Lebetin Peptides, A New Class of Potent Platelet Aggregation Inhibitors: Chemical Synthesis, Biological Activity and NMR Spectroscopic Study

  • Amor MosbahEmail author
  • Naziha Marrakchi
  • Pascal Mansuelle
  • Soumaya Kouidhi
  • Ernest Giralt
  • Mohamed El Ayeb
  • Gaëtan Herbette
  • Ameur Cherif
  • Didier Gigmes
  • Hervé Darbon
  • Kamel MabroukEmail author


Platelets have a well-established role in atherosclerosis and related diseases. Lebetins from the venom of Vipera lebetina, lacking the RGD sequence, emerged as a new family of platelet aggregation inhibitors. However, the interaction sites and precise mechanism between lebetin and its substrate remain unclear. Here, we successfully synthesized two peptide analogs, which differ only by one glycine residue at the N-terminus: lebetin 2α (sL2α residues) and lebetin 2β (sL2ββ residues) were produced in sufficient quantities for structural and functional studies. NMR structure determination showed that the sL2α peptide adopts a compact ring conformation stabilized by a disulfide bond, from which emerge one loop and two extended regions, the C- and N-termini. Interestingly, two RGD-like motifs were identified in the structure of the peptides, suggesting an anti-platelet aggregation effect of the two isoforms. Indeed, activity was demonstrated on human and rabbit platelet-rich plasma where sL2α and sL2β showed more potent inhibitory effect on platelet aggregation compared to the previously described native lebetin 1. Synthetic lebetin 2 peptides constitute promising candidates for drug design toward chimeric compounds with high anti-platelet and natriuretic effects. These findings contribute to a novel field of research triggering platelet activation and natriuretic action.


NMR structure Peptide Synthesis Anti-platelet Aggregation Natriuretic factor 



The authors thank Dr. Harold de Pomyers for helpful discussion. IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain)., and is included in the CERCA Programme of the Catalan Government.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10989_2019_9812_MOESM1_ESM.docx (713 kb)
Supplementary material 1 (DOCX 712 KB)


