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Thromb Haemost 1994; 72(04): 627-633
DOI: 10.1055/s-0038-1648926
Original Article
Schattauer GmbH Stuttgart

Species Variability in Platelet and other Cellular Responsiveness to Thrombin Receptor-derived Peptides

Thomas M Connolly
1   The Department of biological Chemistry, Merck Research Laboratories, West Point, PA, USA
,
Cindra Condra
1   The Department of biological Chemistry, Merck Research Laboratories, West Point, PA, USA
,
Dong-Mei Feng
3   The Department of Medicinal Chemistry, Merck Research Laboratories, West Point, PA, USA
,
Jacquelynn J Cook
2   The Department of Pharmacology, Merck Research Laboratories, West Point, PA, USA
,
Maria T Stranieri
2   The Department of Pharmacology, Merck Research Laboratories, West Point, PA, USA
,
Christopher F Reilly
2   The Department of Pharmacology, Merck Research Laboratories, West Point, PA, USA
,
Ruth F Nutt
3   The Department of Medicinal Chemistry, Merck Research Laboratories, West Point, PA, USA
,
Robert J Gould
1   The Department of biological Chemistry, Merck Research Laboratories, West Point, PA, USA
› Author Affiliations
Further Information

Publication History

Received 29 April 1994

Accepted after resubmission 20 June 1994

Publication Date:
06 July 2018 (online)

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Summary

The aggregation of platelets from a variety of animal species in response to thrombin receptor-derived activating peptides was evaluated. A series of 14-(SFLLRNPNDKYEPF), 7-(SFLLRNP-NH2), 6-(SFLLRN-HN2) or 5-(SFLLR-NH2) residue peptides, the structures of which were based on the deduced amino acid sequence of the human thrombin receptor, promoted full aggregation of platelets in plasma from humans, African Green and Rhesus monkeys, baboons and guinea pigs at 4-50 μM depending on the peptide used. Platelets in plasma from rabbit, dog, pig, and hamster underwent a shape change but failed to aggregate in response to these peptides over 3 log units of peptide up to 800 μM, despite being fully responsive to human thrombin. However, because the receptor peptides induced shape change in the platelets from these non-aggregating species, they apparently can activate some of the intracellular signaling system(s) usually initiated by thrombin in these platelets. In contrast, platelets from rats did not undergo shape change or aggregate in response to the peptides. A 7-residue receptor-derived peptide based on the deduced amino acid sequence of the clone of the hamster thrombin receptor (SFFLRNP-N2) was nearly as efficacious as the corresponding human receptor-derived 7-residue peptide to promote aggregation of human platelets. However, the hamster peptide could not promote aggregation of hamster platelets in plasma at up to 800 μM peptide, while a shape change response was elicited. Platelets from rats, rabbits and pigs also did not aggregate in response to this peptide derived from the hamster thrombin receptor, but all species except the rat underwent a shape change. Longer 17-residue peptides derived from the sequences of the hamster or mouse thrombin receptors elicited aggregation of human platelets but no aggregation of the hamster platelets. In contrast, the human 14- and 5-residue and the hamster 7-residue thrombin receptor-derived peptides promoted mitogenesis of CCL39 cells, a hamster fibroblast cell line. Finally, the human 6-residue thrombin receptor-derived peptide promoted contraction of normal and de-endothelialized canine coronary artery rings, despite having no pro-aggregatory effect on canine platelets. Taken together, these results demonstrate that the thrombin receptor-derived peptides are able to mimic many, but not all, of the activating effect of thrombin in different tissues from multiple species. The heterogeneity of responsiveness to these peptides should be taken into account in future experiments designed to elucidate the mechanism of thrombin stimulation of platelets and other cells.

 

