Dehydroepiandrosterone Prevents the Aggregation of Platelets Obtained from Postmenopausal Women with Type 2 Diabetes Mellitus through the Activation of the PKC/eNOS/NO Pathway

Abstract

The steroid hormone dehydroepiandrosterone (DHEA), suggested to be a cardioprotector, prevents platelet aggregation in healthy humans. This hormone is reduced in postmenopausal women by 60% of its normal value. Platelets in patients with type 2 diabetes (T2D) are more sensitive to aggregation, which has been attributed to a reduced ability to produce nitric oxide (NO). In light of these precedents and considering that DHEA is able to increase the production of NO in cultured endothelial cells, we suggest that DHEA prevents the aggregation of platelet from postmenopausal women with T2D through the activation of PKC/eNOS/NO/cGMP pathway. To determine the effect of DHEA in platelet aggregation, platelet-rich plasma (PRP) obtained from postmenopausal women with T2D was preincubated with DHEA, and aggregation induced by ADP was determined in the presence or absence of L-NNA (LNG-nitroarginine), Rottlerin, NOS, or PKC delta inhibitors, respectively. Platelet NO production was measured with the fluorescent probe DAF2DA and eNOS activation was determined by Western blot, using an anti-p-eNOS (ser 1177) antibody. DHEA 1) prevented platelet aggregation by 40% compared to control, 2) increased NO production by 63%, 3) increased p-eNOS (phosphorylated endothelial nitric oxide synthase) levels, and 4) increased cGMP production. These effects were reduced in the presence of L-NNA or Rottlerin. DHEA prevents platelet aggregation induced by ADP. This effect is mediated by the activation of the PKCδ/eNOS/NO/cGMP pathway. Our results suggest that DHEA could be considered to be a potential therapeutic tool in the prevention of atherothrombotic processes in postmenopausal women with T2D.

