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Thromb Haemost 1985; 54(02): 402-408
DOI: 10.1055/s-0038-1657749
Original Article
Schattauer GmbH Stuttgart

Studies of Alpha2-Adrenergic Receptors of Intact and Functional Washed Human Platelets by Binding of 3H-Dihydroergocryptine and 3H-Yohimbine-Correlation of 3H-Yohimbine Binding with the Potentiation by Adrenaline of ADP-Induced Aggregation

François Lanza
The Laboratoire de Biologie et de Pharmacologie des Interactions du Sang avec les Vaisseaux et les Biomatériaux, Centre Régional de Transfusion Sanguine, Strasbourg, France
,
Jean-Pierre Cazenave
The Laboratoire de Biologie et de Pharmacologie des Interactions du Sang avec les Vaisseaux et les Biomatériaux, Centre Régional de Transfusion Sanguine, Strasbourg, France
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Publikationsverlauf

Received 20. Februar 1985

Accepted 22. April 1985

Publikationsdatum:
18. Juli 2018 (online)

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Summary

The binding of 3H-dihydroergocryptine and 3H-yohimbine to intact, discoid, functional, washed human platelets resuspended in Tyrode’s buffer containing Ca2+, Mg2+, human albumin and apyrase, was studied at 37° C. The binding of 3H-dihydroergocryptine was rapid, reversible and saturable (KD = 19.3 ± 4.2 nM, Bmax = 2590 ± 670 sites per platelet). The results were difficult to interpret because the bound ligand was not easily dissociated. In contrast, 3H-yohimbine bound in a rapid, reversible and saturable fashion to one class of sites (KD = 8.1 ± 1 nM, Bmax = 395 ± 35 sites/platelet) with the characteristics of alpha2-adrenergic receptors. Adrenaline alone did not aggregate intact platelets but potentiated ADP-induced aggregation. This effect of adrenaline was specifically inhibited by alpha2-antagonists such as yohimbine. The inhibition of 3H-yohimbine binding and the inhibition of the synergistic effect of adrenaline on ADP-induced aggregation by 16 different alpha- and beta-adrenergic compounds was significantly correlated (p <0.001). Thus, intact and functional washed human platelets can be used as a simple pharmacological model to screen alpha-adrenergic antagonists by measuring the inhibition of the potentiation of ADP-induced aggregation by adrenaline which is a direct reflection of the physiological effect of adrenaline on human platelet alpha2-adrenergic receptors. The inhibition constant derived from aggregation studies expresses the affinity of the ligand for its receptor as measured by more cumbersome binding studies with radioactive adrenergic antagonists such as 3H-yohimbine.

Keywords

Platelet - Alpha adrenergic receptor - Aggregation - Adrenaline - ADP - Yohimbine - Dihydroergocryptine
 

