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This study identifies a cysteine-knotted peptide GC38 (sequence: GCSGKGARCAPSKCCSGLSCGRHGGNMYKSCEWNWKTG) derived from the venom gland transcriptome of the Macrothele sp. spider, which exerts thrombus-inhibitory effects by potentiating activated protein C (APC) activity. In vitro assays reveal that GC38 enhances APC activity, prolongs plasma clotting time, and shows no significant cytotoxicity or hemolytic activity. Mechanistically, GC38 interacts allosterically with APC; biolayer interferometry (BLI) confirms this direct interaction, with a dissociation constant KD of 6.16 \u03bcM. Additionally, three in vivo thrombosis models (FeCl3-induced arterial occlusion, stasis-induced DVT, and cortical photothrombotic stroke) consistently demonstrated that GC38 was effective in alleviating thrombus formation, with tail-bleeding assays confirming its low hemorrhagic risk. Collectively, our findings position GC38 as a pioneering spider venom-derived lead molecule that addresses dual arterial and venous antithrombotic actions. This opens new avenues for developing spider venom-derived peptides as therapeutic agents targeting intravascular coagulation in arteries and veins.<\/jats:p>","DOI":"10.3390\/ijms262010154","type":"journal-article","created":{"date-parts":[[2025,10,20]],"date-time":"2025-10-20T08:18:54Z","timestamp":1760948334000},"page":"10154","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Discovery of a Novel Anticoagulant Cystine Knot Peptide from Spider Venom Gland Transcriptome"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3832-5009","authenticated-orcid":false,"given":"Jinai","family":"Gao","sequence":"first","affiliation":[{"name":"Engineering Laboratory of Peptides of Chinese Academy of Sciences, Key Laboratory of Bioactive Peptides of Yunnan Province, KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, National Resource Center for Non-Human 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Diseases, National Resource Center for Non-Human Primates, National Research Facility for Phenotypic & Genetic Analysis of Model Animals (Primate Facility), State Key Laboratory of Genetic Evolution & Animal Models, Sino-African Joint Research Center, and New Cornerstone Science Laboratory, Kunming Institute of Zoology, Chinese Academy of Sciences, No. 17 Longxin Road, Kunming 650201, China"},{"name":"University of Chinese Academy of Sciences, Beijing 100049, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruiyin","family":"Guo","sequence":"additional","affiliation":[{"name":"Center for Evolution and Conservation Biology, Southern Marine Science and Engineering Guangdong Laboratory, Guangzhou 511458, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kaixun","family":"Cao","sequence":"additional","affiliation":[{"name":"Engineering Laboratory of Peptides of Chinese Academy of Sciences, Key Laboratory of Bioactive Peptides of Yunnan Province, KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, National Resource Center for Non-Human Primates, National Research Facility for Phenotypic & Genetic Analysis of Model Animals (Primate Facility), State Key Laboratory of Genetic Evolution & Animal Models, Sino-African Joint Research Center, and New Cornerstone Science Laboratory, Kunming Institute of Zoology, Chinese Academy of Sciences, No. 17 Longxin Road, Kunming 650201, China"},{"name":"University of Chinese Academy of Sciences, Beijing 100049, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9986-9950","authenticated-orcid":false,"given":"Qiumin","family":"Lu","sequence":"additional","affiliation":[{"name":"Engineering Laboratory of Peptides of Chinese Academy of Sciences, Key Laboratory of Bioactive Peptides of Yunnan Province, KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, National Resource Center for 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Research Center, and New Cornerstone Science Laboratory, Kunming Institute of Zoology, Chinese Academy of Sciences, No. 17 Longxin Road, Kunming 650201, China"},{"name":"University of Chinese Academy of Sciences, Beijing 100049, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3123-2336","authenticated-orcid":false,"given":"Ren","family":"Lai","sequence":"additional","affiliation":[{"name":"Engineering Laboratory of Peptides of Chinese Academy of Sciences, Key Laboratory of Bioactive Peptides of Yunnan Province, KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, National Resource Center for Non-Human Primates, National Research Facility for Phenotypic & Genetic Analysis of Model Animals (Primate Facility), State Key Laboratory of Genetic Evolution & Animal Models, Sino-African Joint Research Center, and New Cornerstone Science Laboratory, Kunming Institute of Zoology, Chinese Academy of Sciences, No. 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