Platelet activation contributes to sepsis development, leading to microthrombosis and increased inflammation, which results in disseminated intravascular coagulation and multiple organ dysfunction. Although Cathelicidin can alleviate sepsis, its role in sepsis regulation remains largely unexplored. In this study, we identified Cath-HG, a novel Cathelicidin from Hylarana guentheri skin, and analyzed its structure using nuclear magnetic resonance spectroscopy. The modulatory effect of Cath-HG on the symptoms of mice with sepsis induced by cecal ligation and puncture was evaluated in vivo, and the platelet count, degree of organ damage, and microthrombosis were measured. The antiplatelet aggregation activity of Cath-HG was studied in vitro, and its target was verified. Finally, we further investigated whether Cath-HG could regulate thrombosis in vivo in a FeCl3 injury-induced carotid artery model. The results showed that Cath-HG exhibited an α-helical structure in sodium dodecyl sulfate solution and effectively reduced organ inflammation and damage, improving survival in septic mice. It alleviated sepsis-induced thrombocytopenia and microthrombosis. In vitro, Cath-HG specifically inhibited collagen-induced platelet aggregation and modulated glycoprotein VI (GPVI) signaling pathways. Dot blotting, enzyme-linked immunosorbent assay, and pull-down experiments confirmed GPVI as the target of Cath-HG. Molecular docking and amino acid residue truncations/mutations identified crucial sites of Cath-HG. These findings suggest that GPVI represents a promising therapeutic target for sepsis, and Cath-HG may serve as a potential treatment for sepsis-related thrombocytopenia and thrombotic events. Additionally, identifying Cath-HG as a GPVI inhibitor provides insights for developing novel antithrombotic therapies targeting platelet activation mediated by GPVI.

Department of Biochemistry Cardiovascular Research Institute Maastricht Maastricht University 6229 ER Maastricht Netherlands

Institute of Molecular Biology and Biotechnology Foundation for Research and Technology Hellas 70013 Heraklion Crete Greece

Institute of Parasitology Biology Centre of the Czech Academy of Sciences Branisovska 31 Budweis 37005 Czech Republic

NMPA Key Laboratory for Research and Evaluation of Drug Metabolism Guangdong Provincial Key Laboratory of New Drug Screening School of Pharmaceutical Sciences Southern Medical University Guangzhou 510515 China

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Poll T, Shankar-Hari M, Wiersinga WJ. The immunology of sepsis. Immunity. 2021;54(11):2450–2464. PubMed

Du X, Zhang M, Zhou H, Wang W, Zhang C, Zhang L, Qu Y, Li W, Liu X, Zhao M, et al. . Decoy nanozymes enable multitarget blockade of proinflammatory cascades for the treatment of multi-drug-resistant bacterial sepsis. Research. 2022;2022:9767643. PubMed PMC

Bao W, Xing H, Cao S, Long X, Liu H, Ma J, Guo F, Deng Z, Liu X. Neutrophils restrain sepsis associated coagulopathy via extracellular vesicles carrying superoxide dismutase 2 in a murine model of lipopolysaccharide induced sepsis. Nat Commun. 2022;13(1):4583. PubMed PMC

Iba T, Levy JH, Warkentin TE, Thachil J, Poll T, Levi M, Scientific and Standardization Committee on DIC, and the Scientific and Standardization Committee on Perioperative and Critical Care of the International Society on Thrombosis and Haemostasis . Diagnosis and management of sepsis-induced coagulopathy and disseminated intravascular coagulation. J Thromb Haemost. 2019;17(11):1989–1994. PubMed

Iba T, Watanabe E, Umemura Y, Wada T, Hayashida K, Kushimoto S, Japanese Surviving Sepsis Campaign Guideline Working Group for disseminated intravascular coagulation, Wada H. Sepsis-associated disseminated intravascular coagulation and its differential diagnoses. J Intensive Care. 2019;7(1):32. PubMed PMC

Luong HX, Thanh TT, Tran TH. Antimicrobial peptides – Advances in development of therapeutic applications. Life Sci. 2020;260: Article 118407. PubMed PMC

Jing X, Jin K. A gold mine for drug discovery: Strategies to develop cyclic peptides into therapies. Med Res Rev. 2020;40(2):753–810. PubMed

Vilas Boas LCP, Campos ML, Berlanda RLA, Carvalho NN, Franco OL. Antiviral peptides as promising therapeutic drugs. Cell Mol Life Sci. 2019;76(18):3525–3542. PubMed PMC

Otazo-Pérez A, Asensio-Calavia P, González-Acosta S, Baca-González V, López MR, Morales-delaNuez A, Pérez de la Lastra JM. Antimicrobial activity of cathelicidin-derived peptide from the Iberian mole Talpa occidentalis. Vaccine. 2022;10(7):1105. PubMed PMC

