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Keywords:

  • mean platelet volume;
  • myocardial infarction;
  • interleukin 6;
  • polymorphism

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References

Summary.  After rupture of an arteriosclerotic plaque in a coronary artery, platelets play a crucial role in the subsequent thrombus formation, leading to myocardial infarction. An increased mean platelet volume (MPV), as an indicator of larger, more reactive platelets, may represent a risk factor for myocardial infarction. However, this hypothesis is still controversial and most studies addressing the role of MPV were performed comparing patients suffering from myocardial infarction with healthy controls. We intended to identify patients at high risk of suffering myocardial infarction in a group of patients with known coronary artery disease. One hundred and eighty-five consecutive patients with stable coronary artery disease were compared with 188 individuals who had suffered myocardial infarction. Patients within the highest quintile of MPV (≥ 11·6 fl) had a significantly higher risk of experiencing a myocardial infarction compared with patients within the lowest quintile (OR = 2·6, 95% CI 1·3–5·1) in a multivariate analysis that included sex, age, body mass index, hyperlipidaemia, hypertension, smoking and diabetes mellitus. Our results indicate that patients with pre-existing coronary artery disease and an increased MPV (≥ 11·6 fl) are at higher risk of myocardial infarction. These patients can be easily identified during routine haematological analysis and could possibly benefit from preventive treatment.

Myocardial infarction (MI) is the major cause of morbidity and mortality in industrialized countries. Endogenous and exogenous risk factors, such as smoking, hypercholesterolaemia, diabetes mellitus (DM) and hypertension (McKarns et al, 1995), significantly increase the individual risk for MI. However, they only explain part of the cases (Koenig, 1998). Thus, other relevant risk factors need to be identified for accurate calculation of an individual's risk for MI.

After rupture of arteriosclerotic plaques in coronary arteries, platelet activation plays a crucial role in the prothrombotic events leading to MI. Enhanced platelet reactivity significantly increases the individual's susceptibility to MI. Increased platelet reactivity, as well as shortened bleeding time, are associated with increased platelet volume (Milner & Martin, 1985; Trowbridge & Martin, 1987). Large platelets are metabolically and enzymatically more active than small platelets as assessed by in vitro aggregometry (Corash et al, 1977), and they have a higher thrombotic potential (Karpatkin, 1972). They also express higher levels of procoagulatory surface proteins such as P-selectin (Mathur et al, 2001) and glycoprotein IIIa (Pathansali et al, 2001). Various studies found an association between mean platelet volume (MPV) and coronary artery disease (CAD) or the occurrence of an acute MI (Martin et al, 1983; Martin et al, 1991; Pizzulli et al, 1998; van der Loo & Martin, 1999), while others observed no effect (Halbmayer et al, 1995). The biological and prognostic value of an increased MPV is still controversial and the reasons for increased platelet size are still unclear. Platelet morphology and physiology are determined during, or even before, fragmentation of their precursor cell, the megakaryocyte (Rabellino et al, 1981). Although the mechanism is still unclear, megakaryocyte ploidy seems to correlate closely with platelet volume (Hoffman & Long, 1995). Although ploidy and platelet volume are independent variables, alterations in both parameters usually occur in tandem (Trowbridge & Martin, 1985).

Experimental models show that the MPV is increased when both platelet production and destruction are stimulated by chronic hypoxia or administration of anti-platelet serum (Gladwin & Martin, 1990). Similar observations have been made in patients with diabetes or smokers who seem to have an increased platelet turnover (Tschope et al, 1989; Kario et al, 1992), most probably mediated by cytokines. Among the relevant cytokines, interleukin 3, thrombopoietin and in particular interleukin 6 (IL-6) seem to have a major influence on megakaryocyte ploidy leading to the production of larger and more reactive platelets (Debili et al, 1993; Brown et al, 1997). Recently, a frequent G/C polymorphism in the promoter region of IL-6 at nucleotide position (−174) has been shown to influence IL-6 serum levels (Fishman et al, 1998). In individuals carrying the common G allele, higher IL-6 levels have been found compared with the levels in carriers of the C allele (Fishman et al, 1998).

