P2Y12 ADP receptor-dependent tyrosine phosphorylation of proteins of 27 and 31 kDa in thrombin-stimulated human platelets

Knut Fälker; Danica Lange; Peter Presek

doi:10.1160/TH04-09-0612

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2005; 93(05): 880-888
DOI: 10.1160/TH04-09-0612

Platelets and Blood Cells

Schattauer GmbH

P2Y12 ADP receptor-dependent tyrosine phosphorylation of proteins of 27 and 31 kDa in thrombin-stimulated human platelets

Authors

Knut Fälker^*

¹Martin-Luther-University Halle-Wittenberg, Faculty of Medicine, Department of Pharmacology and Toxicology, Division of Clinical Pharmacology, Halle, Germany
Danica Lange^*

¹Martin-Luther-University Halle-Wittenberg, Faculty of Medicine, Department of Pharmacology and Toxicology, Division of Clinical Pharmacology, Halle, Germany
Peter Presek

¹Martin-Luther-University Halle-Wittenberg, Faculty of Medicine, Department of Pharmacology and Toxicology, Division of Clinical Pharmacology, Halle, Germany

Abstract

Summary

In thrombin-stimulated human platelets several proteins undergo rapid and transient changes in tyrosine phosphorylation. We demonstrate that a set of proteins of 27, 29, 31, 34, and 39 kDa is affected by released ADP and P2Y12 receptor signaling during platelet activation. AR-C69931MX, an antagonist of the Gi₂-coupled P2Y12 ADP receptor, inhibits initial tyrosine phosphorylation of p27 and p31 and prevents subsequent dephosphorylation of p29, p34, and p39. Antagonists of the Gq-coupled P2Y1 ADP receptor have no effect. Precluding integrin α_IIbβ₃ outside-in signaling with RGDS or S1197 does not affect the increase in tyrosine phosphorylation of the set of proteins but inhibits their subsequent dephosphorylation. Besides the ADP analogue 2-MeS-ADP, other platelet agonists such as collagen and the TXA₂-mimetic U46619 also induce p27 and p31 tyrosine phosphorylation in a P2Y12 receptor-dependent manner. Tyrosine phosphorylation of p27 and p31 in response to collagen, but not thrombin, is prevented by aspirin and the TXA₂ receptor antagonist SQ29548, indicating that the effect of collagen strongly relies on TXA₂ signaling. Furthermore, epinephrine, acting via inhibitory Gz-coupled α_2A-adrenoceptors, bypasses the inhibitory effect of AR-C69931MX on thrombin-induced p27 and p31 tyrosine phosphorylation. Finally, we demonstrate that tyrosine phosphorylation of p27 and p31 downstream of P2Y12 receptors is due to the inhibition of adenylyl cyclase but not phosphoinositide 3-kinase (PI 3-K) activation. Elevating cAMP levels with PGI₂ or forskolin precludes thrombin-induced p27 and p31 tyrosine phosphorylation. Moreover, direct inhibition of adenylyl cyclase by SQ22536 reverses the effect of AR-C69931MX. Our data indicate that the observed changes in tyrosine phosphorylation are the result of both primary Gq signaling, initiating the release of ADP, as well as subsequent P2Y12 receptor-mediated Gi coupling.

Keywords

Platelets - protein tyrosine phosphorylation - thrombin - P2Y12 APD receptor - cAMP

