Adenosine and Forskolin Inhibit Platelet Aggregation by Collagen but not the Proximal Signalling EventsFunding This study was supported by a grant from the Centre of Membrane and Protein and Receptors, University of Birmingham and University of Nottingham, Midlands, UK. S.P.W. is a British Heart Foundation Professor (CH 03/003).
30 October 2018
20 March 2019
26 May 2019 (eFirst)
Background The G protein-coupled receptor, adenosine A2A, signals through the stimulatory G protein, Gs, in platelets leading to activation of adenylyl cyclase and elevation of cyclic adenosine monophosphate (cAMP) and inhibition of platelet activation.
Objective This article investigates the effect of A2A receptor activation on signalling by the collagen receptor glycoprotein (GP) VI in platelets.
Methods Washed human platelets were stimulated by collagen or the GPVI-specific agonist collagen-related peptide (CRP) in the presence of the adenosine receptor agonist, 5′-N-ethylcarboxamidoadenosine (NECA) or the adenylyl cyclase activator, forskolin and analysed for aggregation, adenosine triphosphate secretion, protein phosphorylation, spreading, Ca2+ mobilisation, GPVI receptor clustering, cAMP, thromboxane B2 (TxB2) and P-selectin exposure.
Results NECA, a bioactive adenosine analogue, partially inhibits aggregation and secretion to collagen or CRP in the absence or presence of the P2Y12 receptor antagonist, cangrelor and the cyclooxygenase inhibitor, indomethacin. The inhibitory effect in the presence of the three inhibitors is largely overcome at higher concentrations of collagen but not CRP. Neither NECA nor forskolin altered clustering of GPVI, elevation of Ca2+ or spreading of platelets on a collagen surface. Further, neither NECA nor forskolin, altered collagen-induced tyrosine phosphorylation of Syk, LAT nor PLCγ2. However, NECA and forskolin inhibited platelet activation by the TxA2 mimetic, U46619, but not the combination of adenosine diphosphate and collagen.
Conclusion NECA and forskolin have no effect on the proximal signalling events by collagen. They inhibit platelet activation in a response-specific manner in part through inhibition of the feedback action of TxA2.
J.C.C. has performed experiments, analysed data, wrote and edited the manuscript. S.W. performed experiments and analysed the data. D.M.K. provided training in super resolution microscopy experiments and expertise for the study design and cluster analysis. J.A.P. has analysed the data and developed the programmes for image and cluster analysis. N.S.P. provided expertise for super resolution microscopy experiment study design and cluster analysis. R.K.A. and E.E.G. provided key materials. S.J.H. provided supervision, concept, funding and contributed to manuscript editing. S.P.W. provided supervision, funding, study design and concept, reviewed data, wrote and edited the manuscript. All authors have read the manuscript.
- 1 Borst S, Sim X, Poncz M, French DL, Gadue P. Induced pluripotent stem cell-derived megakaryocytes and platelets for disease modeling and future clinical applications. Arterioscler Thromb Vasc Biol 2017; 37 (11) 2007-2013
- 2 Siess W. Cross-talk of cGMP- and cAMP-signaling pathways in human platelets. Blood 2003; 101: 4230
- 3 Schwarz UR, Walter U, Eigenthaler M. Taming platelets with cyclic nucleotides. Biochem Pharmacol 2001; 62 (09) 1153-1161
- 4 Eigenthaler M, Nolte C, Halbrügge M, Walter U. Concentration and regulation of cyclic nucleotides, cyclic-nucleotide-dependent protein kinases and one of their major substrates in human platelets. Estimating the rate of cAMP-regulated and cGMP-regulated protein phosphorylation in intact cells. Eur J Biochem 1992; 205 (02) 471-481
- 5 Geiger J, Nolte C, Walter U. Regulation of calcium mobilization and entry in human platelets by endothelium-derived factors. Am J Physiol 1994; 267 (1 Pt 1): C236-C244
- 6 Butt E, Immler D, Meyer HE, Kotlyarov A, Laass K, Gaestel M. Heat shock protein 27 is a substrate of cGMP-dependent protein kinase in intact human platelets: phosphorylation-induced actin polymerization caused by HSP27 mutants. J Biol Chem 2001; 276 (10) 7108-7113
- 7 Harbeck B, Hüttelmaier S, Schluter K, Jockusch BM, Illenberger S. Phosphorylation of the vasodilator-stimulated phosphoprotein regulates its interaction with actin. J Biol Chem 2000; 275 (40) 30817-30825
- 8 Hettasch JM, Sellers JR. Caldesmon phosphorylation in intact human platelets by cAMP-dependent protein kinase and protein kinase C. J Biol Chem 1991; 266 (18) 11876-11881
- 9 Bennett JS, Zigmond S, Vilaire G, Cunningham ME, Bednar B. The platelet cytoskeleton regulates the affinity of the integrin alpha(IIb)beta(3) for fibrinogen. J Biol Chem 1999; 274 (36) 25301-25307