In this study, we analysed the role of IL-6-promoter polymorphism on platelet size in individuals with established CAD, and evaluated the effect of platelet size in these patients with respect to disease progression.

Patients and methods

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References

Patients.  The study was designed as a prospective cross-sectional study. Three hundred and seventy-three consecutive patients referred to the Department of Cardiology, University of Vienna, for coronary angiography were included in our study. The study was approved by the local ethics committee and all patients gave their informed consent.

Inclusion criteria were an angiographically proven CAD and informed consent. Patients without CAD, who were referred to the Department of Cardiology for reasons other than coronary angiography, or those who refused consent were excluded from the study. Patients were divided in two groups according to clinical data and patient history. The first group (MI group) comprised 188 patients who had a history of prior MI, according to the World Health Organization (WHO) criteria, or presented with MI (median time between admission and MI: 2 years, ranging from 0 to 37 years). The second group (stable CAD) included 185 individuals with stable CAD without prior unstable angina or MI. All individuals were tested for established risk factors for CAD such as diabetes mellitus (DM), body mass index (BMI), smoking (> 20 cigarettes/d for more than 5 years), hypertension, lipid profile, haemoglobin (Hb)A1c, family history and standard laboratory parameters at the time of admission. Hypercholesterolaemia was defined as baseline cholesterol levels above 5·18 mmol/l or serum low-density protein (LDL) levels above 3·36 mmol/l and hypertriglyceridaemia as triglyceride levels above 2·03 mmol/l after overnight fasting. DM was considered present in patients with a known history of DM and in patients with HbA1c values above 6·5%. The extent of CAD (one-, two- or three-vessel disease) was angiographically determined in all patients. It was defined as a stenosis of more than 30% luminal diameter reduction of a main coronary artery (left anterior descending artery, arteria circumflexa, right coronary artery). To allow an individual risk assessment the MPV values of all individuals were divided into quintiles as shown in Table I.

Table I. .  Demographic and clinical characteristics of the study population. Univariate analysis P-values are given on the right.
 Patients without MI (n = 185)Patients with MI (n = 188)P-value
  • *

    Chi-square test for trend.

  • MI, myocardial infarction; IQR, interquartile range; BMI, body mass index ; MPV, mean platelet volume.

Male patients (%)131 (70·8%)139 (73·9%)    0·5
Median age, years (IQR)  66 (58–73)  61 (54–71)    0·003
Median BMI kg/m2 (IQR)  26·6 (23·9–29·1)  27·0 (24·8–29·8)    0·1
Hypercholesterolaemia (%)132 (66·5%)150 (79·8%)    0·04
Hypertriglyceridaemia (%)  82 (44·3%)  84 (44·7%)    0·9
Arterial hypertension (%)111 (60·0%)132 (70·2%)    0·04
Diabetes mellitus (%)  67 (36·2%)  78 (41·5%)    0·3
Current smokers (%)  52 (28·1%)  89 (47·3%)< 0·001
MPV      0·04*
First quintile (7·9 fl−9·8 fl)  47 (25·4%)  31 (16·5%) 
Second quintile (9·9 fl−10·3 fl)  32 (17·3%)  38 (20·2%) 
Third quintile (10·4 fl−10·8 fl)  35 (18·9%)  35 (18·6%) 
Fourth quintile (10·9 fl−11·5 fl)  43 (23·2%)  36 (19·1%) 
Fifth quintile (11·6 fl−13·9 fl)  28 (15·1%)  48 (25·5%) 

Determination of MPV. EDTA blood samples, drawn at admission of the patient, were analysed in an automated haematology analysis system (Sysmex NE 8000 autoanalyser, Sysmex Europe, Norderstedt, Germany) (O'Malley et al, 1996). Daily quality controls showed an intra-assay variation coefficient (CV) of 2·5% and interassay CV of 3·0%.