Volltext

Referenzen

References
1 Jackson SP, Schoenwaelder SM, Yuan Y. et al. Nonreceptor protein tyrosine kinases and phosphatases in human platelets. Thromb Haemost 1996; 76: 640-50.
2 Presek P, Martinson EA. Platelet Protein Tyrosine Kinases. in: Platelet and Their Factors. Handbook Exp Pharm126. Springer 1997; 263-96.
3 Ferrell Jr. JE, Martin GS. Platelet tyrosine-specific protein phosphorylation is regulated by thrombin. Mol Cell Biol 1988; 8: 3603-10.
4 Ferrell Jr. JE, Martin GS. Tyrosine-specific protein phosphorylation is regulated by glycoprotein IIb-IIIa in platelets. Proc Natl Acad Sci USA 1989; 86: 2234-8.
5 Gachet C. ADP receptors of platelets and their inhibition. Thromb Haemost 2001; 86: 222-32.
6 Kunapuli SP, Dorsam RT, Kim S. et al. Platelet purinergic receptors. Curr Opin Pharmacol 2003; 3: 175-80.
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 Nurden P, Savi P, Heilmann E. et al. An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. Its influence on glycoprotein IIb-IIIa complexfunction. JClin Invest 1995; 95: 1612-2.
9 Hollopeter G, Jantzen H-M, Vincent D. et al. Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature 2001; 409: 202-7.
10 Levy-Toledano S, Maclouf J, Rosa JP. et al. Abnormal tyrosine phosphorylation linked to a defective interaction between ADP and its receptor on platelets. Thromb Haemost 1998; 80: 463-8.
11 Savi P, Artcanuthurry V, Bornia J. et al. Effect of clopidogrel treatment on ADP-induced phosphorylations in rat platelets. Br J Haematol 1997; 97: 185-91.
12 Contreres JO, Dupuy E, Job B. et al. Effect of clopidogrel administration to healthy volunteers on platelet phosphorylation events triggered by ADP. Br J Haematol 2003; 120: 633-42.
13 Rosa JP, Artcanuthurry V, Grelac F. et al. Reassessment of protein tyrosine phosphorylation in thrombasthenic platelets: evidence that phosphorylation of cortactin and a 64-k Dprotein is dependent onthrombin activationand integrin αIIbβ3. Blood 1997; 89: 4385-92.
14 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.
15 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.
16 Fälker K, Lange D, Presek P. ADP secretion and subsequent P2Y12 receptor signaling play a crucial role in thrombin-induced ERK2 activation in human platelets. Thromb Haemost 2004; 92: 114-23.
17 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.
18 Karim ZA, Mukhopadhyay S, Ramars ASS. et al. Sustained stimulation of platelet thrombin receptor is associated with tyrosine dephosphorylation of a novel p67 peptide in a manner regulated by extracellular calcium. Biochim Biophys Acta 2004; 1693: 147-57.
19 Born GV. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 1962; 194: 927-9.
20 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.
21 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: 1276-88.
22 Steen VM, Holmsen H, Aarbakke G. The plateletstimulating 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 humanplatelet activation in vitro. Thromb Haemost 1993; 70: 506-13.
23 Arderiu G, Diaz-Ricart M, Domenech P. et al. Phosphotyrosine proteins in platelets from patients with storage pool disease: direct relation between granule defects and defective signal transduction. Haematologica 2002; 87: 629-36.
24 Atkinson BT, Stafford MJ, Pears CJ. et al. Signaling events underlying platelet aggregation induced by the glycoprotein VI agonist convulxin. Eur J Biochem 2001; 268: 5242-48.
25 Roger S, Pawlowski M, Habib A. et al. Costimulation of the Gi-coupled ADP receptor and the Gqcoupled TXA2 receptor is required for ERK 2activation in collagen-induced platelet aggregation. FEBS Lett 2004; 556: 227-35.
26 Offermanns S, Laugwitz K, Spicher K. et al. Gproteins of the G12 family are activated via thromboxane A2 and thrombin receptors in human platelets. Proc Natl Acad Sci USA 1994; 91: 504-8.
27 Nieswandt B, Schulte V, Zywietz A. et al. Costimulation of Gi- and G12/G13-mediated signaling pathways induces integrin αIIbβ3 activation in platelets. J Biol Chem 2002; 277: 39493-8.
28 Offermanns S, Toombs CF, Hu YH. et al. Defective platelet activation in G alpha(q)-deficient mice. Nature 1997; 389: 183-6.
29 Paul BZS, Jin J, Kunapuli SP. Molecular mechanism of thromboxane A2-induced platelet aggregation. J Biol Chem 1999; 274: 29108-14.
