Platelet glycoprotein IIb HPA-3 polymorphism and acute coronary syndromes

International Journal of Cardiology 127 (2008) 46 – 50 www.elsevier.com/locate/ijcard

Platelet glycoprotein IIb HPA-3 polymorphism and acute coronary syndromes John Lekakis a,⁎, Sofia Bisti c , Elias Tsougos a , Athanasios Papathanassiou b , Nikolaos Dagres a , Ignatios Ikonomidis a , Ekaterini Soteriadou c , Alexandros D. Tselepis d , John Goudevenos b , Dimitris Th. Kremastinos a a

d

Department of Cardiology, Attikon University Hospital, Athens University, Greece b Department of Cardiology, University of Ioannina, Greece c Department of Microbiology, Hellenic Pasteur Institute, Athens, Greece Laboratory of Biochemistry, Department of Chemistry, University of Ioannina, Greece

Received 25 July 2006; received in revised form 30 March 2007; accepted 4 April 2007 Available online 11 June 2007

Abstract Background: There is considerable research interest about the platelet GPIIb/IIIa receptor polymorphisms in CAD. Methods: We investigated differences in the frequency of the polymorphism in the GPIIb subunit of the receptor HPA-3 (a and b allele) between patients with more extensive coronary thrombosis such as patients with ST segment elevation (STEMI) and those with less extensive coronary thrombosis such as those with non-ST elevation myocardial infarction (NSTEMI), unstable angina (UA) or chronic CAD. We studied 118 CAD patients, of which 38 suffered from STEMI, 62 from NSTEMI or UA and 18 from chronic CAD and 15 healthy individuals. Patients were followed-up for 21 ± 6 months for occurrence of death, myocardial infarction and revascularization. Results: Seventeen out of 38 (45%) patients with STEMI were homozygous for the HPA-3 b allele compared to 6 out 62 (10%) with NSTEMI-UA , 4 out of 18 (22%) with chronic CAD and 2 out of 15 (13%) healthy controls (χ2 = 16,4, p = 0.03.) Homozygous patients for the HPA-3b exhibited a 5-fold higher risk for STEMI compared to heterozygous patients for HPA-3b or homozygous for HPA-3a allele (OR: 5.90, 95% CI: 2.15–16.54, p = 0.01) after adjustment for age, sex and risk factors. The HPA-3 genotypes were not related with cardiovascular events during follow-up. Conclusions: Among patients with an acute coronary syndrome those being HPA-3b homozygous have a tendency to develop ST segment elevation myocardial infarction instead of non-ST segment elevation infarction or unstable angina. There is no association between the HPA3 genotypes and future cardiovascular events. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Platelets; GPIIb/IIIa receptors; Genepolymorphisms; Acute coronary syndromes

1. Introduction Thrombosis is a key mechanism in the pathogenesis of acute coronary ischemic syndromes and the role of platelet adhesion and aggregation is crucial in this process [1]. Thrombus formation is mediated by the binding of fibrinogen to the activated platelet integrin-receptor αΙΙbβ3, ⁎ Corresponding author. 12 Iridanou str, 115 28 Athens, Greece. Tel.: +30 210 7299200; fax: +30 210 7299201. E-mail address: [email protected] (J. Lekakis). 0167-5273/$ - see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2007.04.039

also known as glycoprotein IIb/IIIa (GPIIb/IIIa). GPIIb/IIa is the most abundant platelet receptor; it recognizes fibrinogen, von Willebrand factor, fibronectin and vitronectin as ligands and it is involved in platelet aggregation and thrombus formation. The receptor consists of two subunits: 1) the α subunit or GPIIb is a 136 kDa polypeptide consisting of a heavy and a light chain and 2) the β subunit or GPIIIa which is a single polypeptide of 92 kDa [2]. Several prothrombotic genetic factors that may influence the individual thrombotic risk have been identified. Platelet membrane glycoproteins are highly polymorphic and can be