Wang Y, Ouyang J, Luo X, Zhang M, Jiang Y, Zhang F, Zhou J, Wang Y. Identification and characterization of novel bi-functional cathelicidins from the black-spotted frog (Pelophylax nigromaculata) with both anti-infective and antioxidant activities. Dev Comp Immunol. 2021;116: Article 103928. PubMed

Wu J, Yang J, Wang X, Wei L, Mi K, Shen Y, Liu T, Yang H, Mu L. A frog cathelicidin peptide effectively promotes cutaneous wound healing in mice. Biochem J. 2018;475(17):2785–2799. PubMed PMC

Chai J, Chen X, Ye T, Zeng B, Zeng Q, Wu J, Kascakova B, Martins LA, Prudnikova T, Smatanova IK, et al. . Characterization and functional analysis of cathelicidin-MH, a novel frog-derived peptide with anti-septicemic properties. Elife. 2021;10:e64411. PubMed PMC

Hao X, Yang H, Wei L, Yang S, Zhu W, Ma D, Yu H, Lai R. Amphibian cathelicidin fills the evolutionary gap of cathelicidin in vertebrate. Amino Acids. 2012;43(2):677–685. PubMed

Gao F, Xu WF, Tang LP, Wang MM, Wang XJ, Qian YC. Characteristics of cathelicidin-Bg, a novel gene expressed in the ear-side gland of Bufo gargarizans. Genet Mol Res. 2016;15(3): 10.4238/gmr.15038481. PubMed

Ling G, Gao J, Zhang S, Xie Z, Wei L, Yu H, Wang Y. Cathelicidins from the bullfrog Rana catesbeiana provides novel template for peptide antibiotic design. PLoS One. 2014;9(3): Article e93216. PubMed PMC

Cao X, Wang Y, Wu C, Li X, Fu Z, Yang M, Bian W, Wang S, Song Y, Tang J, et al. . Cathelicidin-OA1, a novel antioxidant peptide identified from an amphibian, accelerates skin wound healing. Sci Rep. 2018;8(1):943. PubMed PMC

Mu L, Zhou L, Yang J, Zhuang L, Tang J, Liu T, Wu J, Yang H. The first identified cathelicidin from tree frogs possesses anti-inflammatory and partial LPS neutralization activities. Amino Acids. 2017;49(9):1571–1585. PubMed PMC

Zanetti M. Cathelicidins, multifunctional peptides of the innate immunity. J Leukoc Biol. 2004;75(1):39–48. PubMed

Chen Q, Cheng P, Ma C, Xi X, Wang L, Zhou M, Bian H, Chen T. Evaluating the bioactivity of a novel broad-spectrum antimicrobial peptide Brevinin-1GHa from the frog skin secretion of Hylarana guentheri and its analogues. Toxins (Basel). 2018;10(10):413. PubMed PMC

Zeng B, Chai J, Deng Z, Ye T, Chen W, Li D, Chen X, Chen M, Xu X. Functional characterization of a novel lipopolysaccharide-binding antimicrobial and anti-inflammatory peptide in vitro and in vivo. J Med Chem. 2018;61(23):10709–10723. PubMed

Zhou J, McClean S, Thompson A, Zhang Y, Shaw C, Rao P, Bjourson AJ. Purification and characterization of novel antimicrobial peptides from the skin secretion of Hylarana guentheri. Peptides. 2006;27(12):3077–3084. PubMed

Qi R, Chen Y, Guo Z, Zhang F, Fang Z, Huang K, Yu HN, Wang YP. Identification and characterization of two novel cathelicidins from the frog Odorrana livida. Zool Res. 2019;40(2):94–101. PubMed PMC

Rittirsch D, Huber-Lang MS, Flierl MA, Ward PA. Immunodesign of experimental sepsis by cecal ligation and puncture. Nat Protoc. 2009;4(1):31–36. PubMed PMC

Wu J, Guo R, Chai J, Xiong W, Tian M, Lu W, Lu X. The protective effects of Cath-MH with anti-Propionibacterium acnes and anti-inflammation functions on acne vulgaris. Front Pharmacol. 2021;12:788358. PubMed PMC

Weiss LJ, Manukjan G, Pflug A, Winter N, Weigel M, Nagler N, Kredel M, Lâm TT, Nieswandt B, Weismann D, et al. . Acquired platelet GPVI receptor dysfunction in critically ill patients with sepsis. Blood. 2021;137(22):3105–3115. PubMed