30 Wang L, Ostberg O, Wihlborg AK. et al. Quantification of ADP and ATP receptor expression in human platelets. J Thromb Haemost 2003; 1: 330-6.
31 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-kinase-dependent mechanism. FEBS Lett 2001; 505: 281-90.
32 Jantzen HM, Milstone DS, Gousset L. et al. Impaired activation of murine platelets lacking G α(i2). J Clin Invest 2001; 108: 477-83.
33 Gachet C, Cazenave JP, Ohlmann P. et al. The thienopyridine ticlopidine selectively prevents the inhibitory effects of ADP but not of adrenaline on cAMP levels raised by stimulation of the adenylate cyclase of human platelets by PGE1. Biochem Pharmacol 1990; 40: 2683-7.
34 Nakahashi TK, Kambayashi I J, Nakamura T. et al. Platelets in nonresponders to epinephrine stimulation showed reduced response to ADP. Thromb Res 2001; 104: 127-35.
35 Dorsam RT, Kunapuli SP. Central role of the P2Y12 receptor in platelet activation. J Clin Invest 2004; 113: 340-5.
36 Savi P, Pflieger AM, Herbert JM. cAMP is not an important messenger for ADP-induced platelet aggregation. Blood Coagul Fibrinolysis 1996; 7: 249-52.
37 Yang J, Wu J, Jiang H. et al. Signaling through Gi family members in platelets. Redundancy and specificity in the regulation of adenylyl cyclase and other effector. J Biol Chem 2002; 277: 46035-42.
38 Haslam RJ, Davidson MM, Desjardins JV. Inhibition of adenylate cyclase by adenosine analogues in preparations of broken and intact human platelets. Evidence for the unidirectional control of platelet function by cycli cAMP. Biochem J 1978; 176: 83-95.
39 Daniel JL, Dangelmaier C, Jin J. et al. Role of intracellular signaling events in ADP-induced platelet aggregation. Thromb Haemost 1999; 82: 1322-6.
40 Lova P, Paganini S, Hirsch E. et al. Aselective role for phosphatidylinositol 3,4,5-trisphosphate in the Gidependent activation of platelet Rap1B. J Biol Chem 2003; 278: 131-8.
41 Kim S, Jin J, Kunapuli SP. Akt activation inplatelets depends on Gi signaling pathways. J Biol Chem 2004; 279: 4186-95.
42 Dangelmaier C, Jin J, Smith JB. et al. Potentiation of thromboxane A2-induced platelet secretion by Gi signaling through the phosphoinositide-3 kinase pathway. Thromb Haemost 2001; 85: 341-8.
43 Trumel C, Payrastre B, Plantavid M. et al. A key role of adenosine diphosphate in the irreversible platelet aggregation induced by the PAR1-activating peptide through the late activation of phosphoinositide 3-kinase. Blood 1999; 94: 4156-65.
44 Pumiglia KM, Huang CK, Feinstein MB. Elevation of cAMP, but not cGMP, inhibits thrombin-stimulated tyrosine phosphorylation in human platelets. Biochem Biophys Res Commun 1990; 171: 738-45.
45 Smith JB, Dangelmaier C, Daniel JL. Elevation of cAMP in human platelets inhibits thrombin- but not collagen-induced tyrosine phosphorylation. Biochem Biophys Res Commun 1993; 191: 695-700.
46 Ryningen A, Jensen BO, Holmsen H. Role of autocrine stimulation on the effects of cyclic AMP on protein and lipid phosphorylation in collagen-activated and thrombin-activated platelets. Eur J Biochem 1999; 260: 87-96.
47 Hardy AR, Jones ML, Mundell SJ. et al. Reciprocal cross-talk between P2Y1 and P2Y12 receptors at the level of calcium signaling in human platelets. Blood 2004; 104: 1745-52.
48 Bain J, McLauchlan H, Elliott M. et al. The specificities of protein kinase inhibitors: an update. Biochem J 2003; 371: 199-204.
49 Marcus K, Immler D, Sternberger J. et al. Identification of platelet proteins separated by two-dimensional gel electrophoresis and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and detection of tyrosine-phosphorylated proteins. Electrophoresis 2000; 21: 2622-36.
50 Maguire PB, Wynne KJ, Harney DF. et al. Identification of the phosphotyrosine proteome from thrombin activated platelets. Proteomics 2002; 2: 642-8.
51 Marcus K, Moebius J, Meyer HE. Differential analysis of phosphorylated proteins in resting and thrombin-stimulated human platelets. Anal Bioanal Chem 2003; 376: 973-93.
52 Chew CS, Parente Jr. JA, Zhou C. et al. Lasp-1 is a regulated phosphoprotein within the cAMP signaling pathway in the gastric parietal cell. Am J Physiol 1998; 275: C56-67.
53 Kotaka M, Lau YM, Cheung KK. et al. Elfin is expressed during early heart development. J Cell Biochem 2001; 83: 463-72.
54 Cong F, Yuan B, Goff SP. Characterization of a novel member of the DOK family that binds and modulates Abl signaling. Mol Cell Biol 1999; 19: 8314-25.