  • References

  • 1 Shuman MA. Thrombin-cellular interaction. In: Annals of N. Y. Acad. Sci Vol 485 Awlz DA, Fenton IIJW, Shuman MA. eds New York Academy of Sciences; New York, NY: 1986. pp 228-239
  • 2 Vu T-KH, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991; 64: 1057-1068
    CrossrefPubMedSearch in Google Scholar
  • 3 Rasmussen UB, Vouret-Craviari V, Jallat S, Schlessinger Y, Pegès G, Pavirani A, Lecocq J-P, Pouysségur J, Van Obberghen-Schilling E. cDNA cloning and expression of a hamster-thrombin receptor coupled to Ca2+ mobilization. FEBS 1991; 288: 123-128
    CrossrefPubMedSearch in Google Scholar
  • 4 Coughlin SR, Vu T-KH, Hung DT, Wheaton VI. Characterization of a functional thrombin receptor, issues and opportunities. J Clin Invest 1992; 89: 351-355
    CrossrefPubMedSearch in Google Scholar
  • 5 Scarborough RM, Naughton MA, Teng W, Hung DT, Rose J, Vu T-KH, Wheaton VJ, Turck CW, Coughlin SR. Tethered ligand agonist peptides. Structural requirements for thrombin receptor activation reveal a mechanism of proteolytic unmasking of agonist function. J Biol Chem 1992; 267: 13146-13149
    PubMedSearch in Google Scholar
  • 6 Vassallo Jr RR, Kieber-Emmons T, Chichowski K, Brass LF. Structure-function relationships in the activation of platelet thrombin receptors by receptor derived peptides. J Biol Chem 1992; 267: 6081-6085
    PubMedSearch in Google Scholar
  • 7 Chao BH, Kalkunte S, Maraganore JM, Stone SR. Essential groups in synthetic agonist peptides for activation of the platelet thrombin receptor. Biochem 1992; 31: 6175-6178
    CrossrefPubMedSearch in Google Scholar
  • 8 Hui KY, Jakubowski JA, Wyes VL, Angleton EL. Minimal sequence requirement of thrombin receptor agonist peptide. Biochem Biophys Res Comm 1992; 184: 790-796
    CrossrefPubMedSearch in Google Scholar
  • 9 Seiler SM, Michel IM, Fenton IIJW. Involvement of the tethered ligand receptor in thrombin inhibition of platelet adenylate cyclase. Biochem Biophys Res Comm 1992; 182: 1296-1302
    CrossrefPubMedSearch in Google Scholar
  • 10 Hung R-S, Sorisky A, Church WR, Simons ER, Rittenhouse SE. Thrombin receptor-directed ligand accounts for activation by thrombin of platelet phospholipase C and accumulation of 3-phosphorylated phosphoinositides. J Biol Chem 1991; 266: 18435-18438
    PubMedSearch in Google Scholar
  • 11 Connolly TM, Jacobs JW, Condra C. An inhibitor of collagen-stimulated platelet activation from the salivary glands of the Haementeria officinalis leech. J Biol Chem 1992; 267: 6893-6898
    PubMedSearch in Google Scholar
  • 12 Plow EF, Marguerie G. Inhibition of fibrinogen binding to human platelets by the tetrapeptide glycyl-L-prolyl-L-Arginyl-L-proline. Proc Natl Acad Sci 1982; 79: 3711-3715
    CrossrefPubMedSearch in Google Scholar
  • 13 Reilly CF, Connolly TM, Feng D-M, Nutt RF, Mayer EJ. Thrombin receptor agonist peptide induction of mitogenesis in CCL 39 cells. Biochem Biophys Res Comm 1993; 190: 1001-1008
    CrossrefPubMedSearch in Google Scholar
  • 14 Vu T-KH, Wheaton VI, Hung DT, Charo I, Coughlin SR. Domains specifying thrombin-receptor interaction. Nature 1991; 353: 674-677
    CrossrefPubMedSearch in Google Scholar
  • 15 Zhong C, Hayzer DJ, Carson MA, Runge MS. Molecular cloning of the rat vascular smooth muscle thrombin receptor: evidence for in vitro regulation by basic fibroblast growth factor. J Biol Chem 1992; 267: 16975-16979
    PubMedSearch in Google Scholar
  • 16 Fenton JWII. Regulation of thrombin generation and functions. Sem in Thromb and Hemost 1988; 14: 234-240
    PubMedSearch in Google Scholar
  • 17 Esmon CT. The regulation of natural anticoagulant pathways. Science 1987; 235: 1348-1352
    CrossrefPubMedSearch in Google Scholar
  • 18 Kinlough-Rathbone RL, Rand ML, Packham MA. Rabbit and rat platelets do not respond to thrombin receptor peptides that activate human platelets. Blood 1993; 82: 103-106
    PubMedSearch in Google Scholar
  • 19 Catalfamo JL, Anderson TT, Fenton II JW. Thrombin receptor activating peptides unlike thrombin are insufficient for platelet activation in most species. Thromb Haemost 1993; 69: 1195 (abstract)
