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Antimicrobial Peptide Cathelicidin-BF Inhibits Platelet Aggregation by Blocking Protease-Activated Receptor 4

  • Guofang Shu
  • Yahui Chen
  • Tongdan Liu
  • Shenhong Ren
  • Yi KongEmail author
Article
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Abstract

Cathelicidin-BF (BF-30), a peptide isolated from the snake venom of Bungarus fasciatus, exhibits multiple biological functions, including antimicrobial, anticancer and anti-inflammatory. However, the effect of BF-30 on platelet and thrombus formation was reported rarely. In this study, we investigated the antiplatelet and antithrombotic effects of BF-30 and its underlying mechanism. Our results showed that BF-30 potently inhibited thrombin-induced platelet aggregation, and further specifically blocked protease-activated receptor 4 (PAR4). It also reduced P-selectin expression, AktSer473 phosphorylation and platelet spreading on fibrinogen. Furthermore, BF-30 exhibited potent inhibitory activity on thrombus formation in vivo: it decreased death of mice with acute pulmonary thrombosis and attenuated thrombosis weight in arterio-venous shunt model. Additionally, a tail cutting bleeding time assay revealed that BF-30 did not prolong bleeding time in mice at efficient dosage. Taken together, BF-30 is a PAR4 antagonist, and inhibits thrombus formation without obvious bleeding risk in vivo. We believe that this study may provide a source for the development of PAR4 antagonist for the treatment of thrombotic disorders.

Keywords

Cathelicidin-BF Protease-activated receptor 4 Antagonist Platelet aggregation Antithrombosis 

Notes

Acknowledgements

This study was supported by the Chinese National Natural Science Foundation (81273375) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Compliance with Ethical Standards

Conflict of interest

Guofang Shu, Yahui Chen, Tongdan Liu, Shenhong Ren and Yi Kong declares that they have no conflict of interest.

Ethical Approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Informed Consent

Informed consent was obtained from all individual participants included in the study.