The reported platelet swelling (Bath, 1993; Smyth et al, 1993) in EDTA blood samples was evaluated in the blood samples of 10 healthy volunteers at three different timepoints. MPV was measured immediately (within 10 min) after venepuncture, 30 min after venepuncture and 2 h after venepuncture. Blood samples of the 10 healthy volunteers showed a median increase in platelet volume of 0·3 fl (interquartile range (IQR): 0·1–0·4 fl) 30 min after venepuncture and 0·5 fl (IQR: 0·3–0·65 fl) 2 h after sample collection.

All patients' samples were processed within 2 h of venepuncture as recommended by Smyth et al (1993) to avoid bias due to excessive platelet swelling.

Polymerase chain reaction (PCR) analysis of the IL-6 G(−174)C promoter polymorphism.  DNA samples were available from 212 patients (54·6%). DNA isolation was performed according to standard procedures. For determination of the IL-6 promoter polymorphism, we developed a mutagenic-separated PCR (MS PCR) assay, following the general principle described previously (Rust et al, 1993; Endler et al, 2001). Briefly, allele specific primers that differ in length by 8–10 base pairs (bp) with single base mismatches at defined positions were used to minimize crossreactions of the PCR products. In every sample, one or two different products are generated depending on the genotype. PCR amplification was carried out in 50 µl volumes containing 1·5 U of AmpliTaq Gold (Perkin Elmer Cetus, Norwalk, CT, USA), 1·5 mmol/l MgCl2, 200 μmol/l of each dNTP (Amersham Pharmacia Biotech, Uppsala, Sweden), 8 pmol IL-6–174G forward primer (5′-TCC CCC TAG TTG TGT CTC GCG-3′), 12 pmol IL-6–174C forward primer (5′-CTG CAC TTT ATC CCC TAG TTG TGT CAT GCC-3′), 10 pmol IL-6 reverse primer (5′-TGA GGG TGG GGC CAG AGC-3′; all MWG Biotech, Ebersberg Germany, GenBank accession number: Y00081) and approximately 50 ng of DNA. Amplifications were performed in a Perkin Elmer 480 DNA Thermo Cycler (Perkin Elmer Cetus, Emeryville, CA, USA). A 10-min denaturation period at 95°C was followed by 37 cycles of 95°C for 1 min, 58°C for 2 min and 72°C for 1 min. A final extension step of 10 min at 72°C completed the reaction. For the IL-6–174G allele a PCR product with a length of 94 bp and for the IL-6–174C allele a product with 103 bp were generated, which were separated on 6% precast Tris-borate-EDTA (TBE) polyacrylamide gel (Novex, San Diego, CA, USA), during 50 min at 160 V. After staining with Sybr Green (Molecular Probes, Eugene, Oregon, USA) for 20 min, bands were visualized on an ultraviolet (UV) transilluminator at 306 nm and photographed using a polaroid land camera. In each experiment, a known heterozygous individual for IL-6 G(−174)C was included as positive control to ensure amplification of both alleles. A reagent control without DNA served as a negative control.

Statistical analysis.  Continuous data are presented as the median and IQR (range from the 25th to the 75th percentile). Percentages were calculated for dichotomous variables. The Mann–Whitney U-test or, if adequate, the Kruskall–Wallis test were used for univariate comparison of continuous data; the chi-square test was applied to compare proportions. Multivariate logistic regression was used to assess the independent association of MPV with the occurrence of MI and to adjust for confounding factors. Variables which showed a trend towards a difference (P < 0·2) between patients with and without MI in univariate analysis were included in the model as possible confounders. Results of the logistic regression model were expressed as the odds ratio (OR) and the 95% confidence interval (95% CI). The Hosmer–Lemshow test was used to assess the model fit. Spearmans rank correlation coefficient was used for the measurement of correlation. All P-values were two-sided, a P-value < 0·05 was considered statistically significant. For statistical analysis, we used the Statistical Package for the Social Sciences (SPSS) version 10·0 software (SPSS, Illinois, USA).