Key words

steroid hormone - cardioprotection - atherothrombosis

• References

• 1 Vokonas PS, Kannel WB. Diabetes mellitus and coronary heart disease in the elderly. Clin Geriatr Med 1996; 12: 69-78
• 2 Nathan DM. Long-term complications of diabetes mellitus. N Engl J Med 1993; 328: 1676-1685
• 3 Beckman JA, Creager MA, Libby P. Diabetes and atherosclerosis: epidemiology, pathophysiology, and management. JAMA 2002; 287: 2570-2581
• 4 Nestler JE, Clore JN, Blackard WG. Metabolism and actions of dehydroepiandrosterone in humans. J Steroid Biochem Mol Biol 1991; 40: 599-605
• 5 Labrie F. DHEA, important source of sex steroids in men and even more in women. Prog Brain Res 2010; 182: 97-148
• 6 Labrie F, Luu-The V, Bélanger A, Lin SX, Simard J, Pelletier G, Labrie C. Is dehydroepiandrosterone a hormone?. J Endocrinol 2005; 187: 169-196
• 7 Labrie F, Luu-The V, Labrie C, Bélanger A, Simard J, Lin SX, Pelletier G. Endocrine and intracrine sources of androgens in women: inhibition of breast cancer and other roles of androgens and their precursor dehydroepiandrosterone. Endocr Rev 2003; 24: 152-182
• 8 Kanazawa I, Yamaguchi T, Yamamoto M, Yamauchi M, Kurioka S, Yano S, Sugimoto T. Serum DHEA-S level is associated with the presence of atherosclerosis in postmenopausal women with type 2 diabetes mellitus. Endocr J 2008; 55: 667-675
• 9 Mitchell LE, Sprecher DL, Borecki IB, Rice T, Laskarzewski PM, Rao DC. Evidence for an association between dehydroepiandrosterone sulfate and nonfatal, premature myocardial infarction in males. Circulation 1994; 89: 89-93
• 10 Alexandersen P, Haarbo J, Christiansen C. The relationship of natural androgens to coronary heart disease in males: a review. Atherosclerosis 1996; 125: 1-13
• 11 Panjari M, Davis SR. DHEA for postmenopausal women: a review of the evidence. Maturitas 2010; 66: 172-179
• 12 Colwell JA, Nesto RW. The platelet in diabetes: focus on prevention of ischemic events. Diabetes Care 2003; 26: 2181-2188
• 13 Vinik AI, Erbas T, Park TS, Nolan R, Pittenger GL. Platelet dysfunction in type 2 diabetes. Diabetes Care 2001; 24: 1476-1485
• 14 Tschope D, Rosen P, Gries FA. Increase in the cytosolic concentration of calcium in platelets of diabetics type II. Thromb Res 1991; 62: 421-428
• 15 Martina V, Bruno GA, Trucco F, Zumpano E, Tagliabue M, Di Bisceglie C, Pescarmona G. Platelet cNOS activity is reduced in patients with IDDM and NIDDM. Thromb Haemost 1998; 79: 520-522
• 16 Ferroni P, Basili S, Falco A, Davì G. Platelet activation in type 2 diabetes mellitus. J Thromb Haemost 2004; 2: 1282-1291
• 17 Hamet P, Skuherska R, Pang SC, Tremblay J. Abnormalities of platelet function in hypertension and diabetes. Hypertension 1985; 7: II135-II142
• 18 Tschoepe D, Roesen P, Kaufmann L, Schauseil S, Kehrel B, Ostermann H, Gries FA. Evidence for abnormal platelet glycoprotein expression in diabetes mellitus. Eur J Clin Invest 1990; 20: 166-170
• 19 Radomski MW, Palmer RM, Moncada S. Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet 1987; 2: 1057-1058
• 20 Freedman JE, Loscalzo J, Barnard MR, Alpert C, Keaney JF, Michelson AD. Nitric oxide released from activated platelets inhibits platelet recruitment. J Clin Invest 1997; 100: 350-356
• 21 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
• 22 Rabini RA, Staffolani R, Fumelli P, Mutus B, Curatola G, Mazzanti L. Decreased nitric oxide synthase activity in platelets from IDDM and NIDDM patients. Diabetologia 1998; 41: 101-104
• 23 Chirkov Y, Tyshchuk IA, Severina IS. Guanylate cyclase in human platelets with different aggregability. Experientia 1990; 46: 697-699
• 24 Ikeda H, Takajo Y, Murohara T, Ichiki K, Adachi H, Haramaki N, Katoh A, Imaizumi T. Platelet-derived nitric oxide and coronary risk factors. Hypertension 2000; 35: 904-907
• 25 Mundell SJ, Jones ML, Hardy AR, Barton JF, Beaucourt SM, Conley PB, Poole AW. Distinct roles for protein kinase C isoforms in regulating platelet purinergic receptor function. Mol Pharmacol 2006; 70: 1132-1142
• 26 Harper MT, Poole AW. Isoform-specific functions of protein kinase C: the platelet paradigm. Biochem Soc Trans 2007; 35: 1005-1008
• 27 Jesse RL, Loesser K, Eich DM, Qian YZ, Hess ML, Nestler JE. Dehydro-epi-androsterone inhibits human platelet aggregation in vitro and in vivo. Ann N Y Acad Sci 1995; 774: 281-290
• 28 Formoso G, Chen H, Kim JA, Montagnani M, Consoli A, Quon MJ. Dehydroepiandrosterone mimics acute actions of insulin to stimulate production of both nitric oxide and endothelin 1 via distinct phosphatidylinositol 3-kinase- and mitogen-activated protein kinase-dependent pathways in vascular endothelium. Mol Endocrinol 2006; 20: 1153-1163
• 29 Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985; 28: 412-419
• 30 Mustard JF, Perry DW, Ardlie NG, Packham MA. Preparation of suspensions of washed platelets from humans. Br J Haematol 1972; 22: 193-204
• 31 Panes O, Matus V, Sáez CG, Quiroga T, Pereira J, Mezzano D. Human platelets synthesize and express functional tissue factor. Blood 2007; 109: 5242-5250
