designed to mechanistically rationalise the experimentally observed inhibitory potency.<\/jats:p>","DOI":"10.3390\/molecules31050848","type":"journal-article","created":{"date-parts":[[2026,3,4]],"date-time":"2026-03-04T16:17:31Z","timestamp":1772641051000},"page":"848","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Computational and Molecular Dynamics Insights into the Antithrombotic Mechanism of Triterpenes Derived from Melaleuca bracteata var. Revolution Gold (Myrtaceae)"],"prefix":"10.3390","volume":"31","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5904-3051","authenticated-orcid":false,"given":"Patrick","family":"Appiah-Kubi","sequence":"first","affiliation":[{"name":"Department of Chemistry, University of Pretoria, Hatfield, Pretoria 0002, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6465-1385","authenticated-orcid":false,"given":"Foluso Oluwagbemiga","family":"Osunsanmi","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Microbiology, University of Zululand, KwaDlangezwa, Empangeni 3886, South Africa"},{"name":"Research Center, International School Development Organization (ISDO), Krugersdorp 1739, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8049-0761","authenticated-orcid":false,"given":"Andrew Rowland","family":"Opoku","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Microbiology, University of Zululand, KwaDlangezwa, Empangeni 3886, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6497-0207","authenticated-orcid":false,"given":"Ashona","family":"Singh","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Pretoria, Hatfield, Pretoria 0002, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Di Cesare, M., Perel, P., Taylor, S., Kabudula, C., Bixby, H., Gaziano, T.A., McGhie, D.V., Mwangi, J., Pervan, B., and Narula, J. 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