The Platelet-Stimulating Effect of Adrenaline Through α₂-Adrenergic Receptors Requires Simultaneous Activation by a True Stimulatory Platelet Agonist

Summary

The stimulating effect of adrenaline on human platelet phospholipase C (PLC) activation and responses in vitro (shape change, aggregation and dense granule secretion) was investigated with respect to its dependence on exogenously added agonists. All experiments were performed with human gel-filtered platelets pretreated with acetylsalicylic acid to prevent endogenous stimulation by the arachidonate pathway. (1) Preliminary experiments demonstrated the presence of trace amounts of extracellular ADP (0.05-0.58 μM) in non-stimulated platelet suspensions; ADP was effectively converted to ATP by the enzyme system creatine phosphate (CP)/creatine phosphokinase (CPK). (2) The adrenaline-induced optical aggregation and single particle (platelet) disappearance in the presence of trace amounts of ADP were almost abolished by the ADP-scavanger system CP/ CPK. (3) The response of CP/CPK-treated thrombin- or platelet-activating factor (PAF)-stimulated platelets was markedly increased by a subsequent addition of adrenaline. When hirudin or BN 50726 was added just prior to adrenaline to terminate the activation by thrombin or PAF, respectively, the stimulating effect of adrenaline was also abolished. (4) CP/CPK-treated, PAF-stimulated platelets rapidly developed decreased responsiveness to a subsequent addition of PAF. When adrenaline was added instead of a second addition of PAF, the stimulating effect of adrenaline was gradually decreased and prevented in parallel with the homologous desensitization of PAF. (5) The weak platelet agonist serotonin by itself induced only shape change in CP/CPK-treated platelets. Adrenaline failed to enhance the extent of this serotonin-induced platelet activation. (6) These results strongly suggest that adrenaline per se is not a platelet agonist in vitro but acts to enhance the stimulation induced by true agonists.