• 32 Born GV. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 1962; 194: 927-929
• 33 Liu D, Dillon JS. Dehydroepiandrosterone activates endothelial cell nitric-oxide synthase by a specific plasma membrane receptor coupled to Galpha(i2,3). J Biol Chem 2002; 277: 21379-21388
• 34 Simoncini T, Mannella P, Fornari L, Varone G, Caruso A, Genazzani AR. Dehydroepiandrosterone modulates endothelial nitric oxide synthesis via direct genomic and nongenomic mechanisms. Endocrinology 2003; 144: 3449-3455
• 35 Hirafuji M, Nezu A, Shinoda H, Minami M. Involvement of platelet cyclic GMP but not cyclic AMP suppression in leukocyte-dependent platelet adhesion to endothelial cells induced by platelet-activating factor in vitro. Br J Pharmacol 1996; 117: 299-304
• 36 Moore PK, al-Swayeh OA, Chong NW, Evans RA, Gibson A. L-NG-nitro arginine (L-NOARG), a novel, L-arginine-reversible inhibitor of endothelium-dependent vasodilatation in vitro. Br J Pharmacol 1990; 99: 408-412
• 37 da Silva CG, Specht A, Wegiel B, Ferran C, Kaczmarek E. Mechanism of purinergic activation of endothelial nitric oxide synthase in endothelial cells. Circulation 2009; 119: 871-879
• 38 Gschwendt M, Müller HJ, Kielbassa K, Zang R, Kittstein W, Rincke G, Marks F. Rottlerin, a novel protein kinase inhibitor. Biochem Biophys Res Commun 1994; 199: 93-98
• 39 Sugimoto K, Fujii S, Takemasa T, Yamashita K. Detection of intracellular nitric oxide using a combination of aldehyde fixatives with 4,5-diaminofluorescein diacetate. Histochem Cell Biol 2000; 113: 341-347
• 40 Cooper ME. Interaction of metabolic and haemodynamic factors in mediating experimental diabetic nephropathy. Diabetologia 2001; 44: 1957-1972
• 41 Libby P. Inflammation in atherosclerosis. Nature 2002; 420: 868-874
• 42 Schafer A, Wiesmann F, Neubauer S, Eigenthaler M, Bauersachs J, Channon KM. Rapid regulation of platelet activation in vivo by nitric oxide. Circulation 2004; 109: 1819-1822
• 43 Hayashi T, Matsui-Hirai H, Miyazaki-Akita A, Fukatsu A, Funami J, Ding QF, Kamalanathan S, Hattori Y, Ignarro LJ, Iguchi A. Endothelial cellular senescence is inhibited by nitric oxide: implications in atherosclerosis associated with menopause and diabetes. Proc Natl Acad Sci USA 2006; 103: 17018-17023
• 44 Tchernof A, Labrie F. Dehydroepiandrosterone, obesity and cardiovascular disease risk: a review of human studies. Eur J Endocrinol 2004; 151: 1-14
• 45 Dhatariya K, Bigelow ML, Nair KS. Effect of dehydroepiandrosterone replacement on insulin sensitivity and lipids in hypoadrenal women. Diabetes 2005; 54: 765-769
• 46 Hsu WC. Consequences of delaying progression to optimal therapy in patients with type 2 diabetes not achieving glycemic goals. South Med J 2009; 102: 67-76
• 47 de Lemos JA, Zirlik A, Schönbeck U, Varo N, Murphy SA, Khera A, McGuire DK, Stanek G, Lo HS, Nuzzo R, Morrow DA, Peshock R, Libby P. Associations between soluble CD40 ligand, atherosclerosis risk factors, and subclinical atherosclerosis: results from the Dallas Heart Study. Arterioscler Thromb Vasc Biol 2005; 25: 2192-2196
• 48 Osuka K, Watanabe Y, Usuda N, Atsuzawa K, Aoshima C, Yamauchi K, Takayasu M, Yoshida J. Phosphorylation of neuronal nitric oxide synthase at Ser847 in the nucleus intermediolateralis after spinal cord injury in mice. Neuroscience 2007; 145: 241-247
• 49 Li J, Yang C, Han S, Zu P, Wu J, Xu Q, Fang L. Increased phosphorylation of neurogranin in the brain of hypoxic preconditioned mice. Neurosci Lett 2006; 391: 150-153
• 50 Gachet C. The platelet P2 receptors as molecular targets for old and new antiplatelet drugs. Pharmacol Ther 2005; 108: 180-192
• 51 Zuccollo A, Shi C, Mastroianni R, Maitland-Toolan KA, Weisbrod RM, Zang M, Xu S, Jiang B, Oliver-Krasinski JM, Cayatte AJ, Corda S, Lavielle G, Verbeuren TJ, Cohen RA. The thromboxane A2 receptor antagonist S18886 prevents enhanced atherogenesis caused by diabetes mellitus. Circulation 2005; 112: 3001-3008
• 52 Offermanns S. Activation of platelet function through G protein-coupled receptors. Circ Res 2006; 99: 1293-1304
• 53 Reid HM, Kinsella BT. The alpha, but not the beta, isoform of the human thromboxane A2 receptor is a target for nitric oxide-mediated desensitization. Independent modulation of Tp alpha signaling by nitric oxide and prostacyclin. J Biol Chem 2003; 278: 51190-51202
• 54 Tolhurst G, Vial C, Léon C, Gachet C, Evans RJ, Mahaut-Smith MP. Interplay between P2Y(1), P2Y(12), and P2X(1) receptors in the activation of megakaryocyte cation influx currents by ADP: evidence that the primary megakaryocyte represents a fully functional model of platelet P2 receptor signaling. Blood 2005; 106: 1644-1651
• 55 Halbrugge M, Walter U. Purification of a vasodilator-regulated phosphoprotein from human platelets. Eur J Biochem 1989; 185: 41-50
• 56 Pula G, Schuh K, Nakayama K, Nakayama KI, Walter U, Poole AW. PKC delta regulates collagen-induced platelet aggregation through inhibition of VASP-mediated filopodia formation. Blood 2006; 108: 4035-4044