Plant food anthocyanins inhibit platelet granule secretion in hypercholesterolaemia: Involving the signalling pathway of PI3K–Akt

Fenglin Song; Yanna Zhu; Zhenyin Shi; Jinju Tian; Xiujuan Deng; Jing Ren; Marc C. Andrews; Heyu Ni; Wenhua Ling; Yan Yang

doi:10.1160/th13-12-1002

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2014; 112(05): 981-991
DOI: 10.1160/th13-12-1002

Platelets and Blood Cells

Schattauer GmbH

Plant food anthocyanins inhibit platelet granule secretion in hypercholesterolaemia: Involving the signalling pathway of PI3K–Akt

Authors

Fenglin Song

¹Guangdong Provincial Key Laboratory of Food, Nutrition, and Health, Guangzhou, People’s Republic of China

²Department of Nutrition, School of Public Health, Sun Yat-Sen University, Guangzhou, People’s Republic of China
Yanna Zhu

³Department of Maternal and Child Health, School of Public Health, Sun Yat-Sen University, Guangzhou, People’s Republic of China
Zhenyin Shi

⁴Disease Control and Prevention Center of Gansu Province, Lanzhou, People’s Republic of China
Jinju Tian

¹Guangdong Provincial Key Laboratory of Food, Nutrition, and Health, Guangzhou, People’s Republic of China

²Department of Nutrition, School of Public Health, Sun Yat-Sen University, Guangzhou, People’s Republic of China
Xiujuan Deng

¹Guangdong Provincial Key Laboratory of Food, Nutrition, and Health, Guangzhou, People’s Republic of China

²Department of Nutrition, School of Public Health, Sun Yat-Sen University, Guangzhou, People’s Republic of China
Jing Ren

¹Guangdong Provincial Key Laboratory of Food, Nutrition, and Health, Guangzhou, People’s Republic of China

²Department of Nutrition, School of Public Health, Sun Yat-Sen University, Guangzhou, People’s Republic of China
Marc C. Andrews

⁵Toronto Platelet Immunobiology Group, Toronto, Ontario, Canada

⁶Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael’s Hospital, Toronto, Ontario, Canada

⁷Department of Physiology, University of Toronto, Toronto, Ontario, Canada
Heyu Ni

⁵Toronto Platelet Immunobiology Group, Toronto, Ontario, Canada

⁶Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael’s Hospital, Toronto, Ontario, Canada

⁷Department of Physiology, University of Toronto, Toronto, Ontario, Canada

⁸Canadian Blood Services, Toronto, Ontario, Canada

⁹Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada

¹⁰Department of Medicine, University of Toronto, Toronto, Ontario, Canada
Wenhua Ling

¹Guangdong Provincial Key Laboratory of Food, Nutrition, and Health, Guangzhou, People’s Republic of China

²Department of Nutrition, School of Public Health, Sun Yat-Sen University, Guangzhou, People’s Republic of China
Yan Yang

¹Guangdong Provincial Key Laboratory of Food, Nutrition, and Health, Guangzhou, People’s Republic of China

²Department of Nutrition, School of Public Health, Sun Yat-Sen University, Guangzhou, People’s Republic of China

Abstract

Summary

Controlling platelet granule secretion has been considered an effective strategy to dampen thrombosis and prevent atherosclerosis. Anthocyanins are natural plant pigments and possess a wide range of biological activities, including cardiovascular protective activity. In the present study we explored the effects and the potential mechanisms of anthocyanins on platelet granule secretion in hypercholesterolemia. In a randomised, double-blind clinical trial, 150 hypercholesterolaemic individuals were treated with purified anthocyanins (320 mg/day) or placebo for 24 weeks. Anthocyanins consumption significantly reduced plasma levels of β-thromboglobulin (β-TG), soluble P-selectin, and of Regulated on Activation Normal T cell Expressed and Secreted (RANTES) as compared with the placebo. A minor reduction in platelet factor 4 (PF4) and transforming growth factor β1 (TGF-β1) levels were also observed. In in vitro experiments, we observed that purified anthocyanin mixture, as well as its two main anthocyanin components, delphinidin-3-glucoside (Dp-3-g) and cyanidin-3-glucoside (Cy-3g) directly inhibited platelet á-granule, dense granule, and lysosome secretion evaluated by P-selectin, RANTES, β-TG, PF4, TGF-β1, serotonin, ATP, and CD63 release. Further, anthocyanins inhibited platelet PI3K/Akt activation and consequently attenuated eNOS phosphorylation and cGMP production, thus interrupting MAPK activation. LY294002, a PI3K inhibitor, did not cause additional inhibitory efficacy, indicating that anthocyanin-induced effects may be involved in inhibition of the PI3K/Akt signalling pathway. These results provide evidence that by inhibiting platelet granule secretion, anthocyanins may be a potent cardioprotective agent.