  1. Adamson K, Dolan C, Moran N, Forster RJ, Keyes TE (2014) RGD labeled Ru(II) polypyridyl conjugates for platelet integrin αIIbβ3 recognition and as reporters of integrin conformation. Bioconjug Chem 25(5):928–944CrossRefGoogle Scholar
  2. Amininasab M, Elmi MM, Endlich N, Endlich K, Parekh N, Naderi-Manesh H, Schaller J, Mostafavi H, Sattler M, Sarbolouki MN, Muhle-Goll C (2004) Functional and structural characterization of a novel member of the natriuretic family of peptides from the venom of Pseudocerastes persicus. FEBS Lett 557(1–3):104–108CrossRefGoogle Scholar
  3. Barbouche R, Marrakchi N, Mansuelle P, Krifi M, Fenouillet E, Rochat H, Ayeb M (1996) Novel anti-platelet aggregation polypeptides from Vipera lebetina venom: isolation and characterization. FEBS Lett 392(1):6–10CrossRefGoogle Scholar
  4. Barbouche R, Marrakchi N, Mabrouk K, Krifi MN, Van Rietschoten J, Fenouillet E, El Ayeb M, Rochat H (1998) Anti-platelet activity of the peptides composing the lebetin 1 family, a new class of inhibitors of platelet aggregation. Toxicon 36(12):1939–1947CrossRefGoogle Scholar
  5. Bartels C, Xia TH, Billeter M, Güntert P, Wüthrich K (1995) The program XEASY for computer-supported NMR spectral analysis of biological macromolecules. J Biomol NMR 6(1):1–10CrossRefGoogle Scholar
  6. Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr Sect D 54(Pt 5):905–921CrossRefGoogle Scholar
  7. Brunne RM, Berndt KD, Guntert P, Wuthrich K, van Gunsteren WF (1995) Structure and internal dynamics of the bovine pancreatic trypsin inhibitor in aqueous solution from long-time molecular dynamics simulations. Proteins 23(1):49–62CrossRefGoogle Scholar
  8. Chiba A, Watanabe-Takano H, Terai K, Fukui H, Miyazaki T, Uemura M, Hashimoto H, Hibi M, Fukuhara S, Mochizuki N (2017) Osteocrin, a peptide secreted from the heart and other tissues, contributes to cranial osteogenesis and chondrogenesis in zebrafish. Development 144(2):334–344CrossRefGoogle Scholar
  9. Clemetson KJ, Lu Q, Clemetson JM (2007) Snake venom proteins affecting platelets and their applications to anti-thrombotic research. Curr Pharm Des 13(28):2887–2892CrossRefGoogle Scholar
  10. Coccheri S (2010) Antiplatelet drugs–do we need new options? With a reappraisal of direct thromboxane inhibitors. Drugs 70(7):887–908CrossRefGoogle Scholar
  11. Dretzke J, Riley RD, Lordkipanidzé M, Jowet S, O’Donnell J, Ensor J, Moloney E, Price M, Raichand S, Hodgkinson J, Bayliss S, Fitzmaurice D, Moore D (2015) The prognostic utility of tests of platelet function for the detection of ‘aspirin resistance’ in patients with established cardiovascular or cerebrovascular disease: a systematic review and economic evaluation. Health Technol Assess 19(37):1–366CrossRefGoogle Scholar
  12. Drouet L, Bal dit Sollier C, Henry P (2010) The basis of platelets: platelets and atherothrombosis: an understanding of the lack of efficacy of aspirin in peripheral arterial disease (PAD) and diabetic patients. Drugs 70(Suppl 1):9–14CrossRefGoogle Scholar
  13. Fairbrother WJ, McDowell RS, Cunningham BC (1994) Solution conformation of an atrial natriuretic peptide variant selective for the type A receptor. Biochemistry 33(30):8897–8904CrossRefGoogle Scholar
  14. Gairí M, Dyachenko A, González MT, Feliz M, Pons M, Giralt E (2015) An optimized method for (15)N R(1) relaxation rate measurements in non-deuterated proteins. J Biomol NMR 62(2):209–220CrossRefGoogle Scholar
  15. Golebiewska EM, Poole AW (2015) Platelet secretion: from haemostasis to wound healing and beyond. Blood Rev 29(3):153–162CrossRefGoogle Scholar
  16. Guntert P, Wuthrich K (1991) Improved efficiency of protein structure calculations from NMR data using the program DIANA with redundant dihedral angle constraints. J Biomol NMR 1(4):447–456CrossRefGoogle Scholar
  17. Guntert P, Braun W, Wuthrich K (1991) Efficient computation of three-dimensional protein structures in solution from nuclear magnetic resonance data using the program DIANA and the supporting programs CALIBA, HABAS and GLOMSA. J Mol Biol 217(3):517–530CrossRefGoogle Scholar
  18. Hanes J, Jermutus L, Plückthun A (2000) Selecting and evolving functional proteins in vitro by ribosome display. Methods Enzymol 328:404–430CrossRefGoogle Scholar
  19. He XL, Dukkipati A, Garcia KC (2006) Structural determinants of natriuretic peptide receptor specificity and degeneracy. J Mol Biol 361(4):698–714CrossRefGoogle Scholar
  20. Holm L, Rosenstrom P (2010) Dali server: conservation mapping in 3D. Nucleic Acids Res 38:W545–W549CrossRefGoogle Scholar
  21. Jennings LK (2009) Mechanisms of platelet activation: need for new strategies to protect against platelet-mediated atherothrombosis. Thromb Haemost 102(2):248–257Google Scholar
  22. Keller TT, Squizzato A, Middeldorp S (2007) Clopidogrel plus aspirin versus aspirin alone for preventing cardiovascular disease. Cochrane Database Syst Rev 3:CD005158Google Scholar
  23. Koehl P, Delarue M (2010) AQUASOL: an efficient solver for the dipolar Poisson-Boltzmann-Langevin equation. J Chem Phys 132(6):064101CrossRefGoogle Scholar