Results

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References

Three hundred and seventy-three patients were included in the study; median age was 64 years (IQR 56–77 years). Two hundred and seventy patients (72%) were male. One hundred and forty-eight patients had a history of MI and 40 patients had suffered recent events. The comparison group comprised 185 patients, presenting with stable CAD without a history of MI. Baseline demographic data are given in Table I. MPV values in the group with stable CAD did not differ significantly from patients without a history of ischaemic heart disease (data not shown).

As expected univariate comparison between patients with and without MI showed that the known risk factors, hypercholesterolaemia, hypertension and smoking had a significant influence on the risk of suffering MI. Interestingly, only within the fifth quintile of MPV was a significant difference between the stable CAD and the MI group observed (Table I).

Multivariate logistical regression analysis was performed to assess the independent association of MPV and MI and to adjust for confounding factors (Table II). The association between MPV values within the highest quintile and an increased risk for MI remained significant in a binary logistical regression model, including other known cardiovascular risk factors (Table II). Hypertriglyceridaemia and DM did not contribute significantly to the risk of MI (P > 0·2) in our model and were thus excluded.

Table II.  (A) Univariate analysis of the quintiles of MPV and the relative risk for MI. (B) Multivariate analysis of the quintiles of MPV adjusted for sex and age. (C) After adjustment for sex, age, hypertension, BMI, smoking and hypercholesterolemia * , no significant changes in the relative risk for MI were observed, indicating that MPV values within the fifth quintile might constitute an independent risk factor for suffering MI.
 OR95% CIP-value
 first quintile (7·9 fl−9·8 fl)1·0
 second quintile (9·9 fl−10·3 fl)1·80·9–3·50·1
 third quintile (10·4 fl−10·8 fl)1·50·8–2·90·2
 fourth quintile (10·9 fl−11·5 fl)1·30·7–2·40·5
 fifth quintile (11·6 fl−13·9 fl)2·61·4–5·00·01
 OR95% CIP-value
 first quintile (7·9 fl−9·8 fl)1·0
 second quintile (9·9 fl−10·3 fl)1·80·9–3·40·1
 third quintile (10·4 fl−10·8 fl)1·40·7–2·80·3
 fourth quintile (10·9 fl−11·5 fl)1·30·7–2·40·5
 fifth quintile (11·6 fl−13·9 fl)2·61·3–5·00·01
 OR95% CIP-value
  • –, denotes reference group.

  • *

    Hosmer–Lemeshow test: C = 4·1, df = 8, P = 0·9.

  • MPV, mean platelet volume; OR, odds ratio; 95% CI, 95% confidence intervals.

C.
MPV
 first quintile (7·9 fl−9·8 fl)1·0
 second quintile (9·9 fl−10·3 fl)1·70·8–3·30·1
 third quintile (10·4 fl−10·8 fl)1·30·7–2·60·4
 fourth quintile (10·9 fl−11·5 fl)1·30·6–2·40·5
 fifth quintile (11·6 fl−13·9 fl)2·61·3–5·10·01

Although MPV values increase because of platelet swelling when using EDTA as anticoagulant, in our hands MPV increased less than ∼0·5 fl when the analysis was performed within 2 h of venepuncture. Nevertheless, the imprecision of the testing system used should also be taken into account and at least a second independent determination of MPV would seem to be indicated.

Additionally, we found no correlation between MPV and the timespan between MI and measurement of MPV (Spearmans correlation coefficient r = 0·027; P = 0·7). MPV in patients with acute MI did not differ significantly from MPV in patients with a history of MI (Table III).

Table III.  Influence of timespan between MI and determination of MI. IQR ranges are given in parenthesis and median MPV values are shown.
Patients with acute MI, at time of determination (n = 40)Patients with MI ≤1 year before determination (n = 39)Patients with MI > 1 year before determination (n = 109)P-value 
  • *

    Kruskal–Wallis test.

  • MI, myocardial infarction; MPV, mean platelet volume; IQR, interquartile range.

10·7 fl (10·0–11·4 fl)10·8 fl (10·3–11·3 fl)10·8 fl (10·0–11·6 fl)0·8

No significant association of the MPV and IL-6 genotype, or the IL-6 genotype and the risk for suffering MI, was observed (Table IV).

