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Increased platelet alpha2B-adrenergic receptor gene expression in well-controlled hypertensives: the effect of arterial stiffness
Journal of the American Society of Hypertension
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(2017) 1–7
Research Article
Increased platelet alpha2B-adrenergic receptor gene expression in well-controlled hypertensives: the effect of arterial stiffness Maria E. Marketou, MD, PhDa,*, Joanna E. Kontaraki, PhDb, John A. Papadakis, MDc, George E. Vrentzos, MDc, Alexandros Patrianakos, MD, PhDa, Konstantinos Fragkiadakis, MDa, Panagiotis Tsiverdis, MDa, Dimitris Lempidakis, MDa, Gregory Chlouverakis, PhDd, Panos E. Vardas, MD, PhDa, and Fragiskos I. Parthenakis, MD, PhDa a
b
Department of Cardiology, Heraklion University Hospital, Heraklion, Crete, Greece; Division of Biostatistics, Department of Social Medicine, Molecular Cardiology Laboratory, School of Medicine, University of Crete, Heraklion, Crete, Greece; c Department of Internal Medicine, Heraklion University Hospital, Crete, Heraklion, Crete, Greece; and d Division of Biostatistics, School of Medicine, University of Crete, Heraklion, Crete, Greece Manuscript received June 15, 2017 and accepted August 18, 2017
Abstract Catecholamines play a major role in atherothrombotic mechanisms in essential hypertension. Alpha2B-adrenergic receptors (a2BARs) are implicated in the pathophysiology of platelet aggregation. In this study, we evaluated platelet a2B-AR gene expression levels in patients with well-controlled essential hypertension compared with normal individuals and investigated their association with increased arterial stiffness. Fifty-nine patients with well-controlled essential hypertension (34 men, mean age 65 9 years) and 26 normotensives (19 men, mean age 64 8 years) were included in the study. For each patient, carotid-femoral pulse wave velocity (PWV) and carotid-radial PWV were evaluated. In addition, blood samples were obtained and platelets were isolated. The a2B-AR gene expression levels in platelets were examined by real-time polymerase chain reaction for each participant. Wellcontrolled hypertensive patients showed significantly higher gene expression levels of a2B-Rs in platelets compared with normotensives (34.7 29.5 vs 17.6 12.5, respectively, P ¼ .005). Interestingly, we found that carotid-femoral PWV and carotid-radial PWV were positively correlated with platelet a2B-R gene expression levels (r ¼ 0.59, P < .001, and r ¼ 0.39, P ¼ .002, respectively).Platelet a2B-R gene expression levels are increased in patients with well-controlled essential hypertension compared with normotensives and are correlated with increased PWV in those patients. Our data indicate an association of arterial stiffness and platelet a2B-Rs gene expression and indicate the need for further research. J Am Soc Hypertens 2017;-(-):1–7. Ó 2017 American Society of Hypertension. All rights reserved. Keywords: Essential hypertension; pulse wave velocity; thrombosis.
Introduction Essential hypertension is associated with an increased incidence of atherothrombotic events, such as myocardial Conflict of interest: None. *Corresponding author: Maria E. Marketou, MD, PhD, Department of Cardiology, Heraklion University Hospital, P.O. Box 1352, Heraklion 71110, Greece. Tel: þ30 2810 394855; Fax: þ30 2810 542055. E-mail: [email protected]
infarction and stroke.1,2 The precise pathophysiological mechanisms that connect increased arterial pressure with thrombogenesis and thrombotic episodes still remain unclear at many levels. We know that, to some degree, the prothrombotic status in hypertension is associated with a number of factors, such as hemodynamic disturbances, vascular dysfunction, and an imbalance between procoagulant and fibrinolytic activity, which ultimately lead to platelet activation and thrombotic complications.3,4 In addition, a significant factor in thrombogenic abnormalities, especially in younger patients, appears to be the activation of the
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sympathetic nervous system (SNS). Overactivity of the SNS has already been demonstrated in patients with essential hypertension,5 and this is widely acknowledged to be a critical component in the pathogenesis of thrombosis.6 Catecholamines play an important role in thrombogenesis, and their effects on platelet aggregation are exerted via the alpha2-adrenergic receptors (a2-ARs).7 Alpha adrenergic activation is certainly a factor associated with the contraction of the arterial wall, which is innervated by the sympathetic nerves and may contribute to the control of arterial wall tone. It has been known for a long time that a-ARs can mediate vasoconstriction in most vascular beds and are implicated in the pathophysiology of hypertension.8,9 In addition, previous studies have indicated an altered expression of a2-ARs in cardiovascular diseases, with an increased prevalence of stroke and coronary thrombosis and have highlighted the significant role of a2-ARs in platelet aggregation.9 Notably, the alpha2B-adrenergic receptor (a2B-AR) subtype in platelets is an important regulator of aggregation and a2B-AR inhibition has a significant antiaggregant effect. Interestingly, even in patients who were receiving dual antiplatelet therapy, the inhibition of a2B-ARs in platelets had an additional antiaggregant effect.10 On the other hand, arterial stiffness is a hallmark of vascular dysfunction and has been proposed as an independent risk factor for fatal and nonfatal cardiovascular events in patients with hypertension. It may also be implicated in the pathophysiology of increased thrombogenicity in hypertensives. In the present study, we sought to evaluate a2B-ARs in the platelets of well-controlled hypertensive patients, compared with a normotensive population, and to investigate their association with arterial stiffness. For the measurement of arterial stiffness, we used pulse wave velocity (PWV), which is widely accepted as the ‘‘gold standard’’ measure for this purpose.11
Methods Hypertensive patients with well-controlled essential hypertension from our outpatient hypertension clinic were included in the study. The diagnosis of hypertension was based on three outpatient measurements of blood pressure (BP) >140/90 mm Hg at intervals of no longer than 2 weeks, in accordance with the recommendations of the European Society of Hypertension/European Society of Cardiology.12 To be eligible for inclusion in the study as well-controlled, patients had to have achieved the target BP of 140/90 mm Hg by their second visit, confirmed by 24-hour ambulatory BP monitoring showing a mean 24-hour BP < 130/80 mm Hg. These patients were compared with sex- and aged-matched individuals who visited our outpatient clinic either complaining about atypical chest pain, or having other cardiovascular risk factors, were not hypertensives and served as control group.