    PubMedSearch in Google Scholar
  • 20 Coller BS, Ward P, Ceruso M, Scudder LE, Springer K, Kutok J, Prestwich GD. Thrombin receptor activating peptides: importance of the N-terminal serine and its ionization state as judged by pH dependence, nuclear magnetic resonance spectroscopy, and cleavage by aminopeptidase M. Biochem 1992; 31: 11713-11720
    CrossrefPubMedSearch in Google Scholar
  • 21 Connolly TM, Condra C, Feng D-M, Nutt RF, Gould RJ. Activation of human platelets by potent thrombin receptor-derived peptides. Thromb Haemost 1993; 69: 787 (abstract)
    Thieme ConnectPubMedSearch in Google Scholar
  • 22 Vouret-Craviari V, Van Obberghen-Schilling E, Rasmusssen UB, Pavirani A, Lecocq J-P, Pouysségur J. Synthetic-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis. Mol Biol of Cell 1992; 3: 95-102
    PubMedSearch in Google Scholar
  • 23 Hung DT, Vu T-HK, Nelken NA, Coughlin SR. Thrombin-induced events in non-platelets cells are mediated by the unique proteolytic mechanism established for the cloned platelet thrombin receptor. J Cell Biol 1992; 116: 827-832
    CrossrefPubMedSearch in Google Scholar
  • 24 Simonet S, Bonhomme E, Laubie M, Thurieau C, Fauchère J-L, Verbeuren TJ. Venous and arterial endothelial cells respond differently to thrombin and its endogenous receptor agonist. Eur J Pharmacol 1992; 216: 135-137
    CrossrefPubMedSearch in Google Scholar
  • 25 Hollenberg MD, Yang S-G, Laniyonu AA, Moore GJ, Saifeddine M. Action of a thrombin receptor polypeptide in gastric smooth muscle: identification of a core pentapeptide retaining full thrombin-mimetic activity. Mol Pharmacol 1992; 42: 186-191
    PubMedSearch in Google Scholar
  • 26 Muramatsu I, Laniyonu AA, Moore GJ, Hollenberg MD. Vascular actions of thrombin receptor peptide. Can J Physiol Pharmacol 1992; 70: 996-1003
    CrossrefPubMedSearch in Google Scholar
  • 27 Antonaccio MJ, Normandin D, Serafino R, Moreland S. Effects of thrombin and thrombin receptor activating peptides on rat aortic vascular smooth muscle. J Pharmacol Exp Ther 1993; 266: 125-132
    PubMedSearch in Google Scholar
  • 28 McNamara CA, Sarembock IJ, Gimple LW, Fenton IIJW, Coughlin SR, Owens GK. Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor. J Clin Invest 1993; 91: 94-98
    CrossrefPubMedSearch in Google Scholar
  • 29 Simons ER, Davies TA, Greenberg SM, Larsen NE. Thrombin receptors on human platelets. In: Methods Enzymol Hawiger J. ed Academic Press; San Diego, CA: 1992. 215 155-176
  • 30 Hung DT, Vu T-KH, Wheaten VI, Coughlin SR. Cloned platelet receptor is necessary for thrombin-induced platelet activation. J Clin Invest 1992; 89: 1350-1353
    CrossrefPubMedSearch in Google Scholar
  • 31 Brass LF, Vassallo Jr RR, Belmonte E, Ahuja M, Cichowski K, Hoxie JA. Structure and function of the human platelet thrombin receptor. Studies using monoclonal antibodies directed against a defined domain within the receptor N terminus. J Biol Chem 1992; 267: 13795-13798
    PubMedSearch in Google Scholar
  • 32 Bahou WF, Coller BS, Potter CL, Norton KJ, Kutok J. Goligorsky MS. The thrombin receptor extracellular domain contains sites crucial for peptide ligand-induced activation. J Clin Invest 1993; 91: 1405-1413
    CrossrefPubMedSearch in Google Scholar
  • 33 Greco NJ, Tenner Jr TE, Tandon NN, Jamieson GA. PPACK-thrombin inhibits thrombin-induced platelet aggregation and cytoplasmic acidification but does not inhibit platelet shape change. Blood 1990; 75: 1983-1990
    PubMedSearch in Google Scholar
  • 34 Seiler SM, Goldenberg HJ, Michel IM, Hunt JT, Zavoico GB. Multiple pathways of thrombin-induced platelet activation differentiated by desensitization and a thrombin exosite inhibitor. Biochem Biophys Res Comm 1991; 181: 636-643
    CrossrefPubMedSearch in Google Scholar
  • 35 Takamatsu J, Horne IIIMK, Gralnick HR. Identification of the thrombin receptor on human platelets by chemical crosslinking. J Clin Invest 1986; 77: 362-368
    CrossrefPubMedSearch in Google Scholar
  • 36 Harmon JT, Jamieson GA. Thrombin binds to a high affinity ∼900000-dalton site on human platelets. Biochem 1985; 24: 58-64
    CrossrefPubMedSearch in Google Scholar