References

  1. Arbel Y, Weitzman D, Raz R et al (2014) Red blood cell distribution width and the risk of cardiovascular morbidity and all-cause mortality. Thromb Haemost 111(2):300–307Google Scholar
  2. Atsuhiro S, Fumitoshi A, Taketoshi O, Teruhike I, Hiroyuki K (2000) The in vivo pharmacological profile of CS-747, a novel antiplatele agent with platelet ADP receptor antagonist properties. Br J Pharmacol 129:1439–1446Google Scholar
  3. Brass L-F (2003) Thrombin and platelet activation. CHEST 124:18S–25SGoogle Scholar
  4. Brill A, Chauhan A-K, Canault M, Walsh M-T, Bergmeier W, Wagner D-D (2009) Oxidative stress activates ADAM17/TACE and induces its target receptor shedding in platelets in a p38-dependent fashion. Cardiovasc Res 84:137–144Google Scholar
  5. Canobbio I, Balduini C, Torti M (2004) Signalling through the platelet glycoprotein Ib-V–IX complex. Cell Signal 16(12):1329–1344Google Scholar
  6. Catella-Lawson F, Reilly MP, Kapoor SC et al (2001) Cyclooxygenase inhibitors and the antiplatelet effects of aspirin. New Engl J Med 345(25):1809–1817Google Scholar
  7. Chackalamannil S (2006) Thrombin receptor (protease activated receptor-1) antagonists as potent antithrombotic agents with strong antiplatelet effects. J Med Chem 49(18):5389–5403Google Scholar
  8. Chen M, Ye X, Ming X et al (2015a) A novel direct factor xa inhibitory peptide with anti-platelet aggregation activity from agkistrodon acutus venom hydrolysates. Sci Rep 5:10846Google Scholar
  9. Chen Y, Wang Y, Xie Z, Ming X, Li Z, Kong Y (2015b) A tryptophan derivative TD-26 attenuates thrombus formation by inhibiting both PI3K/Akt signaling and binding of fibrinogen to integrin alphaIIbbeta3. Biochem Biophys Res Commun 465:516–522Google Scholar
  10. Coughlin SR (2000) Thrombin signalling and protease-activated receptors. Nature 407(6801):258–264Google Scholar
  11. Coughlin SR (2005) Protease-activated receptors in hemostasis, thrombosis and vascular biology. J Thromb Haemost 3(8):1800–1814Google Scholar
  12. Davie EW, Fujikawa K, Kisiel W (1991) The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 30(43):10363–10370Google Scholar
  13. Disdier M, Morrissey JH, Fugate RD, Bainton DF, McEver RP (1992) Cytoplasmic domain of P-selectin (CD62) contains the signal for sorting into the regulated secretory pathway. Mol Biol Cell 3(3):309–321Google Scholar
  14. Elcioglu OC, Ozkok A, Akpınar TS et al (2012) Severe thrombocytopenia and alveolar hemorrhage represent two types of bleeding tendency during tirofiban treatment: case report and literature review. Int J Hematol 96(3):370–375Google Scholar
  15. Engelmann B, Massberg S (2013) Thrombosis as an intravascular effector of innate immunity. Nat Rev Immunol 13:34–45Google Scholar
  16. Furie B, Furie BC (2008) Mechanisms of thrombus formation. New Engl J Med 359(9):938–949Google Scholar
  17. Goldhaber SZ, Bounameaux H (2012) Pulmonary embolism and deep vein thrombosis. Lancet 379:1835–1846Google Scholar
  18. Hankey GJ, Eikelboom JW (2003) Antiplatelet drugs. Med J Australia 178(11):568–574Google Scholar
  19. Holinstat M, Voss B, Bilodeau ML, McLaughlin JN, Cleator J, Hamm HE (2006) PAR4, but not PAR1, signals human platelet aggregation via Ca2+ mobilization and synergistic P2Y12 receptor activation. J Biol Chem 281(36):26665–26674Google Scholar
  20. Huang ZS, Zeng CL, Zhu LJ, Jiang L, Li N, Hu H (2010) Salvianolic acid A inhibits platelet activation and arterial thrombosis via inhibition of phosphoinositide 3-kinase. J Thromb Haemost 8:1383–1393Google Scholar
  21. Jacques S, Kuliopulos A (2003) Protease-activated receptor-4 uses dual prolines and an anionic retention motif for thrombin recognition and cleavage. Biochem J 376:733–740Google Scholar
  22. Khan A, Li D, Ibrahim S, Smyth E, Woulfe DS (2014) The physical association of the P2Y12 receptor with PAR4 regulates arrestin-mediated Akt activation. Mol Pharmacol 86(1):1–11Google Scholar
  23. Kim S, Jin J, Kunapuli SP (2006) Relative contribution of G-protein-coupled pathways to protease-activated receptor-mediated Akt phosphorylation in platelets. Blood 107(3):947–954Google Scholar
  24. Kong Y, Xu C, He ZL et al (2014) A novel peptide inhibitor of platelet aggregation from stiff silkworm, Bombyx batryticatus. Peptides 53:70–78Google Scholar