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All patients underwent a complete physical examination and routine laboratory tests. Patients with any of the following characteristics were excluded: pregnant or lactating women; grade 3 hypertension or secondary hypertension; coronary artery disease; heart failure; cerebrovascular, liver or renal disease; albumin excretion >300 mg/ 24 h; history of drug or alcohol abuse; any chronic inflammatory or other infectious disease during the last 6 months; thyroid gland disease; body mass index (BMI) > 35 kg/m2; or a history of any hematologic disease. Vascular or neoplastic conditions were ruled out in all participants by a careful examination of the history and routine laboratory tests. BMI was calculated as mass/height2 (kg/m2). A full echocardiographic examination was performed in all participants. Once BP levels had been controlled, blood samples were taken for assessment of a2B-AR gene expression levels in platelets. More specifically, in all participants, after a rest of 20 minutes, blood was drawn from a superficial brachial vein via a 21-ga needle, with care to avoid stasis, hemolysis, and contamination by tissue fluids or exposure to glass. The study was carried out in accordance with the Declaration of Helsinki and the protocol was approved by the local ethics committee. All patients gave informed consent to their inclusion in the study.
RNA Isolation and Platelet a2B-AR Messenger RNA Quantification Blood samples were collected into citrated tubes and centrifuged at 150g for 20 minutes. To avoid leukocyte contamination, only the top 75% of the supernatant platelet-rich plasma was collected and platelets were isolated by centrifugation. Total RNA was isolated from platelets using the TRI-Reagent (Ambion; Life Technologies, Carlsbad, CA, USA) and reverse-transcribed using the PrimeScript RT reagent Kit (Takara Bio Inc, Otsu, Shiga, Japan). Measurements of messenger RNA levels were performed by quantitative real-time polymerase chain reaction (qPCR) using the Corbett Research 6000 detection system. qPCR assays were performed using the KAPA SYBR FAST qPCR Kit (Kapa Biosystems, Woburn, MA, USA). All samples were performed in duplicates. The housekeeping gene GAPDH (glyceraldehyde-3-phosphate-dehydrogenase) was used as an endogenous reference gene. Primer sequences for the alpha2B adrenoreceptor were 50 -GAT TTG GAA GGG CAC CGA GGG A-30 (sense) and 50 -GGC CAG CAG AGG GTC ACA GTC AG-30 (antisense); those for GAPDH were 50 -CCA TCT TCC AGG AGC GAG-30 (sense) and 50 -GCA GGA GGC ATT GCT GAT-30 (antisense). The standard curve method was used for absolute quantification of the amplification products and specificity was determined by performing a melting curve analysis.
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Measurement of PWV
Results
PWV was measured at baseline in all patients. On the day of examination, all subjects were asked to refrain from caffeine, alcohol, and smoking during the preceding 12 hours. The study was carried out between 8:00 and 9:00 AM in a quiet room at 22 1 C. Height and weight were measured. Subjects were allowed a further 15 minutes supine rest before baseline measurements. Brachial BP was measured over the brachial artery three times at 5-minute intervals. The mean of the last two measurements was recorded as representative of brachial BP. After brachial BP, the carotid, femoral, and radial arteries were palpated to find the location of the points with the most pronounced pulse pressure waves. Carotid-femoral PWV (c-f PWV) and carotid-radial PWV (c-r PWV) artery waveforms were measured, and PWV (Complior SP; Atech Medical, France) was determined. The distances traveled by the pulse waves were assessed in triplicate over the surface of the body with a nonelastic tape measure. The same examiner, who was blinded to the patient’s history, performed all measurements.
A total of 59 patients with essential hypertension (34 men, mean age 65 9 years) were included in the final analysis and were compared with a group of 26 individuals with normal BP (19 men, mean age 64 8 years). Mean hypertension duration was 6.4 3.2 years. The majority of patients (95%) were taking a renin–angiotensin–aldosterone system blocker, 76% were taking diuretics, 69% a calcium channel blocker, and 37% a beta-blocker. The main demographic, echocardiographic, and clinical characteristics of the patients and controls are reported in Table 1. Patients with essential hypertension had significantly higher office systolic and diastolic BP, BMI, c-f PWV, c-r PWV, and left ventricular mass index compared with normotensives (Table 1). Twenty-six patients (44%) were receiving angiotensinconverting enzyme inhibitor, 25 (42.3%) were taking angiotensin receptor blockers, 21 (35.5%) calcium blockers, five (0.08%) beta-blockers, and 24 (40.6%) diuretics. Hypertensive patients showed significantly higher gene expression levels of a2B-Rs in their platelets, compared with those of normotensives (Figure 1). More specifically, a2B-R platelet gene expression levels were 34.7 29.5 in hypertensive patients compared with 17.6 12.5 in the control group (P ¼ .005). Stepwise regression showed that platelet a2B-R gene expression levels showed a significant correlation with arterial stiffness in hypertensive patients. Our analysis revealed that levels of a2B-R gene expression in the platelets of patients with essential hypertension were positively correlated with c-f PWV and c-r PWV (Figure 2). More specifically, a2B-R platelet gene expression levels were strongly correlated with c-f PWV (r ¼ 0.59, P < .001), whereas a modest
Statistical Analysis Summary descriptive statistics are presented as mean SD, or frequencies (percentage), as appropriate. Comparisons of continuous variables between the normotensive and hypertensive groups were performed with independent samples t-test. Categorical variables between the two groups were compared using the chi-square test. The association between continuous variables was assessed with Pearson’s correlation coefficient and simple linear regression methods. All statistical tests were carried out in IBM-SPSS 21 at the two-sided 5% level of significance.
Table 1 Participants’ main demographic, echocardiographic, and clinical characteristics
Sex (male/female) Age (y) Smokers (%) Cholesterol (mg/dL) Glucose (mg/dL) Uric acid (mg/dL) Creatinine (mg/dL) Heart rate (bpm) Office systolic blood pressure (mm Hg) Office diastolic blood pressure (mm Hg) Body mass index (kg/m2) Left ventricular mass index (g/m2) Ejection fraction (%) Carotid-femoral pulse wave velocity (m/s) Carotid-radial pulse wave velocity (m/s) NS, no statistically significant.
Hypertensive Patients (n ¼ 59)
Control Group (n ¼ 26)
P
34/25 65 61 237 102 6.6 0.98 73 138 84 30.6 106.3 61.6 10.8 11.1
15/11 64 60 248 96 6.7 0.95 71 124 75 29.7 76.4 63.8 9.7 9.9
NS NS NS NS NS NS NS NS .01 .02 .03 <.001 NS .04 .04
9
66 16 3.2 0.3 8 10 8 4.1 24.5 4.8 2.5 2.6
8 71 19 4.9 0.4 10 5 6 2.9 7.6 5.1 1.9 2
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Figure 1. Platelet alpha2B-adrenergic receptors (a2B-AR) gene expression levels (arbitrary units) in hypertensives compared with normotensives.
positive correlation was also found with c-r PWV (r ¼ 0.39, P ¼ .002). Notably, no significant correlation was found between BP levels and PWV in these well-controlled hypertensives. In addition, no significant correlation was found between levels of a2B-R gene expression in the platelets of normotensives and c-f PWV or c-r PWV and no other association was detected between them and any other clinical parameter. Finally, the drugs-adjusted correlations do not differ from the unadjusted; in other words there is no indication that drug use affects the correlations between a2B-R gene expression levels and c-f PWV or c-r PWV.