J. Lekakis et al. / International Journal of Cardiology 127 (2008) 46–50 Table 1 Patient characteristics

Age (years) Sex (male) Total cholesterol (mg/dl) HDL cholesterol (mg/dl) Triglycerides (mg/dl) Hypertension Smoking Diabetes Family history of CAD No. of vessels in angiography with stenosis N70% No. of total occlusions 0 1 2 3 Non-significant stenosis (b50%) LMS LAD CX RCA HP-3 polymorphisms a/a a/b b/b CV events at f/u

STEMI (n = 38)

NSTEMI-UA Stable CAD p (n = 62) (n = 18)

67 ± 12 25 (66%) 194 ± 53 42 ± 9 129 ± 48 23 (61%) 20 (53%) 10 (26%) 5 (14%) 2.2 ± 0.8

64 ± 10 51 (82%) 201 ± 48 46 ± 9 141 ± 56 47 (76%) 30 (48%) 24 (39%) 13 (21%) 1.7 ± 0.8

69 ± 8 13 (72%) 219 ± 98 47 ± 13 186 ± 136 13 (72%) 8 (44%) 3 (17%) 3 (17%) 1.8 ± 0.9

0.21 0.17 0.87 0.19 0.32 0.26 0.84 0.15 0.64 0.016

25 (66%) 10 (26%) 3 (8%) 0 (0%) 1 (3%)

49 (79%) 9 (14%) 3 (5%) 1 (2%) 1 (2%)

12 (66%) 4 (22%) 1 (6%) 1 (6%) 1 (6%)

0.65

0 (0%) 19 (50%) 29 (76%) 16 (42%)

2 (6%) 41(66%) 34 (55%) 24 (39%)

1 (6%) 9 (50%) 13 (72%) 3 (17%)

0.41 0.21 0.37 0.15

10 (26%) 11 (29%) 17 (45%) 5 (13%)

23 (37%) 33 (53%) 6 (10%) 6 (10%)

3 (17%) 11 (61%) 4 (22%) 2 (11%)

b0.01

0.18

0.43

Data are expressed as number (%) of patients or mean value ± SD. STEMI = ST segment elevation myocardial infarction, NSTEMI-UA = unstable angina/non-ST segment elevation infarction, LMS = left main stem, LAD = left anterior descending artery, CX = circumflex coronary artery, RCA = right coronary artery, CV = cardiovascular.

recognized as alloantigens or autoantigens [3]. In particular, there is considerable research interest in the association between the GPIIb/IIIa receptor polymorphism and coronary acute ischemic syndromes as well as the response to the antiplatelet therapy. A common polymorphism results from a single nucleotide mutation (C1565T) in the GPIIIa gene encoding for a single aminoacid substitution (Leu/Pro at position 33) in the GPIIIa subunit of the receptor. This change results in two alleles; the HPA-1a allele, and the HPA-1b allele. A single nucleotide change (T2622G) in the GPIIb gene results in another common polymorphism in the GPIIb subunit of the receptor named HPA-3. The HPA3a and 3b alleles encode for subunits that only differ at position 843 where Ile substitutes Ser. Most, but not all, of the studies agree that the HPA-1b allele represents an inherited risk predisposing to acute coronary syndromes [4–6], while the reports on the significance of the polymorphisms of the a subunit are rare [6–9]. Moreover there are no data on the significance of HPA-3 alleles in the genesis of ST segment elevation myocardial infarction (STEMI) or unstable angina/ non-ST segment elevation infarction (NSTEMI-UA).