  • References

  • 1 Scrutton MC, Wallis RB. Catecholamine receptors. In: Platelets in Biology and Pathology. vol. 2. Gordon J L. (ed). Elsevier; North-Holland: 1981: 179-210
    Suche in Google Scholar
  • 2 O'Brien JR. Some effects of adrenaline and antiadrenaline compounds on platelets in vitro and in vivo. Nature 1963; 200: 763-765
    CrossrefPubMedSuche in Google Scholar
  • 3 Mills DCB, Robert GCK. Effects of adrenaline on human blood platelets. J Physiol (Lond) 1967; 193: 443-453
    CrossrefPubMedSuche in Google Scholar
  • 4 Ardlie NG, Glew G, Schwartz CJ. Influence of catecholamines on nucleotide-induced platelet aggregation. Nature 1966; 212: 415-417
    CrossrefPubMedSuche in Google Scholar
  • 5 Thomas DP. The role of platelet catecholamines in the aggregation of platelets by collagen and thrombin. Exp Biol Med 1968; 3: 129-134
    PubMedSuche in Google Scholar
  • 6 Vargaftig BB, Fouque F, Benveniste J, Odiot J. Adrenaline and PAF-acether synergize to trigger cyclooxygenase-independent activation of plasma-free human platelets. Thromb Res 1982; 28: 557-573
    CrossrefPubMedSuche in Google Scholar
  • 7 Beretz A, Lanza F, Cazenave JP. Adrenaline potentiates platelet aggregation induced by PAF-acether and endoperoxide analog U-46619 through stimulation of alpha2 receptors independently of the cyclo-oxygenase pathway. Thromb Haemostas 1983; 50: 41 (Abstr)
    PubMedSuche in Google Scholar
  • 8 Rao GHR, White JG. Epinephrine potentiation of arachidonate-induced aggregation of cyclooxygenase-deficient platelets. Am J Hematol 1981; 11: 355-366
    CrossrefPubMedSuche in Google Scholar
  • 9 Kinlough-Rathbone RL, Mustard JF, Packham MA, Perry DW, Reimers HJ, Cazenave JP. Properties of washed human platelets. Thromb Haemostas 1977; 37: 291-308
    PubMedSuche in Google Scholar
  • 10 Kinlough-Rathbone RL, Packham MA, Mustard JF. Platelet aggregation. In: Methods in Hematology. Measurements of Platelet Function. Harker LA, Zimmerman TS. (eds). Churchill Livingstone; Edinburgh: 1983: 64-91
    Suche in Google Scholar
  • 11 Glusa E, Markwardt F. Adrenaline-induced reactions of human platelets in hirudin plasma. Haemostasis 1980; 9: 188-192
    PubMedSuche in Google Scholar
  • 12 Cazenave JP, Sutter A, Hemmendinger S, Wiesel ML, Lanza F, Daver J. Adrenaline activates human platelets but does not cause primary aggregation if thrombin generation is inhibited by hirudin. Thromb Haemostas 1981; 46: 95 (Abstr)
    PubMedSuche in Google Scholar
  • 13 Lages B, Weiss HJ. Dependence of human platelet functional responses on divalent cations. Aggregation and secretion in heparin-anticoagulated and hirudin-anticoagulated platelet-rich plasma and the effects of chelating agents. Thromb Haemostas 1981; 45: 173-179
    PubMedSuche in Google Scholar
  • 14 Glusa E, Markwardt F. Characterization of alpha2-adrenoceptors on blood platelets from various species using 3H-yohimbine. Haemostasis 1983; 13: 96-102
    PubMedSuche in Google Scholar
  • 15 Kerry R, Scrutton MC, Wallis RB. Mammalian platelet adrenoceptors. Br J Pharmacol 1984; 81: 91-102
    CrossrefPubMedSuche in Google Scholar
  • 16 Hoffman BB, Michel T, Kilpatrick DM, Lefkowitz RJ, Tolbert MEM, Gilman M, Fain JN. Agonist versus antagonist binding to alpha-adrenergic receptors. Proc Natl Acad Sci USA 1980; 77: 4569-4573
    CrossrefPubMedSuche in Google Scholar
  • 17 Lynch CJ, Steer ML. Evidence for high and low affinity alpha2 receptors. J Biol Chem 1981; 256: 3298-3303
    PubMedSuche in Google Scholar
  • 18 Shattil SJ, McDonough M, Turnbull J, Insel PA. Characterization of alpha-adrenergic receptors in human platelets using 3H-clonidine. Mol Pharmacol 1981; 19: 179-183
    PubMedSuche in Google Scholar
  • 19 Garcia-Sevilla JA, Hollingsworth PJ, Smith CB. Alpha2-adrenoceptors on human platelets: selective labelling by 3H-clonidine and 3H-yohimbine and competitive inhibition by antidepressant drugs. Eur J Pharmacol 1981; 74: 329-341