Discussion This is the first study to evaluate gene expression levels of a2B-ARs that are implicated in platelet aggregation— in platelets from patients with well-controlled essential hypertension and to compare them with those of normotensive individuals. We found that a2B-AR platelet gene expression levels were significantly higher in patients with essential hypertension compared with normotensives. Notably, we also found a significant correlation between c-f PWV and a2B-AR gene expression levels in platelets from hypertensive subjects. Essential hypertension is widely known to be associated with an increased incidence of arterial thrombotic complications, such as myocardial infarction and ischemic stroke.1,2 The processes of thrombogenesis and atherogenesis in hypertension have a close pathophysiological relation. Although there is a significant pathophysiological and epidemiologic relationship between thrombosis and hypertension, the mechanisms of thrombosis in hypertension have not been fully elucidated.
Figure 2. Association between platelet alpha2B-adrenergic receptors (a2B-AR) gene expression levels (arbitrary units). (A) Carotid-femoral pulse wave velocity (c-f PWV) and (B) carotid-radial pulse wave velocity (c-r PWV) in hypertensive population.
Previous studies have highlighted that the hypercoagulable state observed in patients with hypertension appears to be implicated in the previously mentioned pathophysiological mechanisms, and that this state is more obvious the more elevated the BP.13 The prothrombotic state in hypertension is sustained by a number of factors, such as hemodynamic disturbances, endothelial dysfunction, and an imbalance between procoagulant and fibrinolytic activity in the peripheral blood of hypertensive subjects.3,4 Furthermore, it has been claimed that this prothrombotic status is correlated with the degree and the duration of hypertension14,15 as well as with the presence of target organ damage, such as left ventricular hypertrophy.16 In addition, von Willebrand factor levels are higher in hypertensive patients with target organ damage,17 whereas BP reduction resulted in improvements in indices of hemorheology and endothelial and platelet functions.4
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Platelet activation plays a major role in the thrombotic process, which is triggered by various mechanisms and leads to platelet aggregation and thrombus formation. Experimental studies have shown that hypertension is associated with major changes in morphology, signaling mechanisms, and oxidative stress in blood platelets.18 We know from previous studies that stress enhances platelet function and may influence thrombus formation.19 SNS activation is recognized to be a critical component in the pathogenesis of thrombosis, and platelet reactivity in vivo is influenced by sympathoadrenal activation through an increase in circulating catecholamines.5 In most cases, essential hypertension is associated with sympathetic overactivity, especially in younger patients.20 The sympathetic overactivation contributes to the pathophysiology of hypertension and seems to have additional adverse consequences in hypertensive patients that go beyond the elevation of BP. Norepinephrine enhances platelet aggregability and platelet secretion in vivo21 and may limit the antithrombotic effect of antiplatelet treatment in situations with sympathoadrenal activation.22 According to the literature, the main role of a-ARs is in maintaining vasomotor tone and in the pathophysiology of hypertension. The a1-ARs—through the action of catecholamines— activate vascular smooth muscle contraction, whereas a2-ARs, depending on their location, may act as a feedback mechanism for modulating the release of norepinephrine.8,9 In addition, catecholamines exert significant effects also on platelet aggregation mostly via the a2-ARs.22 It is established that a2-ARs may play a role in platelet aggregation and in the pathophysiology of arterial thrombosis.23,24 It has been highlighted in the literature that marked alterations occur in the platelet alpha2-adrenoceptors of hypertensive subjects.25 The a2B-ARs play a significant role in essential hypertension and are responsible for the central hypertensive sympathoexcitatory response to salt.26 Recently, the a2B-AR subtype has been recognized as an important regulator of aggregation and a2B-AR inhibition in platelets has a significant antiaggregant effect.10 Interestingly, even in patients who were receiving dual antiplatelet therapy, the inhibition of a2B-ARs had an additional antiaggregant effect.10 We have shown that hypertensive patients have significantly elevated levels of a2B-AR gene expression in their platelets that are likely to be implicated in the mechanisms and the pathophysiology of atherothrombosis in essential hypertension. These findings may contribute to a better risk stratification and better selection of individuals who are at especially high risk of thrombotic events. In addition, they may offer future perspectives in the development of novel therapeutic targets, providing strategies for reducing the prothrombotic state.
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On the other hand, alterations in arterial properties and remodeling of the arterial wall have an impact on the cardiovascular risk in hypertensives.27 Importantly, increased arterial stiffness is an independent factor for the development of atherosclerosis and an adverse cardiovascular outcome in essential hypertension.28,29 Increased arterial stiffness in a hypertensive population was associated with platelet activation, as has been shown by an increased mean platelet volume.30 Our findings demonstrated a positive correlation between arterial stiffness and platelet a2B-AR gene expression levels. We hypothesize that endothelial damage and adverse vascular remodeling in hypertension might be a triggering factor of platelet activation, and furthermore a predisposing condition for atherothrombotic events. This still applies even in hypertensive patients who have achieved their BP targets, like those in our study population. This indicates that when the arterial wall has already suffered damage, BP control is not in itself sufficient to eliminate the atherothrombotic risk. Our study had several limitations. The patient population in this study was not large; however, our findings were clear and indicated that other mechanisms associated with pathologic vascular remodeling are involved in the pathology of thrombosis in hypertensive patients. However, we cannot rule out the possibility that the small number of participants might have been obscured other significant correlations. The control group was normotensives but not completely healthy individuals who had high cholesterol levels. We cannot exclude that this may have affected our results. Antihypertensive agents such as beta-blockers did not show any significant effects on our findings. Unfortunately, the number of patients included is not adequately powered to draw safe conclusions for this question. PWV is defined as the velocity at which the BP wave propagates along the arterial tree and provides information about its elastic properties. However, only c-f PWV is established as a parameter with clinical and prognostic value.12 Our data regarding c-r PWV are less meritable but still indicative of the regional arterial stiffness. We focused our analysis in hypertensives. This was because the value of PWV as a prognostic index of cardiovascular events in healthy controls has not been documented. Therefore, our PWV findings could not have been evaluated in the normotensive group. In conclusion, platelet a2B-R gene expression levels are increased in patients with well-controlled essential hypertension compared with normotensives and are strongly associated with increased arterial stiffness in those patients. A possible implication of adverse arterial remodeling in platelet a2B-Rs gene expression should be further investigated. Future research is needed to clarify the pathophysiological pathways that link our findings with platelet aggregation and cardiovascular events.