Cuccurullo A, Greco E, Lupia E, De Giuli P, Bosco O, Martin-Conte E, Spatola T, Turco E, Montrucchio G. Blockade of thrombopoietin reduces organ damage in experimental endotoxemia and polymicrobial sepsis. PLoS One. 2016;11(3): Article e0151088. PubMed PMC

Steven S, Jurk K, Kopp M, Kröller-Schön S, Mikhed Y, Schwierczek K, Roohani S, Kashani F, Oelze M, Klein T, et al. . Glucagon-like peptide-1 receptor signalling reduces microvascular thrombosis, nitro-oxidative stress and platelet activation in endotoxaemic mice. Br J Pharmacol. 2017;174(12):1620–1632. PubMed PMC

Stoppelaar SF, Veer C, TAM Claushuis, BJA Albersen, JJTH Roelofs, Poll T. Thrombocytopenia impairs host defense in gram-negative pneumonia–derived sepsis in mice. Blood. 2014;124(25):3781–3790. PubMed PMC

Jiao Y, Li W, Wang W, Tong X, Xia R, Fan J, du J, Zhang C, Shi X. Platelet-derived exosomes promote neutrophil extracellular trap formation during septic shock. Crit Care. 2020;24(1):380. PubMed PMC

Sygitowicz G, Sitkiewicz D. Molecular mechanisms of organ damage in sepsis: An overview. Brazilian J Infect Dis. 2020;24(6):552–560. PubMed PMC

Fleischmann C, Scherag A, Adhikari NKJ, Hartog CS, Tsaganos T, Schlattmann P, Angus DC, Reinhart K, International Forum of Acute Care Trialists . Assessment of global incidence and mortality of hospital-treated sepsis. Current estimates and limitations. Am J Respir Crit Care Med. 2016;193(3):259–272. PubMed

Xu X, Lai R. The chemistry and biological activities of peptides from amphibian skin secretions. Chem Rev. 2015;115(4):1760–1846. PubMed

Wu J, Zhang H, Chen X, Chai J, Hu Y, Xiong W, Lu W, Tian M, Chen X, Xu X. FM-CATH, A novel cathelicidin from Fejervarya multistriata, shows therapeutic potential for treatment of CLP-induced sepsis. Front Pharmacol. 2021;12:731056. PubMed PMC

Xiong W, Zhou C, Yin S, Chai J, Zeng B, Wu J, Li Y, Li L, Xu X. Fejerlectin, a lectin-like peptide from the skin of Fejervarya limnocharis, inhibits HIV-1 entry by targeting gp41. ACS Omega. 2021;6(9):6414–6423. PubMed PMC

Chai J, Liu J, Tian M, Liao H, Wu J, Xie J, Lai S, Mo G, Chen X, Xu X. Multiple mechanistic action of brevinin-1FL peptide against oxidative stress effects in an acute inflammatory model of carrageenan-induced damage. Oxidative Med Cell Longev. 2022;2022:1–21. PubMed PMC

Li J, Karakas D, Xue F, Chen Y, Zhu G, Yucel YH, MacParland SA, Zhang H, Semple JW, Freedman J, et al. . Desialylated platelet clearance in the liver is a novel mechanism of systemic immunosuppression. Research. 2023;6:0236. PubMed PMC

Li C, Li J, Ni H. Crosstalk between platelets and microbial pathogens. Front Immunol. 2020;11:1962. PubMed PMC

Ma X, Liang J, Zhu G, Bhoria P, Shoara AA, MacKeigan DT, Khoury CJ, Slavkovic S, Lin L, Karakas D, et al. . SARS-CoV-2 RBD and its variants can induce platelet activation and clearance: Implications for antibody therapy and vaccinations against COVID-19. Research. 2023;6:0124. PubMed PMC

Wang Y, Ouyang Y, Liu B, Ma X, Ding R. Platelet activation and antiplatelet therapy in sepsis: A narrative review. Thromb Res. 2018;166:28–36. PubMed

Seidel M, Winning J, Claus RA, Bauer M, Lösche W. Beneficial effect of clopidogrel in a mouse model of polymicrobial sepsis. J Thromb Haemost. 2009;7(6):1030–1032. PubMed

Wang X, Deng H, Li T, Miao S, Xiao Z, Liu M, Liu K, Xiao XZ. Clopidogrel reduces lipopolysaccharide-induced inflammation and neutrophil-platelet aggregates in an experimental endotoxemic model. J Biochem Mol Toxicol. 2019;33(4): Article e22279. PubMed

Halbach JL, Wang AW, Hawisher D, Cauvi DM, Lizardo RE, Rosas J, Reyes T, Escobedo O, Bickler SW, Coimbra R, et al. . Why antibiotic treatment is not enough for sepsis resolution: An evaluation in an experimental animal model. Infect Immun. 2017;85(12):e00664-17. PubMed PMC