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Therefore, the aim of the present study was to investigate the frequency of HPA-3 polymorphisms of the platelet receptor GPIIb/IIIa in patients developing coronary thrombosis leading to STEMI in comparison to patients with less extensive coronary thrombosis causing NSTEMI-UA or patients with stable coronary artery disease (CAD) without evidence of previous clinical coronary thrombosis. 2. Methods 2.1. Study population We studied 100 consecutive patients presenting with an acute coronary syndrome in our institutions (ACS group) during a 6-month screening period. The diagnosis of ACS and classification STEMI (n = 38), NSTEMI (n = 22) and UA (n = 40) were performed according to the definitions proposed recently by the European Society of Cardiology and the American College of Cardiology [10]. Patients with NSTEMI and patients with UA were characterized as NSTEMI-UA group. The results of the ACS group were compared with a) a group of 18 consecutive patients with stable angina without history of ACS in the past who were referred for coronary angiography (Stable CAD) and b) a group of 15 healthy individuals (mean age 56 ± 4 years) without history of cardiac disease and negative for ischemia treadmill test. Thus, the study population consisted of a total of 133 subjects. Patient characteristics of the ACS and the Stable group are given in Table 1. Arterial hypertension was defined as blood pressure N 140/90 mm Hg requiring medical treatment; smoking was defined as current or previous smoking. Total cholesterol, high-density lipoprotein (HDL) cholesterol and triglycerides were measured using standard techniques. Hyperlipidemia was defined as fasting total cholesterol N 5.17 mmol/l (200 mg/dl) or LDL cholesterol N 3.36 mmol/l (130 mg/dl) or fasting triglycerides N2.66 mmol/l (200 mg/dl) or current treatment with lipid-lowering medication. DM was considered to be present in patients with a known history of DM or a fasting glucose N7 nmol/L (125 mg/dl). In brief, the ACS and the Stable group did not differ with regard to age, sex and risk factors. All patients in the ACS and the Stable group underwent coronary angiography with standard techniques. The number of major coronary vessels with luminal stenosis N 70% or b 50% (non-significant stenosis), the number of occluded vessel, the diseased vessel (left main stem, left anterior descending, circumflex, right coronary artery) in the three groups is also given in Table 1. Patients with STEMI had a moderately higher number of diseased vessels. Patients with CAD were followed up for a mean period of 21 ± 6 months to record the occurrence of death, MI (STEMI or Non-STEMI) and revascularisation (PCI or CABG). 2.2. Genomic DNA analysis Genomic DNA was extracted from anticoagulated whole blood collected in EDTA-coated vacuum tubes (BD

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Table 2 Univariate logistic regression analysis assessing the association between clinical presentation of CAD and HPA-3 genotypes Dependent variables

STEMI OR (95% CI)

p

NSTEMI-UA OR (95% CI)

p

Stable CAD OR (95% CI)

p

HPA-3 a/a HPA-3 a/b HPA-3 b/b

0.742 (0.314–1.54) 0.33 (0.146–0.763) 5.67 (2.26–14.23)

0.496 0.009 b0.01

1.951 (0.871–4.369) 1.759 (0.845–3.658) 0.179 (0.066–0.486)

0.104 0.131 b0.01

0.375 (0.102–1.379) 2.233 (0.810–6.150) 0.881 (0.266–2.918)

0.144 0.120 0.88

OR = odds ratio, CI = confidence intervals, STEMI = ST segment elevation myocardial infarction, NSTEMI-UA = unstable angina/non-ST segment elevation infarction.

Vacutainer, K3E) using a DNA isolation kit (Nucleospin blood, Macherey-Nagel) and processed according to the manufacturer's instructions. Sequence Specific Primers (SSPs) were used for the genotyping of the HPA-3 (alleles: HPA-3a and HPA-3b) polymorphisms as described previously [11]. Allele specific primer sequences used for HPA-3 polymorphisms were: HPA-3a (5′-GGG GGA GGG GCT GGG GA-3′), HPA-3b (5′GGG GGA GGG GCT GGG GC-3′) and HPA-3-AS (antisense) (5′-GAC CTG CTC TAC ATC CTG GA-3′). Specific primers amplifying a fragment of HGH (Human Growth Hormone) were used as positive control: HGH-S (sense) (5′-TGC CTT CCC AAC CAT TCC CTT A-3′) and HGH-AS (antisense) (5′-CCA CTC ACG GAT TTC TGT TGT GTT TC-3′). The size of the PCR products for HPA-3 polymorphisms was 230 base pairs and 434 base pairs for the positive control (HGH). PCR were performed in a 50-μl reaction volume and contained 50 ng of genomic DNA, 5 μl enzyme buffer, 1 μl BSA, 1,5 mM MgCl2, allele specific primer pairs (0.4 μÌ of each primer), 200 μÌ of dNTPs (Promega) and 2.5 units of DNA polymerase (AmpliTaq Gold, Applied Biosystems). PCR cycling conditions were the following: After denaturation for 11 min at 95 °C the amplification cycle (denaturation step at 95 °C for 30 s, annealing step at 63 °C for 30 s and extension step at 74 °C for 30 s) was repeated 32 times and followed by final extension for 10 min at 74 °C. Analysis of PCR products was carried out by electrophoresis in ethidium bromide-stained 2.5% agarose gel run at 150 V for 30 min, visualized under UV illumination and documented by photography. To control for potential contamination by previous PCR products, samples without DNA were amplified and analyzed in the same manner as the samples above. 2.3. Statistical analysis All variables are expressed as mean ± SD. Continuous variables were tested for normal distribution using the Kolmogorov–Smirnov test. Comparisons between patient subgroups and healthy controls were performed using the standard χ2 or the unpaired t-test. The association between HPA-3 genotypes (a/a, a/b, b/b) and clinical characteristics was assessed by ANOVA or Kruskal Wallis test for nonparametric data. The distribution of the examined genotypes among of patients with STEMI, NSTEMI-UA, stable CAD or healthy controls was assessed by χ2 contingency tables.