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References 1. MacMahon S, Peto R, Cutler J, Collins R, Sorlie P, Neaton J, et al. Blood pressure, stroke, and coronary heart disease. Part 1, prolonged differences in blood pressure: prospective observational studies corrected for the regression dilution bias. Lancet 1990;335: 765–74. 2. Lewington S, Clarke R, Qizilbash N, Peto R, Collins R; Prospective Studies Collaboration. Agespecific relevance of usual blood pressure to vascular mortality: a meta-analysis of individual data for one million adults in 61 prospective studies. Lancet 2002;360:1903–13. 3. Poli KA, Tofler GH, Larson MG, Evans JC, Sutherland PA, Lipinska I, et al. Association of blood pressure with fibrinolytic potential in the Framingham offspring population. Circulation 2000;101: 264–9. 4. Spencer CG, Gurney D, Blann AD, Beevers DG, Lip GY; ASCOT Steering Committee; Anglo-Scandinavian Cardiac Outcomes Trial. Von Willebrand factor, soluble P-selectin, and target organ damage in hypertension: a substudy of the AngloScandinavian Cardiac Outcomes Trial (ASCOT). Hypertension 2002;40: 61–6. 5. Esler M, Kaye D. Sympathetic nervous system activation in essential hypertension, cardiac failure and psychosomatic heart disease. J Cardiovasc Pharmacol 2000;35:S1–7. 6. von K€anel R, Dimsdale JE. Effects of sympathetic activation by adrenergic infusions on hemostasis in vivo. Eur J Haematol 2000;65:357–69. 7. Wang JS, Cheng LJ. Effect of strenuous, acute exercise on a2-adrenergic agonist-potentiated platelet activation. Arterioscler Thromb Vasc Biol 1999;19:1559–65. 8. Timmermans PB, Chiu AT, Thoolen MJ. Calcium handling in vasoconstriction to stimulation of alpha 1and alpha 2-adrenoceptors. Can J Physiol Pharmacol 1987;65:1649–57. 9. Gavras H, Handy D, Gavras I. Alpha-adrenergic receptors in hypertension. In: Laragh JH, Brenner BM, editors. Pathophysiology, Diagnosis and Management. New York: Raven Press; 1995. P. 853–61. 10. Marketou ME, Kintsurashvili E, Androulakis NE, Kontaraki J, Alexandrakis MG, Gavras I, et al. Blockade of platelet alpha2B-adrenergic receptors: a novel antiaggregant mechanism. Int J Cardiol 2013; 168:2561–6. 11. Laurent S, Cockcroft J, Van Bortel L, Boutouyrie P, Giannattasio C, Hayoz D, et al. European network for non-invasive investigation of large arteries expert consensus document on arterial stiffness: methodological issues and clinical applications. Eur Heart J 2006;27:2588–605.
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12. Mancia G, Fagard R, Narkiewicz K, Red on J, Zanchetti A, B€ohm M, et al; Task Force Members. 2013 ESH/ESC Guidelines for the management of arterial hypertension: the Task Force for the management of arterial hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC). J Hypertens 2013;31:1281–357. 13. Yang P, Liu YF, Yang L, Wei Q, Zeng H. Mechanism and clinical significance of the prothrombotic state in patients with essential hypertension. Clin Cardiol 2010;33:E81–6. 14. Lip GY, Blann AD, Jones AF, Lip PL, Beevers DG. Relation of endothelium, thrombogenesis, and hemorheology in systemic hypertension to ethnicity and left ventricular hypertrophy. Am J Cardiol 1997;80: 1566–71. 15. Weir MR, Dzau VJ. The renin-angiotensin-aldosterone system: a specific target for hypertension management. Am J Hypertens 1999;12:205S–13S. 16. Preston RA, Jy W, Jimenez JJ, Mauro LM, Horstman LL, Valle M, et al. Effects of severe hypertension on endothelial and platelet microparticles. Hypertension 2003;41:211–7. 17. Alexandrou N, Popov D, Dragan E, Andrei E, Georgescu A. Platelet activation in hypertension associated with hypercholesterolemia: effects of irbesartan. J Thromb Haemost 2010;9:173–84. 18. Hjemdahl P, Larsson PT, Wallen NH. Effects of stress and beta-blockade on platelet function. Circulation 1991;84:VI44–V161. 19. Anfossi G, Trovati M. Role of catecholamines in platelet function: pathophysiological and clinical significance. Eur J Clin Invest 1996;26:353–70. 20. Larsson PT, Wallen NH, Hjemdahl P. Norepinephrineinduced human platelet activation in vivo is only partly counteracted by aspirin. Circulation 1994;89:1951–7. 21. O’ Brien JR. Some effects of adrenaline and antiadrenaline compounds in platelets in vitro and in vivo. Nature 1963;200:763–4. 22. Berlin I, Crespo-Laumonnier B, Cournot A, Landault C, Aubin F, Legrand JC, et al. The alpha 2adrenergic receptor antagonist yohimbine inhibits epinephrine-induced platelet aggregation in healthy subjects. Clin Pharmacol Ther 1991;49:362–9. 23. Grant JA, Scrutton MC. Novel a2-adrenoceptors primarily responsible for inducing human platelet aggregation. Nature 1979;277:659–61. 24. Varani K, Gessi S, Caiazza A, Rastelli G, Portaluppi F, Borea PA. Platelet alpha2-adrenoceptor alterations in patients with essential hypertension. Br J Clin Pharmacol 1999;47:167–72. 25. Gavras I, Gavras H. Role of alpha2-adrenergic receptors in hypertension. Am J Hypertens 2001;14:171S–7S. 26. Gavras I, Manolis AJ, Gavras H. The alpha2 -adrenergic receptors in hypertension and heart failure:
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experimental and clinical studies. J Hypertens 2001;19: 2115–24. 27. Shantsila E, Lim HS. Targeting the arterial wall, but what is the target? J Hum Hypertens 2009;23:1–3. 28. Meaume S, Benetos A, Henry OF, Rudnichi A, Safar ME. Aortic pulse wave velocity predicts cardiovascular mortality in subjects >70 years of age. Arterioscler Thromb Vasc Biol 2001;21:2046–50.
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29. Cruickshank K, Riste L, Anderson SG, Wright JS, Dunn G, Gosling RG. Aortic pulse-wave velocity and its relationship to mortality in diabetes and glucose intolerance: an integrated index of vascular function? Circulation 2002;106:2085–90. 30. Wang RT, Li Y, Zhu XY, Zhang YN. Increased mean platelet volume is associated with arterial stiffness. Platelets 2011;22:447–51.
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Research Article
Increased platelet alpha2B-adrenergic receptor gene expression in well-controlled hypertensives: the effect of arterial stiffness Maria E. Marketou, MD, PhDa,*, Joanna E. Kontaraki, PhDb, John A. Papadakis, MDc, George E. Vrentzos, MDc, Alexandros Patrianakos, MD, PhDa, Konstantinos Fragkiadakis, MDa, Panagiotis Tsiverdis, MDa, Dimitris Lempidakis, MDa, Gregory Chlouverakis, PhDd, Panos E. Vardas, MD, PhDa, and Fragiskos I. Parthenakis, MD, PhDa a
b
Department of Cardiology, Heraklion University Hospital, Heraklion, Crete, Greece; Division of Biostatistics, Department of Social Medicine, Molecular Cardiology Laboratory, School of Medicine, University of Crete, Heraklion, Crete, Greece; c Department of Internal Medicine, Heraklion University Hospital, Crete, Heraklion, Crete, Greece; and d Division of Biostatistics, School of Medicine, University of Crete, Heraklion, Crete, Greece Manuscript received June 15, 2017 and accepted August 18, 2017
Abstract Catecholamines play a major role in atherothrombotic mechanisms in essential hypertension. Alpha2B-adrenergic receptors (a2BARs) are implicated in the pathophysiology of platelet aggregation. In this study, we evaluated platelet a2B-AR gene expression levels in patients with well-controlled essential hypertension compared with normal individuals and investigated their association with increased arterial stiffness. Fifty-nine patients with well-controlled essential hypertension (34 men, mean age 65 9 years) and 26 normotensives (19 men, mean age 64 8 years) were included in the study. For each patient, carotid-femoral pulse wave velocity (PWV) and carotid-radial PWV were evaluated. In addition, blood samples were obtained and platelets were isolated. The a2B-AR gene expression levels in platelets were examined by real-time polymerase chain reaction for each participant. Wellcontrolled hypertensive patients showed significantly higher gene expression levels of a2B-Rs in platelets compared with normotensives (34.7 29.5 vs 17.6 12.5, respectively, P ¼ .005). Interestingly, we found that carotid-femoral PWV and carotid-radial PWV were positively correlated with platelet a2B-R gene expression levels (r ¼ 0.59, P < .001, and r ¼ 0.39, P ¼ .002, respectively).Platelet a2B-R gene expression levels are increased in patients with well-controlled essential hypertension compared with normotensives and are correlated with increased PWV in those patients. Our data indicate an association of arterial stiffness and platelet a2B-Rs gene expression and indicate the need for further research. J Am Soc Hypertens 2017;-(-):1–7. Ó 2017 American Society of Hypertension. All rights reserved. Keywords: Essential hypertension; pulse wave velocity; thrombosis.
Introduction Essential hypertension is associated with an increased incidence of atherothrombotic events, such as myocardial Conflict of interest: None. *Corresponding author: Maria E. Marketou, MD, PhD, Department of Cardiology, Heraklion University Hospital, P.O. Box 1352, Heraklion 71110, Greece. Tel: þ30 2810 394855; Fax: þ30 2810 542055. E-mail: [email protected]
infarction and stroke.1,2 The precise pathophysiological mechanisms that connect increased arterial pressure with thrombogenesis and thrombotic episodes still remain unclear at many levels. We know that, to some degree, the prothrombotic status in hypertension is associated with a number of factors, such as hemodynamic disturbances, vascular dysfunction, and an imbalance between procoagulant and fibrinolytic activity, which ultimately lead to platelet activation and thrombotic complications.3,4 In addition, a significant factor in thrombogenic abnormalities, especially in younger patients, appears to be the activation of the
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sympathetic nervous system (SNS). Overactivity of the SNS has already been demonstrated in patients with essential hypertension,5 and this is widely acknowledged to be a critical component in the pathogenesis of thrombosis.6 Catecholamines play an important role in thrombogenesis, and their effects on platelet aggregation are exerted via the alpha2-adrenergic receptors (a2-ARs).7 Alpha adrenergic activation is certainly a factor associated with the contraction of the arterial wall, which is innervated by the sympathetic nerves and may contribute to the control of arterial wall tone. It has been known for a long time that a-ARs can mediate vasoconstriction in most vascular beds and are implicated in the pathophysiology of hypertension.8,9 In addition, previous studies have indicated an altered expression of a2-ARs in cardiovascular diseases, with an increased prevalence of stroke and coronary thrombosis and have highlighted the significant role of a2-ARs in platelet aggregation.9 Notably, the alpha2B-adrenergic receptor (a2B-AR) subtype in platelets is an important regulator of aggregation and a2B-AR inhibition has a significant antiaggregant effect. Interestingly, even in patients who were receiving dual antiplatelet therapy, the inhibition of a2B-ARs in platelets had an additional antiaggregant effect.10 On the other hand, arterial stiffness is a hallmark of vascular dysfunction and has been proposed as an independent risk factor for fatal and nonfatal cardiovascular events in patients with hypertension. It may also be implicated in the pathophysiology of increased thrombogenicity in hypertensives. In the present study, we sought to evaluate a2B-ARs in the platelets of well-controlled hypertensive patients, compared with a normotensive population, and to investigate their association with arterial stiffness. For the measurement of arterial stiffness, we used pulse wave velocity (PWV), which is widely accepted as the ‘‘gold standard’’ measure for this purpose.11
Methods Hypertensive patients with well-controlled essential hypertension from our outpatient hypertension clinic were included in the study. The diagnosis of hypertension was based on three outpatient measurements of blood pressure (BP) >140/90 mm Hg at intervals of no longer than 2 weeks, in accordance with the recommendations of the European Society of Hypertension/European Society of Cardiology.12 To be eligible for inclusion in the study as well-controlled, patients had to have achieved the target BP of 140/90 mm Hg by their second visit, confirmed by 24-hour ambulatory BP monitoring showing a mean 24-hour BP < 130/80 mm Hg. These patients were compared with sex- and aged-matched individuals who visited our outpatient clinic either complaining about atypical chest pain, or having other cardiovascular risk factors, were not hypertensives and served as control group.
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All patients underwent a complete physical examination and routine laboratory tests. Patients with any of the following characteristics were excluded: pregnant or lactating women; grade 3 hypertension or secondary hypertension; coronary artery disease; heart failure; cerebrovascular, liver or renal disease; albumin excretion >300 mg/ 24 h; history of drug or alcohol abuse; any chronic inflammatory or other infectious disease during the last 6 months; thyroid gland disease; body mass index (BMI) > 35 kg/m2; or a history of any hematologic disease. Vascular or neoplastic conditions were ruled out in all participants by a careful examination of the history and routine laboratory tests. BMI was calculated as mass/height2 (kg/m2). A full echocardiographic examination was performed in all participants. Once BP levels had been controlled, blood samples were taken for assessment of a2B-AR gene expression levels in platelets. More specifically, in all participants, after a rest of 20 minutes, blood was drawn from a superficial brachial vein via a 21-ga needle, with care to avoid stasis, hemolysis, and contamination by tissue fluids or exposure to glass. The study was carried out in accordance with the Declaration of Helsinki and the protocol was approved by the local ethics committee. All patients gave informed consent to their inclusion in the study.
RNA Isolation and Platelet a2B-AR Messenger RNA Quantification Blood samples were collected into citrated tubes and centrifuged at 150g for 20 minutes. To avoid leukocyte contamination, only the top 75% of the supernatant platelet-rich plasma was collected and platelets were isolated by centrifugation. Total RNA was isolated from platelets using the TRI-Reagent (Ambion; Life Technologies, Carlsbad, CA, USA) and reverse-transcribed using the PrimeScript RT reagent Kit (Takara Bio Inc, Otsu, Shiga, Japan). Measurements of messenger RNA levels were performed by quantitative real-time polymerase chain reaction (qPCR) using the Corbett Research 6000 detection system. qPCR assays were performed using the KAPA SYBR FAST qPCR Kit (Kapa Biosystems, Woburn, MA, USA). All samples were performed in duplicates. The housekeeping gene GAPDH (glyceraldehyde-3-phosphate-dehydrogenase) was used as an endogenous reference gene. Primer sequences for the alpha2B adrenoreceptor were 50 -GAT TTG GAA GGG CAC CGA GGG A-30 (sense) and 50 -GGC CAG CAG AGG GTC ACA GTC AG-30 (antisense); those for GAPDH were 50 -CCA TCT TCC AGG AGC GAG-30 (sense) and 50 -GCA GGA GGC ATT GCT GAT-30 (antisense). The standard curve method was used for absolute quantification of the amplification products and specificity was determined by performing a melting curve analysis.
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Measurement of PWV
Results
PWV was measured at baseline in all patients. On the day of examination, all subjects were asked to refrain from caffeine, alcohol, and smoking during the preceding 12 hours. The study was carried out between 8:00 and 9:00 AM in a quiet room at 22 1 C. Height and weight were measured. Subjects were allowed a further 15 minutes supine rest before baseline measurements. Brachial BP was measured over the brachial artery three times at 5-minute intervals. The mean of the last two measurements was recorded as representative of brachial BP. After brachial BP, the carotid, femoral, and radial arteries were palpated to find the location of the points with the most pronounced pulse pressure waves. Carotid-femoral PWV (c-f PWV) and carotid-radial PWV (c-r PWV) artery waveforms were measured, and PWV (Complior SP; Atech Medical, France) was determined. The distances traveled by the pulse waves were assessed in triplicate over the surface of the body with a nonelastic tape measure. The same examiner, who was blinded to the patient’s history, performed all measurements.
A total of 59 patients with essential hypertension (34 men, mean age 65 9 years) were included in the final analysis and were compared with a group of 26 individuals with normal BP (19 men, mean age 64 8 years). Mean hypertension duration was 6.4 3.2 years. The majority of patients (95%) were taking a renin–angiotensin–aldosterone system blocker, 76% were taking diuretics, 69% a calcium channel blocker, and 37% a beta-blocker. The main demographic, echocardiographic, and clinical characteristics of the patients and controls are reported in Table 1. Patients with essential hypertension had significantly higher office systolic and diastolic BP, BMI, c-f PWV, c-r PWV, and left ventricular mass index compared with normotensives (Table 1). Twenty-six patients (44%) were receiving angiotensinconverting enzyme inhibitor, 25 (42.3%) were taking angiotensin receptor blockers, 21 (35.5%) calcium blockers, five (0.08%) beta-blockers, and 24 (40.6%) diuretics. Hypertensive patients showed significantly higher gene expression levels of a2B-Rs in their platelets, compared with those of normotensives (Figure 1). More specifically, a2B-R platelet gene expression levels were 34.7 29.5 in hypertensive patients compared with 17.6 12.5 in the control group (P ¼ .005). Stepwise regression showed that platelet a2B-R gene expression levels showed a significant correlation with arterial stiffness in hypertensive patients. Our analysis revealed that levels of a2B-R gene expression in the platelets of patients with essential hypertension were positively correlated with c-f PWV and c-r PWV (Figure 2). More specifically, a2B-R platelet gene expression levels were strongly correlated with c-f PWV (r ¼ 0.59, P < .001), whereas a modest
Statistical Analysis Summary descriptive statistics are presented as mean SD, or frequencies (percentage), as appropriate. Comparisons of continuous variables between the normotensive and hypertensive groups were performed with independent samples t-test. Categorical variables between the two groups were compared using the chi-square test. The association between continuous variables was assessed with Pearson’s correlation coefficient and simple linear regression methods. All statistical tests were carried out in IBM-SPSS 21 at the two-sided 5% level of significance.
Table 1 Participants’ main demographic, echocardiographic, and clinical characteristics
Sex (male/female) Age (y) Smokers (%) Cholesterol (mg/dL) Glucose (mg/dL) Uric acid (mg/dL) Creatinine (mg/dL) Heart rate (bpm) Office systolic blood pressure (mm Hg) Office diastolic blood pressure (mm Hg) Body mass index (kg/m2) Left ventricular mass index (g/m2) Ejection fraction (%) Carotid-femoral pulse wave velocity (m/s) Carotid-radial pulse wave velocity (m/s) NS, no statistically significant.
Hypertensive Patients (n ¼ 59)
Control Group (n ¼ 26)
P
34/25 65 61 237 102 6.6 0.98 73 138 84 30.6 106.3 61.6 10.8 11.1
15/11 64 60 248 96 6.7 0.95 71 124 75 29.7 76.4 63.8 9.7 9.9
NS NS NS NS NS NS NS NS .01 .02 .03 <.001 NS .04 .04
9
66 16 3.2 0.3 8 10 8 4.1 24.5 4.8 2.5 2.6
8 71 19 4.9 0.4 10 5 6 2.9 7.6 5.1 1.9 2
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Figure 1. Platelet alpha2B-adrenergic receptors (a2B-AR) gene expression levels (arbitrary units) in hypertensives compared with normotensives.
positive correlation was also found with c-r PWV (r ¼ 0.39, P ¼ .002). Notably, no significant correlation was found between BP levels and PWV in these well-controlled hypertensives. In addition, no significant correlation was found between levels of a2B-R gene expression in the platelets of normotensives and c-f PWV or c-r PWV and no other association was detected between them and any other clinical parameter. Finally, the drugs-adjusted correlations do not differ from the unadjusted; in other words there is no indication that drug use affects the correlations between a2B-R gene expression levels and c-f PWV or c-r PWV.
Discussion This is the first study to evaluate gene expression levels of a2B-ARs that are implicated in platelet aggregation— in platelets from patients with well-controlled essential hypertension and to compare them with those of normotensive individuals. We found that a2B-AR platelet gene expression levels were significantly higher in patients with essential hypertension compared with normotensives. Notably, we also found a significant correlation between c-f PWV and a2B-AR gene expression levels in platelets from hypertensive subjects. Essential hypertension is widely known to be associated with an increased incidence of arterial thrombotic complications, such as myocardial infarction and ischemic stroke.1,2 The processes of thrombogenesis and atherogenesis in hypertension have a close pathophysiological relation. Although there is a significant pathophysiological and epidemiologic relationship between thrombosis and hypertension, the mechanisms of thrombosis in hypertension have not been fully elucidated.
Figure 2. Association between platelet alpha2B-adrenergic receptors (a2B-AR) gene expression levels (arbitrary units). (A) Carotid-femoral pulse wave velocity (c-f PWV) and (B) carotid-radial pulse wave velocity (c-r PWV) in hypertensive population.
Previous studies have highlighted that the hypercoagulable state observed in patients with hypertension appears to be implicated in the previously mentioned pathophysiological mechanisms, and that this state is more obvious the more elevated the BP.13 The prothrombotic state in hypertension is sustained by a number of factors, such as hemodynamic disturbances, endothelial dysfunction, and an imbalance between procoagulant and fibrinolytic activity in the peripheral blood of hypertensive subjects.3,4 Furthermore, it has been claimed that this prothrombotic status is correlated with the degree and the duration of hypertension14,15 as well as with the presence of target organ damage, such as left ventricular hypertrophy.16 In addition, von Willebrand factor levels are higher in hypertensive patients with target organ damage,17 whereas BP reduction resulted in improvements in indices of hemorheology and endothelial and platelet functions.4
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Platelet activation plays a major role in the thrombotic process, which is triggered by various mechanisms and leads to platelet aggregation and thrombus formation. Experimental studies have shown that hypertension is associated with major changes in morphology, signaling mechanisms, and oxidative stress in blood platelets.18 We know from previous studies that stress enhances platelet function and may influence thrombus formation.19 SNS activation is recognized to be a critical component in the pathogenesis of thrombosis, and platelet reactivity in vivo is influenced by sympathoadrenal activation through an increase in circulating catecholamines.5 In most cases, essential hypertension is associated with sympathetic overactivity, especially in younger patients.20 The sympathetic overactivation contributes to the pathophysiology of hypertension and seems to have additional adverse consequences in hypertensive patients that go beyond the elevation of BP. Norepinephrine enhances platelet aggregability and platelet secretion in vivo21 and may limit the antithrombotic effect of antiplatelet treatment in situations with sympathoadrenal activation.22 According to the literature, the main role of a-ARs is in maintaining vasomotor tone and in the pathophysiology of hypertension. The a1-ARs—through the action of catecholamines— activate vascular smooth muscle contraction, whereas a2-ARs, depending on their location, may act as a feedback mechanism for modulating the release of norepinephrine.8,9 In addition, catecholamines exert significant effects also on platelet aggregation mostly via the a2-ARs.22 It is established that a2-ARs may play a role in platelet aggregation and in the pathophysiology of arterial thrombosis.23,24 It has been highlighted in the literature that marked alterations occur in the platelet alpha2-adrenoceptors of hypertensive subjects.25 The a2B-ARs play a significant role in essential hypertension and are responsible for the central hypertensive sympathoexcitatory response to salt.26 Recently, the a2B-AR subtype has been recognized as an important regulator of aggregation and a2B-AR inhibition in platelets has a significant antiaggregant effect.10 Interestingly, even in patients who were receiving dual antiplatelet therapy, the inhibition of a2B-ARs had an additional antiaggregant effect.10 We have shown that hypertensive patients have significantly elevated levels of a2B-AR gene expression in their platelets that are likely to be implicated in the mechanisms and the pathophysiology of atherothrombosis in essential hypertension. These findings may contribute to a better risk stratification and better selection of individuals who are at especially high risk of thrombotic events. In addition, they may offer future perspectives in the development of novel therapeutic targets, providing strategies for reducing the prothrombotic state.
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On the other hand, alterations in arterial properties and remodeling of the arterial wall have an impact on the cardiovascular risk in hypertensives.27 Importantly, increased arterial stiffness is an independent factor for the development of atherosclerosis and an adverse cardiovascular outcome in essential hypertension.28,29 Increased arterial stiffness in a hypertensive population was associated with platelet activation, as has been shown by an increased mean platelet volume.30 Our findings demonstrated a positive correlation between arterial stiffness and platelet a2B-AR gene expression levels. We hypothesize that endothelial damage and adverse vascular remodeling in hypertension might be a triggering factor of platelet activation, and furthermore a predisposing condition for atherothrombotic events. This still applies even in hypertensive patients who have achieved their BP targets, like those in our study population. This indicates that when the arterial wall has already suffered damage, BP control is not in itself sufficient to eliminate the atherothrombotic risk. Our study had several limitations. The patient population in this study was not large; however, our findings were clear and indicated that other mechanisms associated with pathologic vascular remodeling are involved in the pathology of thrombosis in hypertensive patients. However, we cannot rule out the possibility that the small number of participants might have been obscured other significant correlations. The control group was normotensives but not completely healthy individuals who had high cholesterol levels. We cannot exclude that this may have affected our results. Antihypertensive agents such as beta-blockers did not show any significant effects on our findings. Unfortunately, the number of patients included is not adequately powered to draw safe conclusions for this question. PWV is defined as the velocity at which the BP wave propagates along the arterial tree and provides information about its elastic properties. However, only c-f PWV is established as a parameter with clinical and prognostic value.12 Our data regarding c-r PWV are less meritable but still indicative of the regional arterial stiffness. We focused our analysis in hypertensives. This was because the value of PWV as a prognostic index of cardiovascular events in healthy controls has not been documented. Therefore, our PWV findings could not have been evaluated in the normotensive group. In conclusion, platelet a2B-R gene expression levels are increased in patients with well-controlled essential hypertension compared with normotensives and are strongly associated with increased arterial stiffness in those patients. A possible implication of adverse arterial remodeling in platelet a2B-Rs gene expression should be further investigated. Future research is needed to clarify the pathophysiological pathways that link our findings with platelet aggregation and cardiovascular events.
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References 1. MacMahon S, Peto R, Cutler J, Collins R, Sorlie P, Neaton J, et al. Blood pressure, stroke, and coronary heart disease. Part 1, prolonged differences in blood pressure: prospective observational studies corrected for the regression dilution bias. Lancet 1990;335: 765–74. 2. Lewington S, Clarke R, Qizilbash N, Peto R, Collins R; Prospective Studies Collaboration. Agespecific relevance of usual blood pressure to vascular mortality: a meta-analysis of individual data for one million adults in 61 prospective studies. Lancet 2002;360:1903–13. 3. Poli KA, Tofler GH, Larson MG, Evans JC, Sutherland PA, Lipinska I, et al. Association of blood pressure with fibrinolytic potential in the Framingham offspring population. Circulation 2000;101: 264–9. 4. Spencer CG, Gurney D, Blann AD, Beevers DG, Lip GY; ASCOT Steering Committee; Anglo-Scandinavian Cardiac Outcomes Trial. Von Willebrand factor, soluble P-selectin, and target organ damage in hypertension: a substudy of the AngloScandinavian Cardiac Outcomes Trial (ASCOT). Hypertension 2002;40: 61–6. 5. Esler M, Kaye D. Sympathetic nervous system activation in essential hypertension, cardiac failure and psychosomatic heart disease. J Cardiovasc Pharmacol 2000;35:S1–7. 6. von K€anel R, Dimsdale JE. Effects of sympathetic activation by adrenergic infusions on hemostasis in vivo. Eur J Haematol 2000;65:357–69. 7. Wang JS, Cheng LJ. Effect of strenuous, acute exercise on a2-adrenergic agonist-potentiated platelet activation. Arterioscler Thromb Vasc Biol 1999;19:1559–65. 8. Timmermans PB, Chiu AT, Thoolen MJ. Calcium handling in vasoconstriction to stimulation of alpha 1and alpha 2-adrenoceptors. Can J Physiol Pharmacol 1987;65:1649–57. 9. Gavras H, Handy D, Gavras I. Alpha-adrenergic receptors in hypertension. In: Laragh JH, Brenner BM, editors. Pathophysiology, Diagnosis and Management. New York: Raven Press; 1995. P. 853–61. 10. Marketou ME, Kintsurashvili E, Androulakis NE, Kontaraki J, Alexandrakis MG, Gavras I, et al. Blockade of platelet alpha2B-adrenergic receptors: a novel antiaggregant mechanism. Int J Cardiol 2013; 168:2561–6. 11. Laurent S, Cockcroft J, Van Bortel L, Boutouyrie P, Giannattasio C, Hayoz D, et al. European network for non-invasive investigation of large arteries expert consensus document on arterial stiffness: methodological issues and clinical applications. Eur Heart J 2006;27:2588–605.
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12. Mancia G, Fagard R, Narkiewicz K, Red on J, Zanchetti A, B€ohm M, et al; Task Force Members. 2013 ESH/ESC Guidelines for the management of arterial hypertension: the Task Force for the management of arterial hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC). J Hypertens 2013;31:1281–357. 13. Yang P, Liu YF, Yang L, Wei Q, Zeng H. Mechanism and clinical significance of the prothrombotic state in patients with essential hypertension. Clin Cardiol 2010;33:E81–6. 14. Lip GY, Blann AD, Jones AF, Lip PL, Beevers DG. Relation of endothelium, thrombogenesis, and hemorheology in systemic hypertension to ethnicity and left ventricular hypertrophy. Am J Cardiol 1997;80: 1566–71. 15. Weir MR, Dzau VJ. The renin-angiotensin-aldosterone system: a specific target for hypertension management. Am J Hypertens 1999;12:205S–13S. 16. Preston RA, Jy W, Jimenez JJ, Mauro LM, Horstman LL, Valle M, et al. Effects of severe hypertension on endothelial and platelet microparticles. Hypertension 2003;41:211–7. 17. Alexandrou N, Popov D, Dragan E, Andrei E, Georgescu A. Platelet activation in hypertension associated with hypercholesterolemia: effects of irbesartan. J Thromb Haemost 2010;9:173–84. 18. Hjemdahl P, Larsson PT, Wallen NH. Effects of stress and beta-blockade on platelet function. Circulation 1991;84:VI44–V161. 19. Anfossi G, Trovati M. Role of catecholamines in platelet function: pathophysiological and clinical significance. Eur J Clin Invest 1996;26:353–70. 20. Larsson PT, Wallen NH, Hjemdahl P. Norepinephrineinduced human platelet activation in vivo is only partly counteracted by aspirin. Circulation 1994;89:1951–7. 21. O’ Brien JR. Some effects of adrenaline and antiadrenaline compounds in platelets in vitro and in vivo. Nature 1963;200:763–4. 22. Berlin I, Crespo-Laumonnier B, Cournot A, Landault C, Aubin F, Legrand JC, et al. The alpha 2adrenergic receptor antagonist yohimbine inhibits epinephrine-induced platelet aggregation in healthy subjects. Clin Pharmacol Ther 1991;49:362–9. 23. Grant JA, Scrutton MC. Novel a2-adrenoceptors primarily responsible for inducing human platelet aggregation. Nature 1979;277:659–61. 24. Varani K, Gessi S, Caiazza A, Rastelli G, Portaluppi F, Borea PA. Platelet alpha2-adrenoceptor alterations in patients with essential hypertension. Br J Clin Pharmacol 1999;47:167–72. 25. Gavras I, Gavras H. Role of alpha2-adrenergic receptors in hypertension. Am J Hypertens 2001;14:171S–7S. 26. Gavras I, Manolis AJ, Gavras H. The alpha2 -adrenergic receptors in hypertension and heart failure:
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experimental and clinical studies. J Hypertens 2001;19: 2115–24. 27. Shantsila E, Lim HS. Targeting the arterial wall, but what is the target? J Hum Hypertens 2009;23:1–3. 28. Meaume S, Benetos A, Henry OF, Rudnichi A, Safar ME. Aortic pulse wave velocity predicts cardiovascular mortality in subjects >70 years of age. Arterioscler Thromb Vasc Biol 2001;21:2046–50.
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(2017) 1–7
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29. Cruickshank K, Riste L, Anderson SG, Wright JS, Dunn G, Gosling RG. Aortic pulse-wave velocity and its relationship to mortality in diabetes and glucose intolerance: an integrated index of vascular function? Circulation 2002;106:2085–90. 30. Wang RT, Li Y, Zhu XY, Zhang YN. Increased mean platelet volume is associated with arterial stiffness. Platelets 2011;22:447–51.