ADP secretion and subsequent P2Y12 receptor signalling play a crucial role in thrombin-induced ERK2 activation in human platelets

Knut Fälker; Danica Lange; Peter Presek

doi:10.1160/TH03-12-0729

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2004; 92(01): 114-123
DOI: 10.1160/TH03-12-0729

Platelets and Blood Cells

Schattauer GmbH

ADP secretion and subsequent P2Y12 receptor signalling play a crucial role in thrombin-induced ERK2 activation in human platelets

Knut Fälker

¹Martin-Luther-University Halle-Wittenberg, Halle, Germany

,

Danica Lange

¹Martin-Luther-University Halle-Wittenberg, Halle, Germany

,

Peter Presek

¹Martin-Luther-University Halle-Wittenberg, Halle, Germany

› Author Affiliations

Abstract

Summary

Stimulating human platelets with thrombin induces the activation of the extra cellular signal-regulated kinase 2 (ERK2). We demonstrate that this effect is highly dependent on ADP secretion and P2Y12 receptor signalling. AR-C69931MX (10 µM), a specific antagonist of the Gi-coupled P2Y12 ADP receptor, inhibits ERK2 activation induced by thrombin. Antagonists of the Gq-coupled P2Y1 ADP receptor, A3P5P (500 µM) and MRS2179 (100 µM), have no effect. ADP and its more potent analogue 2-methylthio-ADP alone (both up to 100 µM) do not induce ERK2 activation. Furthermore, we show that the inhibitory effect of AR-C69931MX on ERK2 activation induced by 0.1 U/ml thrombin as well as on platelet aggregation can be bypassed by epinephrine (1 and 10 µM), whereas epinephrine alone has no effect. Epinephrine acts on platelets mainly via α_2A-adrenergic receptors, which, like P2Y12 receptors, couple to inhibitory G proteins. In addition, 2-methylthioADP as well as epinephrine provoke ERK2 activation at a thrombin concentration that alone has no detectable effect (0.05 U/ml). Thromboxane A₂ (TXA₂), which, like ADP, is released by activated platelets, acts as a positive feedback mediator. Stimulating the Gq-coupled TXA₂-receptor with U46619 (10 µM), which leads to ADP secretion and P2Y12 receptor dependent platelet aggregation, also induces P2Y12-related ERK2 activation. The inhibition of U46619-induced ERK2 activation and platelet aggregation by AR-C69931MX are also rescued by epinephrine. Pretreatment with aspirin inhibits ERK2 activation induced by 0.1 U/ml thrombin, but has no effect at high concentrations of thrombin. The combination of U46619 and thrombin, at concentrations which alone have no effect, provokes ERK2 activation, suggesting that thrombin and released TXA₂ act synergistically. Our data indicate that both primary signalling through Gq, which evokes ADP secretion, as well as subsequent coupling via Gi by the P2Y12 receptor are required for ERK2 activation.