Keywords

Hypercholesterolaemia - platelet granule - anthocyanins - PI3K

Full Text

References

References
1 Ruggeri ZM. Platelets in atherothrombosis. Nat Med 2002; 8: 1227-1234.
2 Ni H, Freedman J. Platelets in haemostasis and thrombosis: role of integrins and their ligands. Transfus Apher Sci 2003; 28: 257-264.
3 Li C, Li J, Li Y. et al. Crosstalk between Platelets and the Immune System: Old Systems with New Discoveries. Adv Hematol 2012; 2012: 384685.
4 Murphy AJ, Bijl N, Yvan-Charvet L. et al. Cholesterol efflux in megakaryocyte progenitors suppresses platelet production and thrombocytosis. Nat Med 2013; 19: 586-594.
5 Blair P, Flaumenhaft R. Platelet alpha-granules: basic biology and clinical correlates. Blood Rev 2009; 23: 177-189.
6 McNicol A, Israels SJ. Platelet dense granules: structure, function and implications for haemostasis. Thromb Res 1999; 95: 1-18.
7 Li J, Callum JL, Lin Y. et al. Severe platelet desialylation in a patient with glyco-protein Ib/IX antibody-mediated immune thrombocytopenia and fatal pulmonary haemorrhage. Haematologica 2014; 99: e61-63.
8 Rendu F, Brohard-Bohn B. The platelet release reaction: granules’ constituents, secretion and functions. Platelets 2001; 12: 261-273.
9 Shirakawa R, Yoshioka A, Horiuchi H. et al. Small GTPase Rab4 regulates Ca²⁺-induced alpha-granule secretion in platelets. J Biol Chem 2000; 275: 33844-33849.
10 Cipollone F, Mezzetti A, Porreca E. et al. Association between enhanced soluble CD40L and prothrombotic state in hypercholesterolaemia: effects of statin therapy. Circulation 2002; 106: 399-402.
11 Prisco D, Rogasi PG, Paniccia R. et al. Altered lipid composition and thromboxane A2 formation in platelets from patients affected by IIa hyperlipoproteinemia. Thromb Res 1988; 50: 593-604.
12 King SM, McNamee RA, Houng AK. et al. Platelet dense-granule secretion plays a critical role in thrombosis and subsequent vascular remodeling in athero-sclerotic mice. Circulation 2009; 120: 785-791.
13 Paigen B, Holmes PA, Novak EK. et al. Analysis of atherosclerosis susceptibility in mice with genetic defects in platelet function. Arteriosclerosis 1990; 10: 648-652.
14 Kennedy SG, Wagner AJ, Conzen SD. et al. The PI3-kinase/Akt signalling pathway delivers an anti-apoptotic signal. Genes Dev 1997; 11: 701-713.
15 Morello F, Perino A, Hirsch E. Phosphoinositide 3-kinase signalling in the vascular system. Cardiovasc Res 2009; 82: 261-271.
16 Li Z, Delaney MK, O’Brien KA. et al. Signalling during platelet adhesion and activation. Arterioscler Thromb Vasc Biol 2010; 30: 2341-2349.
17 Wallace TC. Anthocyanins in cardiovascular disease. Adv Nutr 2011; 2: 1-7.
18 Karlsen A, Retterstol L, Laake P. et al. Anthocyanins inhibit nuclear factor-kappaB activation in monocytes and reduce plasma concentrations of pro-inflammatory mediators in healthy adults. J Nutr 2007; 137: 1951-1954.
19 Yang Y, Shi Z, Reheman A. et al. Plant food delphinidin-3-glucoside significantly inhibits platelet activation and thrombosis: novel protective roles against cardiovascular diseases. PLoS One 2012; 7: e37323.
20 Rechner AR, Kroner C. Anthocyanins and colonic metabolites of dietary polyphenols inhibit platelet function. Thromb Res 2005; 116: 327-334.
21 Yang Y, Andrews MC, Hu Y. et al. Anthocyanin extract from black rice significantly ameliorates platelet hyperactivity and hypertriglyceridemia in dyslipidaemc rats induced by high fat diets. J Agric Food Chem 2011; 59: 6759-6764.
22 Zhu Y, Xia M, Yang Y. et al. Purified anthocyanin supplementation improves endothelial function via NO-cGMP activation in hypercholesterolaemic individuals. Clin Chem 2011; 57: 1524-1533.
23 Zhu Y, Ling W, Guo H. et al. Anti-inflammatory effect of purified dietary anthocyanin in adults with hypercholesterolaemia: a randomized controlled trial. Nutr Metab Cardiovasc Dis 2013; 23: 843-849.
24 Yang H, Reheman A, Chen P. et al. Fibrinogen and von Willebrand factor-independent platelet aggregation in vitro and in vivo. J Thromb Haemost 2006; 4: 2230-2237.
