Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1992 Feb;89(2):444–450. doi: 10.1172/JCI115604

"Mirror image" antagonists of thrombin-induced platelet activation based on thrombin receptor structure.

D T Hung 1, T K Vu 1, V I Wheaton 1, I F Charo 1, N A Nelken 1, N Esmon 1, C T Esmon 1, S R Coughlin 1
PMCID: PMC442871  PMID: 1310695

Abstract

Platelet activation by thrombin plays a critical role in hemostasis and thrombosis. Based on structure-activity studies of a cloned platelet thrombin receptor, we designed two "mirror image" antagonists of thrombin and thrombin receptor function. First, "uncleavable" peptides mimicking the receptor domain postulated to interact with thrombin were found to be potent thrombin inhibitors. Second, proteolytically inactive mutant thrombins designed to bind but not cleave the thrombin receptor were found to be specific antagonists of receptor activation by thrombin. The effectiveness of these designed antagonists in blocking thrombin-induced platelet activation suggests a model for thrombin-receptor interaction and possible strategies for the development of novel antithrombotic agents.

Full text

PDF
450

Images in this article

447Image
on p.447

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baenziger N. L., Majerus P. W. Isolation of human platelets and platelet surface membranes. Methods Enzymol. 1974;31:149–155. doi: 10.1016/0076-6879(74)31015-4. [DOI] [PubMed] [Google Scholar]
  2. Bode W., Mayr I., Baumann U., Huber R., Stone S. R., Hofsteenge J. The refined 1.9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J. 1989 Nov;8(11):3467–3475. doi: 10.1002/j.1460-2075.1989.tb08511.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bode W., Turk D., Stürzebecher J. Geometry of binding of the benzamidine- and arginine-based inhibitors N alpha-(2-naphthyl-sulphonyl-glycyl)-DL-p-amidinophenylalanyl-pipe ridine (NAPAP) and (2R,4R)-4-methyl-1-[N alpha-(3-methyl-1,2,3,4-tetrahydro-8- quinolinesulphonyl)-L-arginyl]-2-piperidine carboxylic acid (MQPA) to human alpha-thrombin. X-ray crystallographic determination of the NAPAP-trypsin complex and modeling of NAPAP-thrombin and MQPA-thrombin. Eur J Biochem. 1990 Oct 5;193(1):175–182. doi: 10.1111/j.1432-1033.1990.tb19320.x. [DOI] [PubMed] [Google Scholar]
  4. Charo I. F., Feinman R. D., Detwiler T. C. Interrelations of platelet aggregation and secretion. J Clin Invest. 1977 Oct;60(4):866–873. doi: 10.1172/JCI108841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ehrlich H. J., Grinnell B. W., Jaskunas S. R., Esmon C. T., Yan S. B., Bang N. U. Recombinant human protein C derivatives: altered response to calcium resulting in enhanced activation by thrombin. EMBO J. 1990 Aug;9(8):2367–2373. doi: 10.1002/j.1460-2075.1990.tb07411.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413–7417. doi: 10.1073/pnas.84.21.7413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fenton J. W., 2nd, Fasco M. J., Stackrow A. B. Human thrombins. Production, evaluation, and properties of alpha-thrombin. J Biol Chem. 1977 Jun 10;252(11):3587–3598. [PubMed] [Google Scholar]
  8. Greco N. J., Tenner T. E., Jr, Tandon N. N., Jamieson G. A. PPACK-thrombin inhibits thrombin-induced platelet aggregation and cytoplasmic acidification but does not inhibit platelet shape change. Blood. 1990 May 15;75(10):1989–1990. [PubMed] [Google Scholar]
  9. Hanson S. R., Harker L. A. Interruption of acute platelet-dependent thrombosis by the synthetic antithrombin D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone. Proc Natl Acad Sci U S A. 1988 May;85(9):3184–3188. doi: 10.1073/pnas.85.9.3184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Harmon J. T., Jamieson G. A. Activation of platelets by alpha-thrombin is a receptor-mediated event. D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-thrombin, but not N alpha-tosyl-L-lysine chloromethyl ketone-thrombin, binds to the high affinity thrombin receptor. J Biol Chem. 1986 Dec 5;261(34):15928–15933. [PubMed] [Google Scholar]