  24. Kononova O, Litvinov RI, Blokhin DS, Klochkov VV, Weisel JW, Bennett JS (2017) Mechanistic Basis for the Binding of RGD- and AGDV-Peptides to the Platelet Integrin αIIbβ3. Biochemistry 56(13):1932–1942CrossRefGoogle Scholar
  25. Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8(4):477–486CrossRefGoogle Scholar
  26. Lewis RJ, Garcia ML (2003) Therapeutic potential of venom peptides. Nat Rev Drug Discov 2(10):790–802CrossRefGoogle Scholar
  27. Marion D, Wuthrich K (1983) Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins. Biochem Biophys Res Commun 113(3):967–974CrossRefGoogle Scholar
  28. Martin-Du Pan RC, Ricou F (2003) Use of brain natriuretic peptide (BNP) in the diagnosis and treatment of heart failure. Rev Med Suisse Romande 123(2):125–128Google Scholar
  29. McNicholas S, Potterton E, Wilson KS, Noble MEM (2011) Presenting your structures: the CCP4mg molecular-graphics software. Acta Cryst D67:386–394Google Scholar
  30. Merrifield B (1986) Solid phase synthesis. Science 232(4748):341–347CrossRefGoogle Scholar
  31. Perutelli P (1994) Disintegrins: potent inhibitors of platelet aggregation. Recent Prog Med 86(4):168–174Google Scholar
  32. Piotto M, Saudek V, Sklenar V (1992) Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR 2(6):661–665CrossRefGoogle Scholar
  33. Rocchia W, Sridharan S, Nicholls A, Alexov E, Chiabrera A, Honig B (2002) Rapid grid based construction of the molecular surface and the use of induced surface charge to calculate reaction field energies: applications to the molecular systems and geometric objects. J Comput Chem 23(1):128–137CrossRefGoogle Scholar
  34. Rosenzweig A, Seidman CE (1991) Atrial natriuretic factor and related peptide horiviones. Annu Rev Biochem 60:229–255CrossRefGoogle Scholar
  35. Salles II, Feys HB, Iserbyt BF, De Meyer SF, Vanhoorelbeke K, Deckmyn H (2008) Inherited traits affecting platelet function. Blood Rev 22(3):155–172CrossRefGoogle Scholar
  36. Sánchez-Cortés J, Mrksich M (2009) The platelet integrin αIIbβ3 binds to the RGD and AGD motifs in fibrinogen. Chem Biol 16(9):990–1000CrossRefGoogle Scholar
  37. Squizzato A, Keller T, Romualdi E, Middeldorp S (2011) Clopidogrel plus aspirin versus aspirin alone for preventing cardiovascular disease. Cochrane Database Syst Rev 1:CD005158Google Scholar
  38. Tourki B, Matéo P, Morand J, Elayeb M, GodinRibuot D, Marrakchi N, Belaidi E, Messadi E (2016) Lebetin 2, a snake venom-derived natriuretic peptide, attenuates acute myocardial ischemic injury through the modulation of mitochondrial permeability transition pore at the time of reperfusion. PLoS ONE 11(9):e0162632CrossRefGoogle Scholar
  39. Ulker S, Akgür S, Evinç A, Soykan N, Koşay S (1995) Platelet aggregation and atrial natriuretic peptide. Gen Pharmacol 26(6):1409–1412CrossRefGoogle Scholar
  40. Vaguine AA, Richelle J, Wodak SJ (1999) SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model. Acta Crystallogr Sect D 55(Pt 1):191–205CrossRefGoogle Scholar
  41. Valentine N, Van de Laar FA, van Driel ML (2012) Adenosine-diphosphate (ADP) receptor antagonists for the prevention of cardiovascular disease in type 2 diabetes mellitus. Cochrane Database Syst Rev 11:CD005449Google Scholar
  42. Williams JA (1992) Disintegrins: RGD-containing proteins which inhibit cell/matrix interactions (adhesion) and cell/cell interactions (aggregation) via the integrin receptors. Pathol Biol 40(8):813–821Google Scholar
  43. Wu Y, Ghosh A, Szyperski T (2009) Clean absorption mode NMR data acquisition based on time-proportional phase incrementation. J Struct Funct Genom 10(3):227–232CrossRefGoogle Scholar
  44. Wüthrich K (1986) NMR of proteins and nucleic acids. Wiley, New YorkCrossRefGoogle Scholar
  45. Zhu J, Zhu J, Springer TA (2013) Complete integrin headpiece opening in eight steps. J Cell Biol 201(7):1053–1068CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Amor Mosbah
    • 1
    • 2
    • 3
    Email author
  • Naziha Marrakchi
    • 4
  • Pascal Mansuelle
    • 5
  • Soumaya Kouidhi
    • 1
  • Ernest Giralt
    • 6
    • 7
  • Mohamed El Ayeb
    • 4
  • Gaëtan Herbette
    • 8
  • Ameur Cherif
    • 1
  • Didier Gigmes
    • 3
  • Hervé Darbon
    • 2
  • Kamel Mabrouk
    • 3
    • 9
    Email author
  1. 1.Univ. Manouba, ISBST, BVBGR-LR11ES31, Biotechnopole Sidi ThabetArianaTunisia
  2. 2.UMR 7257, CNRS, Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB)MarseilleFrance
  3. 3.Aix Marseille Univ, CNRS, ICR, Institut de Chimie Radicalaire, UMRMarseilleFrance
  4. 4.Laboratoire des Venins et ToxinesInstitut Pasteur de TunisTunis-BelevédèreTunisia
  5. 5.Plate-forme Protéomique, Marseille Protéomique (MaP) IBiSA labelledInstitut de Microbiologie de la Méditerranée, FR 3479, CNRSMarseille Cedex 20France
  6. 6.Institute for Research in Biomedicine (IRB Barcelona)The Barcelona Institute of Science and TechnologyBarcelonaSpain
  7. 7.Department of Inorganic and Organic ChemistryUniversity of BarcelonaBarcelonaSpain
  8. 8.Aix Marseille Univ, Spectropôle, FR1739MarseilleFrance
  9. 9.Laboratoire de Chimie RadicalaireOrganique et Polymères de SpécialitéMarseilleFrance

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