- 10 Nieswandt B, Watson SP. Platelet-collagen interaction: is GPVI the central receptor?. Blood 2003; 102 (02) 449-461
- 11 Watson SP, Auger JM, McCarty OJ, Pearce AC. GPVI and integrin alphaIIb beta3 signaling in platelets. J Thromb Haemost 2005; 3 (08) 1752-1762
- 12 Miura Y, Takahashi T, Jung SM, Moroi M. Analysis of the interaction of platelet collagen receptor glycoprotein VI (GPVI) with collagen. A dimeric form of GPVI, but not the monomeric form, shows affinity to fibrous collagen. J Biol Chem 2002; 277 (48) 46197-46204
- 13 Cooper JA, Hill SJ, Alexander SP, Rubin PC, Horn EH. Adenosine receptor-induced cyclic AMP generation and inhibition of 5-hydroxytryptamine release in human platelets. Br J Clin Pharmacol 1995; 40 (01) 43-50
- 14 Fuentes E, Pereira J, Mezzano D, Alarcón M, Caballero J, Palomo I. Inhibition of platelet activation and thrombus formation by adenosine and inosine: studies on their relative contribution and molecular modeling. PLoS One 2014; 9 (11) e112741
- 15 Amisten S, Braun OO, Bengtsson A, Erlinge D. Gene expression profiling for the identification of G-protein coupled receptors in human platelets. Thromb Res 2008; 122 (01) 47-57
- 16 Cattaneo M, Schulz R, Nylander S. Adenosine-mediated effects of ticagrelor: evidence and potential clinical relevance. J Am Coll Cardiol 2014; 63 (23) 2503-2509
- 17 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 (02) 695-700
- 18 Smith JB, Dangelmaier C, Selak MA, Ashby B, Daniel J. Cyclic AMP does not inhibit collagen-induced platelet signal transduction. Biochem J 1992; 283 (Pt 3): 889-892
- 19 Ichinohe T, Takayama H, Ezumi Y, Yanagi S, Yamamura H, Okuma M. Cyclic AMP-insensitive activation of c-Src and Syk protein-tyrosine kinases through platelet membrane glycoprotein VI. J Biol Chem 1995; 270 (47) 28029-28036
- 20 Riondino S, Lotti LV, Cutini L, Pulcinelli FM. Collagen-induced platelet shape change is not affected by positive feedback pathway inhibitors and cAMP-elevating agents. J Biol Chem 2005; 280 (08) 6504-6510
- 21 Loyau S, Dumont B, Ollivier V. , et al. Platelet glycoprotein VI dimerization, an active process inducing receptor competence, is an indicator of platelet reactivity. Arterioscler Thromb Vasc Biol 2012; 32 (03) 778-785
- 22 Takayama H, Hosaka Y, Nakayama K. , et al. A novel antiplatelet antibody therapy that induces cAMP-dependent endocytosis of the GPVI/Fc receptor gamma-chain complex. J Clin Invest 2008; 118 (05) 1785-1795
- 23 Al-Tamimi M, Mu F-T, Arthur JF. , et al. Anti-glycoprotein VI monoclonal antibodies directly aggregate platelets independently of FcgammaRIIa and induce GPVI ectodomain shedding. Platelets 2009; 20 (02) 75-82
- 24 Grygielska B, Hughes CE, Watson SP. Molecular basis of platelet activation by an alphaIIbbeta3-CHAMPS peptide. J Thromb Haemost 2009; 7 (02) 339-346
- 25 Berthold M, Cebron N, Dill F. , et al. KNIME - The Konstanz Information Miner: Version 2.0 and Beyond. ACM SIGKDD Explorations Newsletter 2009; 11 (01) 26-31
- 26 Sommer C, Straehle C, Köthe U, Hamprecht FA. , eds. Ilastik: Interactive learning and segmentation toolkit. IEEE International Symposium on Biomedical Imaging: From Nano to Macro; 2011 :230–233
- 27 Poulter NS, Pollitt AY, Owen DM. , et al. Clustering of glycoprotein VI (GPVI) dimers upon adhesion to collagen as a mechanism to regulate GPVI signaling in platelets. J Thromb Haemost 2017; 15 (03) 549-564
- 28 van de Linde S, Löschberger A, Klein T. , et al. Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc 2011; 6 (07) 991-1009
- 29 Ovesný M, Křížek P, Borkovec J, Švindrych Z, Hagen GM. ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 2014; 30 (16) 2389-2390
- 30 Ester M, Kriegel H-P, Sander R, Xu X. A density-based algorithm for discovering clusters a density-based algorithm for discovering clusters in large spatial databases with noise. Proceedings of the Second International Conference on Knowledge Discovery and Data Mining; Portland, Oregon. 3001507: AAAI Press; 1996 :226–231
- 31 Cusack NJ, Hourani SM. 5′-N-ethylcarboxamidoadenosine: a potent inhibitor of human platelet aggregation. Br J Pharmacol 1981; 72 (03) 443-447
- 32 Adnot S, Desmier M, Ferry N, Hanoune J, Sevenet T. Forskolin (a powerful inhibitor of human platelet aggregation). Biochem Pharmacol 1982; 31 (24) 4071-4074
- 33 Smolenski A. Novel roles of cAMP/cGMP-dependent signaling in platelets. J Thromb Haemost 2012; 10 (02) 167-176
- 34 Kroll MH, Schafer AI. Biochemical mechanisms of platelet activation. Blood 1989; 74 (04) 1181-1195
- 35 Quattrin S, Genovese A, Cirillo R, Formisano S, Marone G. Functional and biochemical evidence of a specific adenosine A2/Ra receptor on human platelets. Ric Clin Lab 1988; 18 (2-3): 105-118