Protective mechanisms of adenosine 5′-monophosphate in platelet activation and thrombus formation

Eduardo Fuentes; Lina Badimon; Julio Caballero; Teresa Padró; Gemma Vilahur; Marcelo Alarcón; Pablo Pérez; Iván Palomo

doi:10.1160/TH13-05-0386

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2014; 111(03): 491-507
DOI: 10.1160/TH13-05-0386

Platelets and Blood Cells

Schattauer GmbH

Protective mechanisms of adenosine 5′-monophosphate in platelet activation and thrombus formation

Eduardo Fuentes

¹Department of Clinical Biochemistry and Immunohematology, Faculty of Health Sciences, Interdisciplinary Excellence Research Program on Healthy Aging (PIEI-ES), Universidad de Talca, Talca, Chile

²Centro de Estudios en Alimentos Procesados (CEAP), CONICYT-Regional, Gore Maule, R09I2001, Talca, Chile

,

Lina Badimon

⁴Centro de Investigación Cardiovascular (ICCC-CSIC), Instituto de investigación Biomédica Sant Pau (IIB-Sant Pau), Hospital de la Santa Creu i Sant Pau; Barcelona, España

,

Julio Caballero

³Center for Bioinformatics and Molecular Simulations, Faculty of Engineering in Bioinformatics, Universidad de Talca, Talca, Chile

,

Teresa Padró

⁴Centro de Investigación Cardiovascular (ICCC-CSIC), Instituto de investigación Biomédica Sant Pau (IIB-Sant Pau), Hospital de la Santa Creu i Sant Pau; Barcelona, España

,

Gemma Vilahur

⁴Centro de Investigación Cardiovascular (ICCC-CSIC), Instituto de investigación Biomédica Sant Pau (IIB-Sant Pau), Hospital de la Santa Creu i Sant Pau; Barcelona, España

,

Marcelo Alarcón

¹Department of Clinical Biochemistry and Immunohematology, Faculty of Health Sciences, Interdisciplinary Excellence Research Program on Healthy Aging (PIEI-ES), Universidad de Talca, Talca, Chile

²Centro de Estudios en Alimentos Procesados (CEAP), CONICYT-Regional, Gore Maule, R09I2001, Talca, Chile

,

Pablo Pérez

¹Department of Clinical Biochemistry and Immunohematology, Faculty of Health Sciences, Interdisciplinary Excellence Research Program on Healthy Aging (PIEI-ES), Universidad de Talca, Talca, Chile

,

Iván Palomo

¹Department of Clinical Biochemistry and Immunohematology, Faculty of Health Sciences, Interdisciplinary Excellence Research Program on Healthy Aging (PIEI-ES), Universidad de Talca, Talca, Chile

²Centro de Estudios en Alimentos Procesados (CEAP), CONICYT-Regional, Gore Maule, R09I2001, Talca, Chile

› Author Affiliations

Abstract

Summary

Platelet activation is relevant to a variety of acute thrombotic events. We sought to examine adenosine 5′-monophosphate (AMP) mechanisms of action in preventing platelet activation, thrombus formation and platelet-related inflammatory response. We assessed the effect of AMP on 1) P-selectin expression and GPIIb/IIIa activation by flow cytometry; 2) Platelet aggregation and ATP secretion induced by ADP, collagen, TRAP-6, convulxin and thrombin; 3) Platelet rolling and firm adhesion, and platelet-leukocyte interactions under flow-controlled conditions; and, 4) Platelet cAMP levels, sP-selectin, sCD40L, IL-1β, TGF-β1 and CCL5 release, PDE3A activity and PKA phosphorylation. The effect of AMP on in vivo thrombus formation was also evaluated in a murine model. The AMP docking with respect to A2 adenosine receptor was determined by homology. AMP concentration-dependently (0.1 to 3 mmol/l) inhibited P-selectin expression and GPIIb/IIIa activation, platelet secretion and aggregation induced by ADP, collagen, TRAP-6 and convulxin, and diminished platelet rolling and firm adhesion. Furthermore, AMP induced a marked increase in the rolling speed of leukocytes retained on the platelet surface. At these concentrations AMP significantly decreased inflammatory mediator from platelet, increased intraplatelet cAMP levels and inhibited PDE3A activity. Interestingly, SQ22536, ZM241385 and SCH58261 attenuated the antiplatelet effect of AMP. Docking experiments revealed that AMP had the same orientation that adenosine inside the A2 adenosine receptor binding pocket. These in vitro antithrombotic properties were further supported in an in vivo model of thrombosis. Considering the successful use of combined antiplatelet therapy, AMP may be further developed as a novel antiplatelet agent.