Keywords

Platelets - thrombin - ADP - P2Y12 receptor - ERK2

Full Text

References

References
1 Gachet C. ADP receptors of platelets and their inhibition. Thromb Haemost 2001; 86: 222-32.
2 Kunapuli SP, Dorsam RT, Kim S. et al. Platelet purinergic receptors. Curr Opin Pharmacol 2003; 03: 175-80.
3 Kauffenstein G, Bergmeier W, Eckly A. et al. The P2Y12 receptor induces platelet aggregation through weak activation of the alpha(IIb) beta(3) integrin – a phosphoinositide 3-kinasedependent mechanism. FEBS Lett 2001; 505: 281-90.
4 Jantzen HM, Milstone DS, Gousset L. et al. Impaired activation of murine platelets lacking G alpha(i2). J Clin Invest 2001; 108: 477-83.
5 Nieswandt B, Schulte V, Zywietz A. et al. Costimulation of Gi- and G12/G13-mediated signaling pathways induces integrin alpha II/beta 3 activation in platelets. J Biol Chem 2002; 277: 39493-98.
6 Hechler B, Eckly A, Ohlmann P. et al. The P2Y1 receptor, necessary but not sufficient to support full ADP-induced platelet aggregation, is not the target of the drug clopidogrel. Br J Haematol 1998; 103: 858-66.
7 Nylander S, Mattsson C, Ramstrom S. et al. The relative importance of the ADP receptors, P2Y12 and P2Y1, in thrombin-induced platelet activation. Thromb Res 2003; 111: 65-73.
8 Kim S, Foster C, Lecchi A. et al. Proteaseactivated receptors 1 and 4 do not stimulate Gi signaling pathways in the absence of secreted ADP and cause human platelet aggregation independently of Gi signaling. Blood 2002; 99: 3629-36.
9 Kramer RM, Roberts EF, Strifler BA. et al. Thrombin induces activation of p38 MAP kinase in human platelets. J Biol Chem 1995; 270: 27395-8.
10 Bugaud F, Nadal-Wollbold F, Levy-Toledano S. et al. Regulation of c-Jun-NH2 terminal kinase and extracellular-signal regulated kinase in human platelets. Blood 1999; 94: 3800-5.
11 Papkoff J, Chen R, Blenis J. et al. p42 mitogen-activated protein kinase and p90 ribosomal S6 kinase are selectively phosphorylated and activated during thrombin-induced platelet activation and aggregation. Mol Cell Biol 1994; 14: 463-72.
12 Nakashima S, Chatani Y, Nakamura M. et al. Tyrosine phosphorylation and activation of mitogen-activated protein kinases by thrombin in human platelets: Possible involvement in late arachidonic acid release. Biochem Biophys Res Commun 1994; 198: 497-503.
13 Nadal F, Levy-Toledano S, Grelac F. et al. Negative regulation of mitogen-activated protein kinase activation by integrin alpha II/ beta 3 in platelets. J Biol Chem 1997; 272: 22381-4.
14 McNicol A, Shibou TS, Pampolina C. et al. Incorporation of MAP kinases into the platelet cytoskeleton. Thromb Res 2001; 103: 25-34.
15 Pearson G, Robinson F, Beers TGibson. et al. Mitogen-activated protein (MAP) kinase pathways: Regulation and physiological functions. Endocr Rev 2001; 22: 153-83.
16 Börsch-Haubold AG, Kramer RM, Watson SP. Inhibition of mitogen-activated protein kinase kinase does not impair primary activation of human platelets. Biochem J 1996; 318: 207-12.
17 McNicol A, Jackson ECG. Inhibition of the MEK/ERK pathway has no effect on agonistinduced aggregation of human platelets. Biochem Pharmacol 2003; 65: 1243-50.
18 Nadal-Wollbold F, Pawlowski M, Levy-Toledano S. et al. Platelet ERK2 activation by thrombin is dependent on calcium and conventional protein kinases C but not Raf-1 or B-Raf. FEBS Lett 2002; 531: 475-82.
19 Pawlowski M, Ragab A, Rosa JP. et al. Selective dephosphorylation of the threonine(183) residue of ERK2 upon (alpha)llb (beta)3 engagement in platelets. FEBS Lett 2002; 521: 145-51.
20 Blaukat A, Barac A, Cross MJ. et al. G Protein-coupled receptor-mediated mitogenactivated protein kinase activation through cooperation of Galpha q and Galpha i signals. Mol Cell Biol 2000; 20: 6837-48.