25 Li Z, Zhang G, Le Breton GC. et al. Two waves of platelet secretion induced by thromboxane A2 receptor and a critical role for phosphoinositide 3-kinases. J Biol Chem 2003; 278: 30725-30731.
26 Kroner C, Eybrechts K, Akkerman JW. Dual regulation of platelet protein kinase B. J Biol Chem 2000; 275: 27790-27798.
27 Stojanovic A, Marjanovic JA, Brovkovych VM. et al. A phosphoinositide 3-kinase-AKT-nitric oxide-cGMP signalling pathway in stimulating platelet secretion and aggregation. J Biol Chem 2006; 281: 16333-16339.
28 Li Z, Xi X, Du X. A mitogen-activated protein kinase-dependent signalling pathway in the activation of platelet integrin alpha IIbbeta3. J Biol Chem 2001; 276: 42226-42232.
29 Flevaris P, Li Z, Zhang G. et al. Two distinct roles of mitogen-activated protein kinases in platelets and a novel Rac1-MAPK-dependent integrin outside-in retractile signalling pathway. Blood 2009; 113: 893-901.
30 Kramer RM, Roberts EF, Strifler BA. et al. Thrombin induces activation of p38 MAP kinase in human platelets. J Biol Chem 1995; 270: 27395-27398.
31 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: 3800-3805.
32 Gawaz M, Langer H, May AE. Platelets in inflammation and atherogenesis. J Clin Invest 2005; 115: 3378-3384.
33 King SM, Reed GL. Development of platelet secretory granules. Semin Cell Dev Biol 2002; 13: 293-302.
34 Yang H, Lang S, Zhai Z. et al. Fibrinogen is required for maintenance of platelet intracellular and cell-surface P-selectin expression. Blood 2009; 112: 425-436.
35 Kaplan KL, Owen J. Plasma levels of beta-thromboglobulin and platelet factor 4 as indices of platelet activation in vivo. Blood 1981; 57: 199-202.
36 Lane DA, Ireland H, Wolff S. et al. Detection of enhanced in vivo platelet alpha-granule release in different patient groups--comparison of beta-thromboglobulin, platelet factor 4 and thrombospondin assays. Thromb Haemost 1984; 52: 183-187.
37 Assoian RK, Komoriya A, Meyers CA. et al. Transforming growth factor-beta in human platelets. Identification of a major storage site, purification, and characterization. J Biol Chem 1983; 258: 7155-7160.
38 Ferroni P, Martini F, Riondino S. et al. Soluble P-selectin as a marker of in vivo platelet activation. Clin Chim Acta 2009; 399: 88-91.
39 Podrez EA, Byzova TV, Febbraio M. et al. Platelet CD36 links hyperlipidaema, oxidant stress and a prothrombotic phenotype. Nat Med 2007; 13: 1086-1095.
40 Friend M, Vucenik I, Miller M. Research pointers: Platelet responsiveness to aspirin in patients with hyperlipidaemia. BMJ 2003; 326: 82-83.
41 Hohlfeld T, Scharnowski F, Braun M. et al. Antiplatelet effects of ticlopidine are reduced in experimental hypercholesterolaemia. Thromb Haemost 1994; 71: 112-118.
42 McGhie TK, Walton MC. The bioavailability and absorption of anthocyanins: towards a better understanding. Mol Nutr Food Res 2007; 51: 702-713.
43 Kay CD. Aspects of anthocyanin absorption, metabolism and pharmacokinetics in humans. Nutr Res Rev 2006; 19: 137-146.
44 Garcia-Alonso M, Minihane AM, Rimbach G. et al. Red wine anthocyanins are rapidly absorbed in humans and affect monocyte chaemoattractant protein 1 levels and antioxidant capacity of plasma. J Nutr Biochem 2009; 20: 521-529.
45 Kim K, Bae ON, Lim KM. et al. Novel antiplatelet activity of protocatechuic acid through the inhibition of high shear stress-induced platelet aggregation. J Pharmacol Exp Ther 2012; 343: 704-711.
46 Voss B, McLaughlin JN, Holinstat M. et al. PAR1, but not PAR4, activates human platelets through a Gi/o/phosphoinositide-3 kinase signalling axis. Mol Pharmacol 2007; 71: 1399-1406.
47 Radomski MW, Palmer RM, Moncada S. The role of nitric oxide and cGMP in platelet adhesion to vascular endothelium. Biochem Biophys Res Commun 1987; 148: 1482-1489.
48 Radomski MW, Palmer RM, Moncada S. An L-arginine/nitric oxide pathway present in human platelets regulates aggregation. Proc Natl Acad Sci USA 1990; 87: 5193-5197.
49 Marjanovic JA, Li Z, Stojanovic A. et al. Stimulatory roles of nitric-oxide synthase 3 and guanylyl cyclase in platelet activation. J Biol Chem 2005; 280: 37430-37438.

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