Keywords

Platelets - antithrombotic - adenosine 5′-monophosphate - PDE inhibition - A_2A receptor

Full Text

References

References
1 Badimon L, Vilahur G, Padro T. Lipoproteins, platelets and atherothrombosis. Rev Esp Cardiol 2009; 62: 1161-1178.
2 da Costa Martins PA, van Gils JM, Mol A. et al. Platelet binding to monocytes increases the adhesive properties of monocytes by up-regulating the expression and functionality of beta1 and beta2 integrins. J Leukoc Biol 2006; 79: 499-507.
3 da Costa Martins P, van den Berk N, Ulfman LH. et al. Platelet-monocyte complexes support monocyte adhesion to endothelium by enhancing secondary tethering and cluster formation. Arterioscler Thromb Vasc Biol 2004; 24: 193-199.
4 Fuentes QE, Fuentes QF, Andres V. et al. Role of platelets as mediators that link inflammation and thrombosis in atherosclerosis. Platelets 2013; 24: 255-262.
5 Ridker PM, Buring JE, Rifai N. Soluble P-selectin and the risk of future cardiovascular events. Circulation 2001; 103: 491-495.
6 Huo Y, Schober A, Forlow SB. et al. Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E. Nat Med 2003; 9: 61-67.
7 Aukrust P, Muller F, Ueland T. et al. Enhanced levels of soluble and membrane-bound CD40 ligand in patients with unstable angina. Possible reflection of T lymphocyte and platelet involvement in the pathogenesis of acute coronary syndromes. Circulation 1999; 100: 614-620.
8 Nurden AT. Platelets, inflammation and tissue regeneration. Thromb Haemost 2011; 105 (Suppl. 01) S13-33.
9 Collins B, Hollidge C. Antithrombotic drug market. Nat Rev Drug Discov 2003; 2: 11-12.
10 Palomo I, Toro C, Alarcon M. The role of platelets in the pathophysiology of atherosclerosis (Review). Mol Med Rep 2008; 1: 179-184.
11 Barrett NE, Holbrook L, Jones S. et al. Future innovations in anti-platelet therapies. Br J Pharmacol 2008; 154: 918-939.
12 Raju NC, Eikelboom JW. The aspirin controversy in primary prevention. Curr Opin Cardiol 2012; 27: 499-507.
13 Hu FB. Plant-based foods and prevention of cardiovascular disease: an overview. Am J Clin Nutr 2003; 78 (Suppl. 03) 544S-551S.
14 Fuentes EJ, Astudillo LA, Gutierrez MI. et al. Fractions of aqueous and metha-nolic extracts from tomato (Solanum lycopersicum L.) present platelet antiag-gregant activity. Blood Coagul Fibrinolysis 2012; 23: 109-117.
15 Fuentes E, Carle R, Astudillo L. et al. Antioxidant and Antiplatelet Activities in Extracts from Green and Fully Ripe Tomato Fruits (Solanum lycopersicum) and Pomace from Industrial Tomato Processing. Evid Based Complement Alternat Med 2013; 2013: 1-9.
16 Hubbard GP, Wolffram S, Lovegrove JA. et al. Ingestion of quercetin inhibits platelet aggregation and essential components of the collagen-stimulated platelet activation pathway in humans. J Thromb Haemost 2004; 2: 2138-2145.
17 Fuentes E, Castro R, Astudillo L. et al. Bioassay-Guided Isolation and HPLC Determination of Bioactive Compound That Relate to the Antiplatelet Activity (Adhesion, Secretion, and Aggregation) from Solanum lycopersicum. Evid Based Complement Alternat Med 2012; 2012: 147031.