21 Stilz HU, Guba W, Jablonka B. et al. Discovery of an orally active non-peptide fibrinogen receptor antagonist based on the hydantoin scaffold. J Med Chem 2001; 44: 1158-76.
22 Martinson EA, Scheible S, Greinacher A. et al. Platelet phospholipase D is activated by protein kinase C via an integrin alpha IIb beta 3-independent mechanism. Biochem J 1995; 310: 623-8.
23 Baurand A, Eckly A, Bari N. et al. Desensitization of the platelet aggregation response to ADP: differential down-regulation of the P2Y1 and P2cyc receptors. Thromb Haemost 2000; 84: 484-91.
24 Born GV. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 1962; 194: 927-9.
25 Yang J, Wu J, Kowalska MA. et al. Loss of signaling through the G protein, Gz, results in abnormal platelet activation and altered responses to psychoactive drugs. Proc Natl Acad Sci USA 2000; 97: 9984-9.
26 Lanza F, Beretz A, Stierle A. et al. Epinephrine potentiates human platelet activation but is not an aggregating agent. Am J Physiol Heart Circ Physiol 1988; 255: H1276-88.
27 Steen VM, Holmsen H, Aarbakke G. The platelet-stimulating effect of adrenaline through alpha 2-adrenergic receptors requires simultaneous activation by a true stimulatory platelet agonist. Evidence that adrenaline per se does not induce human platelet activation in vitro. Thromb Haemost 1993; 70: 506-13.
28 Ohkubo S, Nakahata N, Ohizumi Y. Thromboxane A2 stimulates mitogen-activated protein kinase and arachidonic acid liberation in rabbit platelets. Prostaglandins 1996; 52: 403-13.
29 Oury C, Toth-Zsamboki E, Vermylen J. et al. P2X1-mediated activation of extracellular signal-regulated kinase 2 contributes to platelet secretion and aggregation induced by collagen. Blood 2002; 100: 2499-505.
30 Martinson EA, Scheible S, Marx-Grunwitz A. et al. Secreted ADP plays a central role in thrombin-induced phospholipase D activation in human platelets. Thromb Haemost 1998; 80: 976-81.
31 Baurand A, Raboisson P, Freund M. et al. Inhibition of platelet function by administration of MRS2179, a P2Y1 receptor antagonist. Eur J Pharmacol 2001; 412: 213-21.
32 Selheim F, Froyset AK, Strand I. et al. Adrenaline potentiates PI 3-kinase in platelets stimulated with thrombin and SFRLLN: role of secreted ADP. FEBS Lett 2000; 485: 62-6.
33 Offermanns S, Laugwitz K, Spicher K. et al. Proteins of the G12 family are activated via thromboxane A2 and thrombin receptors in human platelets. Proc Natl Acad Sci USA 1994; 91: 504-8.
34 Offermanns S, Toombs CF, Hu YH. et al. Defective platelet activation in G alpha(q)deficient mice. Nature 1997; 389: 183-6.
35 Paul BZS, Jin J, Kunapuli SP. Molecular mechanism of thromboxane A2-induced platelet aggregation. J Biol Chem 1999; 274: 29108-14.
36 Rosado JA, Sage SO. The ERK cascade, a new pathway involved in the activation of store-mediated calcium entry in human platelets. Trends Cardiovas Med 2002; 12: 229-34.
37 Börsch-Haubold AG, Pasquet S, Watson SP. Direct inhibition of cyclooxygenase-1 and -2 by the kinase inhibitors SB 203580 and PD 98059. SB 203580 also inhibits thromboxane synthase. J Biol Chem 1998; 273: 28766-72.
38 Li Z, Xi X, Du X. A Mitogen-activated protein kinase-dependent signaling pathway in the activation of platelet integrin α_IIbβ₃ . J Biol Chem 2001; 276: 42226-32.
39 Roger S, Pawlowski M, Habib A. et al. Costimulation of the Gi-coupled ADP receptor and the Gq-coupled TXA2 receptor is required for ERK2 activation in collagen-induced platelet aggregation. FEBS Lett 2004; 556: 227-35.
40 Soulet C, Sauzeau V, Plantavid M. et al. Gidependent and -independent mechanisms downstream of the P2Y12 ADP-receptor. J Thromb Haemost 2004; 02: 135-46.