Table IV.  Median MPV values (IQR) in relation to the IL-6 G(−174)C-promoter polymorphism.
IL-6 promoter genotype(−174)GG(−174)C(−174)CP-value*
  • *

    Kruskal–Wallis test.

  • MI, myocardial infarction; MPV, mean platelet volume; IQR, interquartile range; IL-6, interleukin 6.

Patients without MI10·6 fl (9·8–11·1 fl)10·4 fl (10·0–11·5 fl)11·1 fl (9·9–11·6 fl)0·4
(n = 103)(n = 29)(n = 55)(n = 19) 
Patients with MI10·4 fl (10·0–11·5 fl)10·7 fl (10·0–11·3 fl)10·3 fl (9·8–11·7 fl)0·7
(n = 104)(n = 37)(n = 49)(n = 18) 
Total10·5 fl (10·0–11·2 fl)10·6 fl (10·0–11·3 fl)10·8 fl (9·9–11·7 fl)0·9
(n = 207)(n = 66)(n = 104)(n = 37) 

Discussion

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References

Our findings indicate that increased platelet volume is associated with a higher risk of suffering an acute coronary event, independent of the extent of CAD. Patients within the highest quintile of MPV (MPV ≥ 11·6 fl) had a significantly increased risk of MI after adjusting for confounding factors. Thus, MPV values above this level may represent an independent risk factor for MI in patients with CAD. In various studies the influence of smoking (Kario et al, 1992), diabetes (Tschope et al, 1989) and hypertension (Pathansali et al, 2001) on platelet size was observed. However, in our patients, MPV remained a significant independent risk factor for MI in a multivariate model that included smoking, diabetes and hypertension.

The mechanisms for an increased platelet volume are not fully understood. Possibly, cytokines may trigger the production of larger more reactive platelets, following platelet destruction in peripheral blood. Whether or not higher serum IL-6 levels correlate with high MPV is still controversial (Bath et al, 1994). In our study, we found no association between MPV and a frequent IL-6 C to G polymorphism at nt(−174) in the IL-6 promoter. Previously, individuals carrying the G allele have been shown to have increased IL-6 serum levels (Fishman et al, 1998). Recent in vitro findings indicate that megakaryocyte expansion is mediated by a combination of various cytokines, especially IL-6, IL-11 and thrombopoietin. Thus, it is reasonable to assume that a single polymorphism will not have a major impact on MPV levels (Lazzari et al, 2000). In a physicians health study, increased serum IL-6 levels have been found to be associated with an increased risk of suffering MI (Ridker et al, 2000). In contrast, others recently reported that the IL-6–174C allele, which leads to lower serum IL-6 levels, was associated with an increased risk of suffering MI (Georges et al, 2001). Undoubtedly, the role of IL-6 and the IL-6–174 polymorphism on the development of MI is still unclear, but does not seem to be mediated via the platelet volume.

Similar to other studies, the timespan between MI and laboratory testing did not influence platelet size, suggesting that MPV does not change during the acute-phase reaction but is determined by other factors. The findings of our study confirm that increased MPV might be responsible for the prethrombotic state that eventually leads to thrombus formation after rupture of a coronary plaque (Gladwin & Martin, 1990; Kario et al, 1992). Our data also suggest that patients with an MPV ≥ 11·6 fl may represent a group at higher risk of MI. For these patients modification of other cardiovascular risk factors could be advisable.

Recently, in vitro data on the therapeutic effects on platelet size of Losartan, an angiotensin II receptor antagonist, or Doxazosin, an alpha1-adrenoreceptor antagonist, have been reported (Jagroop & Mikhailidis, 2001). These observations have not yet been confirmed in vivo (Jagroop & Mikhailidis, 2000). Little is known about the effects of aspirin and other platelet aggregation inhibitors on MPV. In a small study with 30 patients, Sharpe et al (1994) found no effect on MPV values. However, whether interventions with platelet aggregation inhibitors or other drugs are beneficial for patients with high MPV remains to be evaluated.

References

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. References
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