18 Fuentes E, Alarcon M, Astudillo L. et al. Protective Mechanisms of Guanosine from Solanum lycopersicum on Agonist-Induced Platelet Activation: Role of sCD40L. Molecules 2013; 18: 8120-8135.
19 Anfossi G, Russo I, Massucco P. et al. Adenosine increases human platelet levels of cGMP through nitric oxide: possible role in its antiaggregating effect. Thromb Res 2002; 105: 71-78.
20 Frojmovic M, Wong T, van de Ven T. Dynamic measurements of the platelet membrane glycoprotein IIb-IIIa receptor for fibrinogen by flow cytometry. I. Methodology, theory and results for two distinct activators. Biophys J 1991; 59: 815-827.
21 Born GV, Cross MJ. The Aggregation of Blood Platelets. J Physiol 1963; 168: 178-195.
22 Conant CG, Schwartz MA, Nevill T. et al. Platelet adhesion and aggregation under flow using microfluidic flow cells. J Vis Exp 2009; 32: 1644.
23 Appeldoorn CC, Bonnefoy A, Lutters BC. et al. Gallic acid antagonizes P-selec-tin-mediated platelet-leukocyte interactions: implications for the French paradox. Circulation 2005; 111: 106-112.
24 Hung SH, Zhang W, Pixley RA. et al. New insights from the structure-function analysis of the catalytic region of human platelet phosphodiesterase 3A: a role for the unique 44-amino acid insert. J Biol Chem 2006; 281: 29236-29244.
25 Pan C, Wei X, Ye J. et al. BF066, a novel dual target antiplatelet agent without significant bleeding. PLoS One 2012; 7: e40451
26 Przyklenk K, Whittaker P. Adaptation of a photochemical method to initiate recurrent platelet-mediated thrombosis in small animals. Lasers Med Sci 2007; 22: 42-45.
27 Friesner R, Banks J, Murphy R. et al. Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem 2004; 47: 1739-1749.
28 Munoz C, Adasme F, Alzate-Morales JH. et al. Study of differences in the VEGFR2 inhibitory activities between semaxanib and SU5205 using 3D-QSAR, docking, and molecular dynamics simulations. J Mol Graph Model 2012; 32: 39-48.
29 Jorgensen W, Maxwell D, Tirado-Rives J. Development and testing of the OPLS all-atom force field on conformational energetics and properties of organic liquids. J Am Chem Soc 1996; 118: 11225-11236.
30 Eldridge MD, Murray CW, Auton TR. et al. Empirical scoring functions: I. The development of a fast empirical scoring function to estimate the binding affinity of ligands in receptor complexes. J Comput Aided Mol Des 1997; 11: 425-445.
31 Storey RF, Judge HM, Wilcox RG. et al. Inhibition of ADP-induced P-selectin expression and platelet-leukocyte conjugate formation by clopidogrel and the P2Y12 receptor antagonist AR-C69931MX but not aspirin. Thromb Haemost 2002; 88: 488-494.
32 Lievens D, Zernecke A, Seijkens T. et al. Platelet CD40L mediates thrombotic and inflammatory processes in atherosclerosis. Blood 2010; 116: 4317-4327.
33 von Hundelshausen P, Weber KS, Huo Y. et al. RANTES deposition by platelets triggers monocyte arrest on inflamed and atherosclerotic endothelium. Circulation 2001; 103: 1772-1777.
34 Feijge MA, Ansink K, Vanschoonbeek K. et al. Control of platelet activation by cyclic AMP turnover and cyclic nucleotide phosphodiesterase type-3. Biochem Pharmacol 2004; 67: 1559-1567.
35 Libersan D, Rousseau G, Merhi Y. Differential regulation of P-selectin expression by protein kinase A and protein kinase G in thrombin-stimulated human platelets. Thromb Haemost 2003; 89: 310-317.