• References

• 1 Kroll MH, Schafer AI. Biochemical mechanisms of platelet activation. Blood 1989; 74: 1181-1195
• 2 Steen VM, Holmsen H. Current aspects on human platelet activation and responses. Eur J Haematol 1987; 38: 383-399
• 3 Grant JA, Scrutton MC. Novel alpha₂-adrenoceptors primarily responsible for inducing human platelet aggregation. Nature 1979; 277: 659-661
• 4 Motulsky HJ, Shattil SJ, Insel PA. Characterization of alpha₂-adrenergic receptors on human platelets using ³H-yohimbine. Biochem Biophys Res Commun 1980; 97: 1562-1570
• 5 O’Brien JR. A comparison of platelet aggregation produced by seven compounds and a comparison of their inhibitors. J Clin Pathol 1964; 17: 275-281
• 6 Mills DCB, Roberts GCK. Effects of adrenaline on human blood platelets. J Physiol 1967; 193: 443-453
• 7 Steen VM, Holmsen H. Synergism between thrombin and epinephrine in human platelets: different dose-response relationships for aggregation and dense granule secretion. Thromb Haemostas 1985; 54: 680-683
• 8 Thompson NT, Scrutton MC, Wallis RB. Synergistic responses in human platelets. Comparison between aggregation, secretion and cytosolic Ca²⁺ concentration. Eur J Biochem 1986; 161: 399-408
• 9 Steen VM, Tysnes OB, Holmsen H. Synergism between thrombin and adrenaline (epinephrine) in human platelets. Marked potentiation of inositol phospholipid metabolism. Biochem J 1988; 253: 581-586
• 10 Steen VM, Cook CA, Tysnes OB, Holmsen H. Potentiation by adrenaline of thrombin-induced elevation of pH_i is not essential for synergistic activation of human platelets. FEBS Lett 1989; 250: 211-214
• 11 O’Brien JR. Some effects of adrenaline and anti-adrenaline compounds on platelets in vitro and in vivo. Nature 1963; 200: 763-764
• 12 Peerschke EI. Induction of human platelet fibrinogen receptors by epinephrine in the absence of released ADP. Blood 1982; 60: 71-77
• 13 Connolly TM, Limbird LE. The influence of Na⁺ on the alpha₂-adrenergic receptor system of human platelets. A method for removal of extraplatelet Na⁺. Effect of Na⁺ removal on aggregation, secretion and cAMP accumulation. J Biol Chem 1983; 258: 3907-3912
• 14 Shattil SJ, Budzynski A, Scrutton MC. Epinephrine induces platelet fibrinogen receptor expression, fibrinogen binding, and aggregation in whole blood in the absence of other excitatory agonists. Blood 1989; 73: 150-158
• 15 Lanza F, Cazenave JP. Studies on 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. Thromb Haemostas 1985; 54: 402-408
• 16 Aktories K, Jakobs H. Epinephrine inhibits adenylate cyclase and stimulates a GTPase in human platelet membranes via alpha-adrenoceptors. FEBS Lett 1981; 130: 235-238
• 17 Motulsky HJ, Shattil SJ, Ferry N, Rozansky D, Insel PA. Desensitization of epinephrine-initiated platelet aggregation does not alter binding to the alpha₂-adrenergic receptor or receptor coupling to adenylate cyclase. Mol Pharmacol 1986; 29: 1-6
• 18 Siess W, Weber PC, Lapetina EG. Activation of phospholipase C is dissociated from arachidonate metabolism during platelet shape change induced by thrombin or platelet-activating factor. Epinephrine does not induce phospholipase C activation or platelet shape change. J Biol Chem 1984; 259: 8286-8292
• 19 Banga HS, Somins ER, Brass LF, Rittenhouse SE. Activation of phospholipase A and C in human platelets exposed to epinephrine: role of glycoproteins IIb/IIIa and dual role of epinephrine. Proc Natl Acad Sci USA 1986; 83: 9197-9201
• 20 Crouch MF, Lapetina EG. The Na⁺/H⁺ antiporter is not involved in potentiation of thrombin-induced responses by epinephrine. Biochem Biophys Res Commun 1988; 151: 178-186
• 21 Macmillan DC. Secondary clumping effect in human citrated platelet-rich plasma produced by adenosine diphosphate and adrenaline. Nature 1966; 211: 140-144
• 22 Swart SS, Magurie M, Wood JK, Barnett DB. Alpha₂-adrenoceptor coupling to adenylate cyclase in adrenaline insensitive human platelets. Eur J Pharmacol 1985; 116: 113-119
• 23 Charo IF, Feinman RD, Detwiler TC. Interrelations of platelet aggregation and secretion. J Clin Invest 1977; 60: 866-873
• 24 Lages B, Weiss HJ. Dependence of human platelet functional responses on divalent cations: aggregation and secretion in heparin-and hirudin-anticoagulated platelet-rich plasma and the effects of chelating agents. Thromb Haemostas 1981; 45: 173-179
• 25 Lalau KeralyC, Kinlough-Rathbone RL, Packham MA, Suzuki H, Mustard JF. Conditions affecting the responses of human platelets to epinephrine. Thromb Haemostas 1988; 60: 209-216
• 26 Lanza F, Beretz A, Stierle A, Hanau D, Kubina M, Cazenave JP. Epinephrine potentiates human platelet activation but is not an aggregating agent. Am J Physiol 1988; 255: H1276-H1288
• 27 Plow EF, Marguerie GA. Induction of the fibrinogen receptor on human platelets by epinephrine and the combination of epinephrine and ADP. J Biol Chem 1980; 255: 10971-10977
• 28 Figures WR, Scearce LM, Wachtfogel Y, Chen J, Colman RF, Colman RW. Platelet ADP-receptor and alpha₂-adrenoceptor interaction. Evidence for an ADP requirement for epinephrine-induced platelet activation and an influence of epinephrine on ADP binding. J Biol Chem 1986; 261: 5981-5986
• 29 Verhoeven AJM, Tysnes OB, Horvli O, Cook CA, Holmsen H. Changes in specific ³²P labelling of metabolic ATP and polyphosphoinositides. Stimulation of phosphate uptake in human platelets by thrombin and collagen. J Biol Chem 1987; 262: 7047-7052
• 30 Verhoeven AJM, Mommersteeg MM, Akkerman JWN. Quantification of energy consumption during thrombin-induced aggregation and secretion. Tight coupling between platelet responses and the increment in energy consumption. Biochem J 1984; 221: 777-787
• 31 Lages B, Holmsen H, Weiss HJ, Dangelmaier C. Thrombin and ionophore A23187-induced dense granule secretion in storage pool deficient platelets: evidence for impaired nucleotide storage as the primary dense granule defect. Blood 1983; 61: 154-162
• 32 Born GV, Hume M. Effects of the numbers and sizes of platelet aggregates on the optical density of plasma. Nature 1967; 215: 1027-1029
• 33 Tangen O, Andrae ML, Nilsson BE. Nucleotide leakage from platelets in artificial media. Prevention by albumin and other macromolecules and relation to ADP-induced platelet aggregation. Scand J Haematol 1973; 11: 241-248
• 34 Tam SW, Fenton II JW, Detwiler TC. Dissociation of thrombin from platelets by hirudin. Evidence for receptor processing. J Biol Chem 1979; 254: 8723-8725
• 35 Hallam TJ, Scrutton MC. Desensitization. In Platelet responses and metabolism, vol 2. Holmsen H. CRC Press; Boca Raton, FL: 1987: 105-118
• 36 De Clerk FF, Herman AG. 5-hydroxytryptamine and platelet aggregation. Fed Proc 1983; 42: 228-232