    CrossrefPubMedSuche in Google Scholar
  • 20 Mooney JJ, Horne WC, Handin RI, Schildkraut JJ, Alexander RW. Sodium inhibits both adenylate cyclase and high affinity 3H-labeled paminoclonidine binding to alpha2-adrenergic receptors in purified human platelet membranes. Mol Pharmacol 1982; 21: 600-608
    PubMedSuche in Google Scholar
  • 21 Steer ML, Khorana J, Galgoci B. Quantitation and characterization of human platelet alpha-adrenergic receptors using 3H-phentolamine. Mol Pharmacol 1979; 16: 719-728
    PubMedSuche in Google Scholar
  • 22 Kafka MS, Tallman JF, Smith CC, Costa JL. Alpha-adrenergic receptors on human platelets. Life Sci 1977; 21: 1429-1438
    CrossrefPubMedSuche in Google Scholar
  • 23 Newman KD, Williams LT, Bishopric NN, Lefkowitz RJ. Identification of alpha-adrenergic receptors in human platelets by 3H-dihydroergocryptine binding. J Clin Invest 1978; 61: 395-402
    CrossrefPubMedSuche in Google Scholar
  • 24 Alexander RW, Cooper B, Handin RI. Characterization of the human platelet alpha-adrenergic receptor. J Clin Invest 1978; 61: 1136-1144
    CrossrefPubMedSuche in Google Scholar
  • 25 Elliott JM, Grahame-Smith DG. The binding characteristics of 3H-dihydroergocryptine on intact human platelets. Br J Pharmacol 1982; 76: 121-130
    CrossrefPubMedSuche in Google Scholar
  • 26 MacFarlane DE, Wright BL, Stump DC. Use of methyl-3H-yohimbine as a radioligand for alpha2-adrenoreceptors on intact platelets. Comparison with dihydroergocryptine. Thromb Res 1981; 24: 31-43
    CrossrefPubMedSuche in Google Scholar
  • 27 Motulsky HJ, Shattil SJ, Insel PA. Characterization of alpha2-adrenergic receptors of human platelets using 3H-yohimbine. Biochem Biophys Res Commun 1980; 97: 1562-1570
    CrossrefPubMedSuche in Google Scholar
  • 28 Murkherjee A. Characterization of alpha2-adrenergic receptors in human platelets by binding of a radioactive ligand 3H-yohimbine. Biochim Biophys Acta 1981; 676: 148-154
    CrossrefPubMedSuche in Google Scholar
  • 29 Daiguji M, Meltzer HY, U'Prichard DC. Human platelet alpha2-adrenergic receptors: labelling with 3H-yohimbine, a selective antagonist ligand. Life Sci 1981; 28: 2705-2718
    CrossrefPubMedSuche in Google Scholar
  • 30 Cheung YD, Barnett DB, Nahorski SR. 3H-rauwolscine and 3H-yohimbine binding to rat cerebral and human platelet membranes: possible heterogeneity of alpha2-adrenoreceptors. Eur J Pharmacol 1982; 84: 79-85
    CrossrefPubMedSuche in Google Scholar
  • 31 Regan JW, De Marinis RM, Caron MG, Lefkowitz RJ. Identification of the subunit-binding site of alpha2-adrenergic receptors using 3H-phenoxybenzamine. J Biol Chem 1984; 259: 7864-7869
    PubMedSuche in Google Scholar
  • 32 Robinson GA, Arnold A, Hartmann RC. Divergent effects of epinephrine and prostaglandin El on level of cyclic AMP in human blood platelets. Pharmacol Res Commun 1969; 1: 325-334
    CrossrefPubMedSuche in Google Scholar
  • 33 Salzman EW, Neri LL. Cyclic 3′,5′ adenosine monophosphate in human blood platelets. Nature 1969; 224: 609-612
    CrossrefPubMedSuche in Google Scholar
  • 34 Marquis NR, Becker JA, Vigdahl RL. Platelet aggregation. III. An epinephrine induced decrease in cyclic AMP synthesis. Biochem Biophys Res Commun 1970; 39: 783-789
    CrossrefPubMedSuche in Google Scholar
  • 35 Mills DCB. Initial biochemical responses of platelets to stimulation. In: Biochemistry and Pharmacology of Platelets. Ciba Foundation Symposium 35 (New series). Elsevier; North-Holland: 1975: 146-167
    Suche in Google Scholar
  • 36 Jakobs KH, Saur W, Schultz G. Reduction of adenylate cyclase activity in lysates of human platelets by the alpha-adrenergic component of epinephrine. J Cyclic Nucleotide Res 1976; 2: 381-396
    PubMedSuche in Google Scholar