36 Minamino T, Kitakaze M, Asanuma H. et al. Endogenous adenosine inhibits P-selectin-dependent formation of coronary thromboemboli during hypoperfusion in dogs. J Clin Invest 1998; 101: 1643-1653.
37 Jensen BO, Selheim F, Doskeland SO. et al. Protein kinase A mediates inhibition of the thrombin-induced platelet shape change by nitric oxide. Blood 2004; 104: 2775-2782.
38 Lebon G, Warne T, Edwards PC. et al. Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation. Nature 2011; 474: 521-525.
39 Inoue O, Suzuki-Inoue K, Dean WL. et al. Integrin alpha2beta1 mediates out-side-in regulation of platelet spreading on collagen through activation of Src ki-nases and PLCgamma2. J Cell Biol 2003; 160: 769-780.
40 Ludwig RJ, Schultz JE, Boehncke WH. et al. Activated, not resting, platelets increase leukocyte rolling in murine skin utilizing a distinct set of adhesion molecules. J Invest Dermatol 2004; 122: 830-836.
41 Braun OO, Slotta JE, Menger MD. et al. Primary and secondary capture of platelets onto inflamed femoral artery endothelium is dependent on P-selectin and PSGL-1. Eur J Pharmacol 2008; 592: 128-132.
42 Hunter RW, Mackintosh C, Hers I. Protein kinase C-mediated phosphorylation and activation of PDE3A regulate cAMP levels in human platelets. J Biol Chem 2009; 284: 12339-12348.
43 Movsesian MA. PDE3 inhibition in dilated cardiomyopathy: reasons to reconsider. J Card Fail 2003; 9: 475-480.
44 Levy JH, Bailey JM, Deeb GM. Intravenous milrinone in cardiac surgery. Ann Thorac Surg 2002; 73: 325-330.
45 Beebe HG, Dawson DL, Cutler BS. et al. A new pharmacological treatment for intermittent claudication: results of a randomized, multicenter trial. Arch Intern Med 1999; 159: 2041-2050.
46 Chi YW, Lavie CJ, Milani RV. et al. Safety and efficacy of cilostazol in the management of intermittent claudication. Vasc Health Risk Manag 2008; 4: 1197-1203.
47 Yang D, Chen H, Koupenova M. et al. A new role for the A2b adenosine receptor in regulating platelet function. J Thromb Haemost 2010; 8: 817-827.
48 Loyau S, Dumont B, Ollivier V. et al. Platelet glycoprotein VI dimerisation, an active process inducing receptor competence, is an indicator of platelet reactivity. Arterioscler Thromb Vasc Biol 2012; 32: 778-785.
49 Cusack NJ, Hickman ME, Born GV. Effects of D- and L- enantiomers of adeno-sine, AMP and ADP and their 2-chloro- and 2-azido- analogues on human platelets. Proc R Soc Lond B Biol Sci 1979; 206: 139-144.
50 Deussen A, Bading B, Kelm M. et al. Formation and salvage of adenosine by macrovascular endothelial cells. Am J Physiol 1993; 264: H692-700.
51 Koszalka P, Ozuyaman B, Huo Y. et al. Targeted disruption of cd73/ecto-5’-nu-cleotidase alters thromboregulation and augments vascular inflammatory response. Circ Res 2004; 95: 814-821.
52 Zernecke A, Bidzhekov K, Ozuyaman B. et al. CD73/ecto-5’-nucleotidase protects against vascular inflammation and neointima formation. Circulation 2006; 113: 2120-2127.
53 Blume C, Felix A, Shushakova N. et al. Autoimmunity in CD73/Ecto-5’-nu-cleotidase deficient mice induces renal injury. PLoS One 2012; 7: e37100