Peng Z-Y, Wang H-Z, Srisawat N, Wen X, Rimmelé T, Bishop J, Singbartl K, Murugan R, Kellum JA. Bactericidal antibiotics temporarily increase inflammation and worsen acute kidney injury in experimental sepsis. Crit Care Med. 2012;40(2):538–543. PubMed PMC

Patel A, Joseph J, Periasamy H, Mokale S. Azithromycin in combination with ceftriaxone reduces systemic inflammation and provides survival benefit in a murine model of polymicrobial sepsis. Antimicrob Agents Chemother. 2018;62(9):e00752. PubMed PMC

Manon-Jensen T, Kjeld NG, Karsdal MA. Collagen-mediated hemostasis. J Thromb Haemost. 2016;14(3):438–448. PubMed

Martins Lima A, Martins Cavaco AC, Fraga-Silva RA, Eble JA, Stergiopulos N. From patients to platelets and back again: Pharmacological approaches to glycoprotein VI, a thrilling antithrombotic target with minor bleeding risks. Thromb Haemost. 2019;119(11):1720–1739. PubMed

Dubois C, Panicot-Dubois L, Merrill-Skoloff G, Furie B, Furie BC. Glycoprotein VI–dependent and –independent pathways of thrombus formation in vivo. Blood. 2006;107(10):3902–3906. PubMed PMC

Claushuis TAM, Vos AF, Nieswandt B, Boon L, Roelofs JJTH, Boer OJ, van ’t Veer C, van der Poll T. Platelet glycoprotein VI aids in local immunity during pneumonia-derived sepsis caused by gram-negative bacteria. Blood. 2018;131(8):864–876. PubMed

Burkard P, Schonhart C, Vögtle T, Köhler D, Tang L, Johnson D, Hemmen K, Heinze KG, Zarbock A, Hermanns HM, et al. . A key role for platelet GPVI in neutrophil recruitment, migration, and NETosis in the early stages of acute lung injury. Blood. 2023;142(17):1463–1477. PubMed

Teufel F, Almagro Armenteros JJ, Johansen AR, Gíslason MH, Pihl SI, Tsirigos KD, Winther O, Brunak S, von Heijne G, Nielsen H. SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat Biotechnol. 2022;40(7):1023–1025. PubMed PMC

Gonçalves MC, Horewicz VV, Lückemeyer DD, Prudente AS, Assreuy J. Experimental sepsis severity score associated to mortality and bacterial spreading is related to bacterial load and inflammatory profile of different tissues. Inflammation. 2017;40(5):1553–1565. PubMed

Tokalov SV, Bachiller D. IV delivery of fluorescent beads. Chest. 2012;141(3):833–834. PubMed

Wang S, Wang X, Liu C, Chou R, Chen Z, Huang P, Lin SJ. The dipeptidyl peptidase-4 inhibitor linagliptin ameliorates endothelial inflammation and microvascular thrombosis in a sepsis mouse model. Int J Mol Sci. 2022;23(6):3065. PubMed PMC

Rayes J, Lax S, Wichaiyo S, Watson SK, Di Y, Lombard S, Grygielska B, Smith SW, Skordilis K, Watson SP. The podoplanin-CLEC-2 axis inhibits inflammation in sepsis. Nat Commun. 2017;8(1):2239. PubMed PMC

Xiong W, Li J, Feng Y, Chai J, Wu J, Hu Y, Tian M, Lu W, Xu X, Zou M. Brevinin-2GHk, a peptide derived from the skin of Fejervarya limnocharis, inhibits Zika virus infection by disrupting viral integrity. Viruses. 2021;13(12):2382. PubMed PMC

Pierce BG, Wiehe K, Hwang H, Kim B-H, Vreven T, Weng Z. ZDOCK server: Interactive docking prediction of protein-protein complexes and symmetric multimers. Bioinformatics. 2014;30(12):1771–1773. PubMed PMC

Linghu L, Zong W, Liao Y, Chen Q, Meng F, Wang G, Liao Z, Lan X, Chen M. Herpetrione, a new type of PPARα ligand as a therapeutic strategy against nonalcoholic steatohepatitis. Research. 2023;6:0276. PubMed PMC

Zhang Z, Shen C, Fang M, Han Y, Long C, Liu W, Yang M, Liu M, Zhang D, Cao Q, et al. . Novel contact–kinin inhibitor sylvestin targets thromboinflammation and ameliorates ischemic stroke. Cell Mol Life Sci. 2022;79(5):240. PubMed PMC