  25. Kuliopulos A, Covic L (2003) Blocking receptors on the inside: pepducin-based intervention of PAR signaling and thrombosis. Life Sci 74:255–262Google Scholar
  26. Lin H, Liu AP, Smith TH, Trejo J (2013) Cofactoring and dimerization of proteinase-activated receptors. Pharmacol Rev 65(4):1198–1213Google Scholar
  27. Mackman N (2008) Triggers, targets and treatments for thrombosis. Nature 451:914–918Google Scholar
  28. Mumaw MM, Noble DN, Nieman MT (2014) Targeting the anionic region of human protease-activated receptor 4 inhibits platelet aggregation and thrombosis without interfering with hemostasis. J Thromb Haemost 12(8):1331–1341Google Scholar
  29. Nieman MT (2008) Protease-activated receptor 4 uses anionic residues to interact with α-thrombin in the absence or presence of protease-activated receptor 1†. Biochemistry 47(50):13279–13286Google Scholar
  30. Rivera J, Lozano ML, Navarro-Núñez L, Vicente V (2009) Platelet receptors and signaling in the dynamics of thrombus formation. Haematologica 94(5):700–711Google Scholar
  31. Saravanan RA (2007) Hematologic adverse effects of clopidogrel. Am J Ther 14:106–112Google Scholar
  32. Shapiro MJ, Weiss EJ, Faruqi TR, Coughlin SR (2000) Protease-activated receptors 1 and 4 are shut off with distinct kinetics after activation by thrombin. J Biol Chem 275(33):25216–25221Google Scholar
  33. Shen B, Delaney MK, Du X (2012) Inside-out, outside-in, and inside–outside-in: G protein signaling in integrin-mediated cell adhesion, spreading, and retraction. Curr Opin Cell Biol 24(5):600–606Google Scholar
  34. Su X-L, Su W, He Z-L, Ming X, Kong Y (2015) Tripeptide SQL inhibits platelet aggregation and thrombus formation by affecting PI3k/AKT signaling. J Cardiovase Pharmacol 66:254–260Google Scholar
  35. Suen JY, Barry GD, Lohman RJ et al (2012) Modulating human proteinase activated receptor 2 with a novel antagonist (GB88) and agonist (GB110). Br J Pharmacol 165:1413–1423Google Scholar
  36. Tricoci P, Huang Z, Held C et al (2012) Thrombin-receptor antagonist vorapaxar in acute coronary syndromes. New Engl J Med 366(1):20–33Google Scholar
  37. Tzakos AG, Kontogianni VG, Tsoumani M et al (2012) Exploration of the antiplatelet activity profile of betulinic acid on human platelets. J Agr Food Chem 60(28):6977–6983Google Scholar
  38. Vogel GMT, Meuleman DG, Bourgondiën FGM, Hobbelen PMJ (1989) Comparison of two experimental thrombosis models in rats effects of four glycosaminoglycans. Thromb Res 54(5):399–410Google Scholar
  39. Warkentin TE (2004) Bivalent direct thrombin inhibitors: hirudin and bivalirudin. Best Pract Res Clin Haematol 17(1):105–125Google Scholar
  40. Wu CC, Huang SW, Hwang TL, Kuo SC, Lee FY, Teng CM (2000) YD-3, a novel inhibitor of protease-induced platelet activation. Br J Pharmacol 130(6):1289–1296Google Scholar
  41. Xia X, Zhang L, Wang Y (2015) The antimicrobial peptide cathelicidin-BF could be a potential therapeutic for Salmonella typhimurium infection. Microbiol Res 171:45–51Google Scholar
  42. Yasuko K, Yasuhiro K, Mie N et al (1999) In vitro antiplatelet profile of FR171113 a novel non-peptide thrombin receptor antagonist. Eur J Pharmacol 384:197–202Google Scholar
  43. Yin H, Liu J, Li Z, Berndt MC, Lowell CA, Du X (2008) Src family tyrosine kinase Lyn mediates VWF/GPIb-IX-induced platelet activation via the cGMP signaling pathway. Blood 112:1139–1146Google Scholar
  44. Zhang XX, Han F-F et al (2015) Cathelicidin-BF, a novel antimicrobial peptide from Bungarus fasciatus, attenuates disease in a dextran sulfate sodium model of colitis. Mol Pharm 12:1648–1661Google Scholar
  45. Zhou DJ, Wang J et al (2011) The antibacterial activity of BF-30 in vitro and in infected burned rats is through interference with cytoplasmic membrane integrity. Peptides 32:1131–1138Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Guofang Shu
    • 1
  • Yahui Chen
    • 2
  • Tongdan Liu
    • 2
  • Shenhong Ren
    • 2
  • Yi Kong
    • 2
    Email author
  1. 1.Center of Clinical Laboratory Medicine of Zhongda HospitalSoutheast UniversityNanjingPeople’s Republic of China
  2. 2.School of Life Science & TechnologyChina Pharmaceutical UniversityNanjingPeople’s Republic of China

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