  • 37 Tsai BS, Lefkowitz RJ. Agonist-specific effects of monovalent and divalent cations on adenylate cyclase-coupled alpha-adrenergic receptors in rabbit platelets. Mol Pharmacol 1978; 14: 540-548
    PubMedSuche in Google Scholar
  • 38 Tsai BS, Lefkowitz RJ. Agonist-specific effects of guanine nucleotides on alpha-adrenergic receptors in human platelets. Mol Pharmacol 1979; 16: 61-68
    PubMedSuche in Google Scholar
  • 39 Motulsky HJ, Insel PA. Influence of sodium on the alpha2-adrenergic receptor system of human platelets.Role for intraplatelet sodium in receptor binding. J Biol Chem 1983; 258: 3913-3920
    PubMedSuche in Google Scholar
  • 40 Connolly TM, Limbird LE. The influence of Na+ on the alpha2-adrenergic receptor system of human platelets. J Biol Chem 1983; 258: 3907-3926
    PubMedSuche in Google Scholar
  • 41 Jakobs KH, Saur W, Schultz G. Inhibition of platelet adenylate cyclase by epinephrine requires GTP. FEBS Lett 1978; 85: 167-170
    CrossrefPubMedSuche in Google Scholar
  • 42 Steer ML, Wood A. Inhibitory effects of sodium and other monovalent cations on human platelet adenylate cyclase. J Biol Chem 1981; 256: 9990-9993
    PubMedSuche in Google Scholar
  • 43 Boon NA, Elliott JM, Grahame-Smith DG, John-Green TS, Stump K. A comparison of alpha2-adrenoreceptor binding characteristics of intact human platelets identified by 3H-yohimbine and 3H-dihydroer-gocryptine. J Auton Pharmac 1983; 3: 89-95
    CrossrefPubMedSuche in Google Scholar
  • 44 Swart SS, Pearson D, Wood JK, Barnett DB. Human platelet alpha2-adrenoceptors: lack of correlation between radioligand studies and adrenaline induced aggregation. Br J Clin Pharmacol 1983; 15: 620-621
    PubMedSuche in Google Scholar
  • 45 Cazenave JP, Hemmendinger S, Beretz A, Sutter-Bay A, Launay J. L'agrégation plaquettaire: outil d'investigation clinique et d'étude pharmacologique. Méthodologie. Ann Biol Clin 1983; 41: 167-179
    PubMedSuche in Google Scholar
  • 46 Mustard JF, Perry DW, Kinlough-Rathbone RL, Packham MA. Factors responsible for ADP-induced release reaction of human platelets. Am J Physiol 1975; 228: 1757-1765
    PubMedSuche in Google Scholar
  • 47 Williams LT, Lefkowitz RJ. Receptor binding studies in adrenergic pharmacology. Raven Press; New York: 1978: 27-41
    Suche in Google Scholar
  • 48 Laduron PM. Criteria for receptor sites in binding studies. Biochem Pharmacol 1984; 33: 833-839
    CrossrefPubMedSuche in Google Scholar
  • 49 Holmsen H, Salganicoff L, Fukami MH. Platelet behaviour and biochemistry. In: Haemostasis: Biochemistry, Physiology and Pathology. Ogston D, Bennett B. (eds). John Wiley and Sons; London: 1977: 241-319
    Suche in Google Scholar
  • 50 Eber M, Cazenave JP, Grob JC, Abecassis J, Methlin G. 111Indium labelling of human washed platelets: kinetics and in vivo sequestration sites. In: Blood Cells in Nuclear Medicine, part I. Cell Kinetics and Bio-distribution. Hardeman MR, Najean Y. (eds). Martinus Nijhoff Publishers; Boston: 1984: 29-43
    Suche in Google Scholar
  • 51 Sixma JJ, Lips JP. Isolation of platelet membranes. A review. Thromb Haemostas 1978; 39: 328-337
    PubMedSuche in Google Scholar
  • 52 Wilberding CA, Marks BH. Dihydroergocryptine: a “pseudo irreversible” alpha-adrenergic antagonist in the guinea pig vas deferens. Res Commun Chem Pathol Pharmacol 1981; 31: 387-401
    PubMedSuche in Google Scholar
  • 53 Motulsky HJ, O'Connor DT, Insel PA. Platelet alpha2-adrenergic receptors in treated and untreated essential hypertension. Clin Sci 1983; 64: 265-272
    CrossrefPubMedSuche in Google Scholar
  • 54 Lenox RH, Ellis J, Van Riper D, Ehrlich YH. Alpha2-adrenergic receptor-mediated regulation of adenylate cyclase in the intact human platelet. Evidence for a receptor reserve. Mol Pharmacol 1985; 27: 1-9
    PubMedSuche in Google Scholar