  11. Higaki J. N., Gibson B. W., Craik C. S. Evolution of catalysis in the serine proteases. Cold Spring Harb Symp Quant Biol. 1987;52:615–621. doi: 10.1101/sqb.1987.052.01.070. [DOI] [PubMed] [Google Scholar]
  12. Jorgensen M. J., Cantor A. B., Furie B. C., Furie B. Expression of completely gamma-carboxylated recombinant human prothrombin. J Biol Chem. 1987 May 15;262(14):6729–6734. [PubMed] [Google Scholar]
  13. Julius D., MacDermott A. B., Axel R., Jessell T. M. Molecular characterization of a functional cDNA encoding the serotonin 1c receptor. Science. 1988 Jul 29;241(4865):558–564. doi: 10.1126/science.3399891. [DOI] [PubMed] [Google Scholar]
  14. Kettner C., Shaw E. D-Phe-Pro-ArgCH2C1-A selective affinity label for thrombin. Thromb Res. 1979;14(6):969–973. doi: 10.1016/0049-3848(79)90014-8. [DOI] [PubMed] [Google Scholar]
  15. Krishnaswamy S., Church W. R., Nesheim M. E., Mann K. G. Activation of human prothrombin by human prothrombinase. Influence of factor Va on the reaction mechanism. J Biol Chem. 1987 Mar 5;262(7):3291–3299. [PubMed] [Google Scholar]
  16. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  17. Laura R., Robison D. J., Bing D. H. (p-Amidinophenyl)methanesulfonyl fluoride, an irreversible inhibitor of serine proteases. Biochemistry. 1980 Oct 14;19(21):4859–4864. doi: 10.1021/bi00562a024. [DOI] [PubMed] [Google Scholar]
  18. Liu L. W., Vu T. K., Esmon C. T., Coughlin S. R. The region of the thrombin receptor resembling hirudin binds to thrombin and alters enzyme specificity. J Biol Chem. 1991 Sep 15;266(26):16977–16980. [PubMed] [Google Scholar]
  19. Maraganore J. M., Bourdon P., Jablonski J., Ramachandran K. L., Fenton J. W., 2nd Design and characterization of hirulogs: a novel class of bivalent peptide inhibitors of thrombin. Biochemistry. 1990 Jul 31;29(30):7095–7101. doi: 10.1021/bi00482a021. [DOI] [PubMed] [Google Scholar]
  20. Rydel T. J., Ravichandran K. G., Tulinsky A., Bode W., Huber R., Roitsch C., Fenton J. W., 2nd The structure of a complex of recombinant hirudin and human alpha-thrombin. Science. 1990 Jul 20;249(4966):277–280. doi: 10.1126/science.2374926. [DOI] [PubMed] [Google Scholar]
  21. Sprang S., Standing T., Fletterick R. J., Stroud R. M., Finer-Moore J., Xuong N. H., Hamlin R., Rutter W. J., Craik C. S. The three-dimensional structure of Asn102 mutant of trypsin: role of Asp102 in serine protease catalysis. Science. 1987 Aug 21;237(4817):905–909. doi: 10.1126/science.3112942. [DOI] [PubMed] [Google Scholar]
  22. Subramani S., Mulligan R., Berg P. Expression of the mouse dihydrofolate reductase complementary deoxyribonucleic acid in simian virus 40 vectors. Mol Cell Biol. 1981 Sep;1(9):854–864. doi: 10.1128/mcb.1.9.854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Takebe Y., Seiki M., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol. 1988 Jan;8(1):466–472. doi: 10.1128/mcb.8.1.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tollefsen D. M., Feagler J. R., Majerus P. W. The binding of thrombin to the surface of human platelets. J Biol Chem. 1974 Apr 25;249(8):2646–2651. [PubMed] [Google Scholar]
  25. Vu T. K., Hung D. T., Wheaton V. I., Coughlin S. R. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991 Mar 22;64(6):1057–1068. doi: 10.1016/0092-8674(91)90261-v. [DOI] [PubMed] [Google Scholar]
  26. Williams J. A., McChesney D. J., Calayag M. C., Lingappa V. R., Logsdon C. D. Expression of receptors for cholecystokinin and other Ca2+-mobilizing hormones in Xenopus oocytes. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4939–4943. doi: 10.1073/pnas.85.13.4939. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES