Assessment of Mean Platelet Volume in Patients with Resistant Hypertension , Controlled Hypertension and Normotensives

Objective: Patients with resistant hypertension are at increased risk for cardiovascular events. Mean platelet volume (MPV) is an accepted biomarker of platelet activation and considered as a risk factor for cardiovascular disease. The aim of this study was to determine whether MPV levels are higher in resistant hypertensive (RHTN) patients than in controlled hypertensive (CHTN) patients and healthy normotensive controls. Materials and Methods: 279 consecutive patients were included in this study. Patients were divided into three groups: Resistant hypertension patient group [n=78; mean age 56.8±9.8; 42 males (53.8%)]; controlled hypertension patient group [n=121; mean age 54.1±9.6; 49 males (40.5%)]; and normotensive control group [n=80; mean age 49.8±8.5; 34 males (42.5%)]. Physical examination, laboratory workup, and 24-hour ambulatory blood pressure measurement (ABPM) were performed in all participants. Results: The mean platelet volume levels were significantly higher in RHTN group than in the CHTN and normotensive groups (p<0.001). In correlation analysis office systolic and diastolic blood pressure was positively correlated with MPV. Conclusion: Our study demonstrated that MPV, as an important indicator of platelet activation, was statistically higher in RHTN patients than in CHTN and in normotensive subjects. Elevated MPV levels may help to determine a high risk group for atherosclerosis in RHTN patients.


Introduction
Resistant hypertension (RHTN) is an increasingly common clinical problem that studies have suggested is almost always heterogeneous in terms of etiology, risk factors and comorbidities [1].RHTN has an estimated prevalence of 10% to 15% among all participants treated for hypertension [2].It is demonstrated that patients with resistant hypertension are more likely to have cardiovascular disease, manifest as stroke, heart disease, or congestive heart failure when compared to patients with more easily controlled hypertension [3,4].
Previous studies have reported that increased platelet activity is associated with cardiovascular morbidity and mortality [5].Mean platelet volume (MPV) is a marker of platelet size and activation, and its increased level reflects active large platelets.Some clinical studies have shown that active large platelets contain denser granules, which are metabolically and enzymatically more active than small ones and have a higher thrombotic potential [6].Increased MPV has been found to be related with mortality after following acute myocardial infarction and restenosis [7], and this finding has been reported in hypertension background [8].In this study, we aimed to determine whether MPV levels were higher in RHTN patients than in CHNT and healthy normotensive subjects.Physical examination, laboratory work-up, and 24-hour ABPM were performed for all participants.The exclusion criteria of this study were secondary hypertension, heart failure, recent myocardial infarction (MI), coronary artery bypass surgery, stroke, peripheral vascular disease, valvular disease, haemodynamically significant arrhythmias, angina pectoris, chronic renal failure (serum creatine >1.5 mg/dL, blood urea nitrogen >30 mg/dL), chronic liver diseases, thromboembolic disorders, and haematological abnormalities.Patients who were already on an antiplatelet therapy (acetylsalicylic acid or clopidogrel) were also excluded.Informed consent was obtained from the participants, and the study protocol was approved by the local ethics committee.

Ambulatory Blood Pressure Monitoring
In this study, hypertension was defined as an untreated systolic blood pressure (SBP) ≥140 mm Hg and/or diastolic blood pressure (DBP) ≥90 mm Hg in the sitting position on at least three different occasions [9].CHTN, defined as BP below 140/90 mmHg on three or fewer antihypertensive drugs; RHTN, defined as uncontrolled blood pressure (BP) despite the use of ≥3 anti-hypertensive agents from different classes or controlled BP with the use of ≥4 agents [10].Office BP readings were measured by the physicians following a resting period of at least five minutes with the participant remaining in a seated position.An average of three consecutive measurements was taken at five-minute intervals to determine the office SBP and DBP values.
The 24-hour ABPM was performed using a portable compact digital recorder (Tonoport V, Milwaukee, GE Healthcare) and an analyser using customised analytical software that was programmed to measure the blood pressures at 15-minute intervals, from 07:00 until 23:00, and at 30-minute intervals from 23:00 until 07:00.Daytime was defined as the time interval between the hours of 07:00 and 23:00, and night-time was defined as the time interval between the hours of 23:00 and 07:00.The patients were instructed to perform their usual daily activities, but to stay inactive during the measurements.Recordings were accepted if more than 80% of the raw data were valid.
For diagnosis of Diabetes mellitus (DM) and hyperlipidaemia (HL) American Diabetes Association and ATP 3 guidelines were used [11,12].

Blood Sampling
Blood samples were drawn from the antecubital vein between 08.00 and 10.00 AM after overnight fasting into standardised tubes containing dipotassium ethylenedinitrotetraacetic acid (EDTA) to be stored at room temperature.An automatic blood counter (Beckman Coulter; Miami, FL, USA) was used for the whole blood counting.MPV and other haematologic parameters were measured within 30 minutes after the blood collection.Other biochemical analyses were determined by standard methods.

Statistical Analysis
Statistical analyses were performed using SPSS software version 17.0 for Windows (SPSS Inc.; Chicago, IL, USA).The variables were investigated using visual (histograms, probability plots) and analytical (Kolmogorov-Smirnov) methods to determine the normal distribution.Descriptive analyses were presented using means and standard deviation.The categorical variables were expressed as numbers and percentages.Numerical variables were compared using the one-way ANOVA.Tukey's tests were performed to test the significance of pairwise differences by using the Bonferroni correction to adjust for multiple comparisons.Categorical data were compared with the chi-square test.Spearman's correlation coefficients were used to assess the relationship between variables.An overall 5% type I error level was used to infer statistical significance.Eurasian J Med 2015; 47: 79-84

Results
The baseline demographic clinical and laboratory characteristics of the RHTN, and CHTN patients and normotensives were summarized in Table 1.Sex, smoking, DM, and biochemical parameters, except for MPV, were not statistically different among the groups.MPV, age, body mass index (BMI), office systolic and diastolic blood pressure and drug utilization were statistically significant among the groups.
In the subgroup analysis, BMI, except for CHTN and normotensive group (p=0.715), was statistically significant among other groups (for all group p<0.017).In the subgroup analysis, age, except for RHTN and CHTN group (p=0.134), was statistically significant among the groups (for all p<0.017).In the subgroup analysis of office systolic and diastolic blood pressure, both of them were statistically significant among all groups (for all groups p<0.001).In the subgroup analysis, MPV, except for CTHN and normotensive group (p=0.041), was statistically significant among the groups (p<0.001).
As shown in Table 2, all ambulatory blood pressure measurements were exhibiting statistically significance among the groups.
In the subgroup analysis of the 24 hour systolic, daytime systolic, night time systolic BP, all of them were statistically significant among the groups (for all p<0.001).For 24 hour diastolic blood pressure subgroup analysis, except for CHTN and normotensive group (p=0.203),all group combinations showed statistical significant differences (for all, p<0.001).The subgroup analysis of daytime and night time DBP were significantly different among the groups (p<0.001)statistically, except for CHTN and normotensive group (respectively; p=0.464, p=0.306).
In the correlation analysis of MPV with other parameters including sex, BMI, platelet count, creatinine, hematocrit, office systolic and diastolic blood pressure, only office systolic and diastolic BP was positively correlated with MPV (Table 3).

Discussion
Two main findings emerged from this study.First, MPV levels were significantly higher in the RHTN group than in the CHTN and normotensive groups.Second, MPV levels were correlated with office systolic and diastolic blood pressure ABPM recordings.
MPV is one of the important platelet production indices that may relate to platelet function.It has been shown that platelet size, measured as MPV, correlates with their reactivity [13].Larger and hyperreactive platelets accelerate intracoronary thrombus formation, which leads to a cascade of clinical events, such as acute coronary syndromes [14].Some studies have reported a relationship between MPV and hypertension in different patient groups.Nadar et al. [15] demonstrated that hypertensive patients with target organ damage including stroke, previous MI, angina microalbuminuria/proteinuria, and left ventricular hypertrophy, had higher MPV levels than hypertensive patients without target organ damage.
Patients with resistant hypertension have a high prevalence of cardiac and extracardiac target organ damage with an increased prevalence of left ventricular hypertrophy, carotid intima-media thickness, more advanced retinal involvement, and greater urinary albumin excretion compared to patients with well-controlled hypertension [16].Observational studies have shown that RHTN is associated with an increased risk of cardiovascular events as compared to non-resistant HTN [17].Increased platelet activation has been shown in hypertensive patients [18].Shear forces, the renin-angiotensin system, endothelial dysfunction, elevated catecholamine levels, and the presence of comorbid conditions promotes the increased activation of platelets in hypertensive patients [19].RHTN patients exhibit impairment of the autonomic system that includes abnormal parasympathetic and increased sympathetic nervous system activity [20].This increase in sympathetic nervous system in RHTN occurs by different mechanisms.First of all, RHTN patients tend to have higher body mass index (BMI) as observed in our study.In obese patients several mechanisms proposed to explain sympathetic nervous system activation; increased leptin concentration, hyperinsulinemia, decreased arterial baroreflex sensitivity, elevated plasma angiotensin, obesityrelated kidney disease and lack of exercise [21].Furthermore it was demonstrated that individuals with RHT without primary hyperaldosteronism have higher aldosterone levels than normal population [22].The adverse effect of aldosterone excess causes sympathetic activation by stimulation of mineralocorticoid receptors in the paraventricular nuclei [23].
The effects of an over-activated sympathetic nervous system on the haemostatic system occur in two ways.First, platelet activation via alpha2-adrenoreceptor stimulation which [24] causes shape change and thereby increases MPV [25,26].Second, larger, activated platelets which are sequestered in the spleen [27] can be released into the circulatory system following the elevated levels of adrenaline [27] that contribute to increased MPV levels; this might be the first mechanism of how RHTN promotes the increased levels of MPV.
It has been demonstrated that RHTN patients have increased susceptibility of developing cardiovascular damage compared to normal population [28].The exact mechanism by which RHTN increases cardiovascular risk is unknown, but two hypotheses have been postulated; firstly RHTN have likely had a more severe or prolonged elevations in BP over time compared to the patients with non-resistant HTN, and RHTN is reflective of adverse processes (e.g.increased reninangiotensin system stimulation and aldosterone production, increased arterial stiffness, shear stress and atherosclerotic disease) that have been linked with increased cardiovascular risk [29,30].Moreover platelet activation resulting from increased oxidation, and shear stress, which were represented in hypertensive patients, might be second possible mechanism linking the RHTN with the elevated MPV [31].Our study confirms the suggestion of the increase in MPV levels in hypertensive subjects especially in RHTN.This increase in MPV in the RHTN patient group might be related to the increased target organ damage, inflammation, and increased platelet activation [32].This is the first study to demonstrate a relationship between the RHTN and MPV levels.

Study Limitations
This is a single-centre study with a relatively small study population, both of which limit the power of our research findings.In addition, our study provides no information regarding long-term outcomes of the patient groups.We also excluded the patients with clinically overt cardiovascular disease (such as coronary artery disease, cerebrovascular disease, and renal failure), and therefore, our results cannot be extrapolated to all hypertensive subjects.
In conclusion our study showed that MPV, as important indicators of platelet activation, was statistically higher in RHTN patients than in CHTN and in normotensive subjects.MPV levels were positively correlated with office systolic and diastolic blood pressure.Elevated MPV levels may help to determine a high risk group for atherosclerosis in RHTN patients.

Table 1 . The Clinical and laboratory findings of RHTN, CHTN and control normotensive groups
ARB: angiotensin receptor blockers; ACE: angiotensin converting enzyme blockers; CCB: calcium channel blockers; BB: beta blocker; HL: hyperlipidemia; MPV: mean platelet volume