Logistic regression analysis was performed to assess the association between HPA-3 genotypes (a/a, a/b, b/b) and history of STEMI, NSTEMI-UA or stable CAD (dependent variable) and the adjusted-odds ratios (OR) with the corresponding 95% confidence intervals (CI) were calculated after adjusting for age, sex and atherosclerotic risk factors (smoking status, hypertension, hyperlipidemia, parental CAD, diabetes). Interaction between the genotype of interest and other independent variables were also examined in the multivariable model. Each independent variable was also tested separately in the final multivariable model for the prediction of STEMI, NSTEMI or stable CAD to examine whether the adjusted odds ratios for the genotypes of interest were modified significantly. During follow up, the primary outcome variable was the combination of cardiac death, nonfatal MI and revascularisation. Only the first event was counted as an end point. Patients were stratified into 3 groups based on HPA-3 genotypes (a/a, a/b, b/b) and cardiac event free survival curves were constructed by Kaplan–Meier analysis. Differences between the curves were assessed using the Log rank test. Statistical analysis was performed with the statistical software package SPSS 11.5 (SPSS Inc., Chicago, IL, USA). A p b 0.05 was considered statistically significant. 3. Results 3.1. Association between platelet GPIIb/IIIa polymorphisms and clinical presentation of CAD Assessment of the distribution of HPA-3 genotypes among our study population showed that, 17 out of 38 (45%) patients with history of STEMI were homozygous for the HPA-3 b allele compared to 6 out 62 (10%) with NSTEMI-UA, 4 out of 18 (22%) with chronic CAD (χ2 = 16.4, p b 0.01.) and 2 out of 15 (13%) healthy controls (χ2 = 20.1, p = 0.03, Table 1). Homozygous patients for the HPA-3b allele exhibited a 5fold higher risk for STEMI compared to heterozygous patients for HPA-3b or homozygous for HPA-3a allele in univariate logistic regression analysis (OR: 5.67, 95% CI: 2.26–14.23, p b 0.01, Table 2). The odds-ratio of homozygous HPA-3b genotype for the prediction of STEMI remained unchanged after adjustment for age, sex and traditional atherosclerotic risk factors (smoking status, hypertension, hyperlipidemia, parental CAD, diabetes) (OR: 5.90, 95% CI: 2.15–16.54, p = 0.01)

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There was no association between the HPA-3 genotypes and age, sex, smoking, hypertension, hyperlipidemia, diabetes and family history for CAD or angiographic characteristics either among cases or controls. Thirteen patients presented cardiac events within the follow up period. Of these patients, 4 died, 4 suffered from an MI and 5 had a revascularisation procedure (2 PCI and 3 CABG]. There was no association between the HPA-3 genotypes and occurrence of cardiovascular events during follow up (log rank = 0.61, p = 0.43). 4. Discussion The present study suggests that in patients that develop acute coronary syndrome those being HPA-3b homozygous have a tendency to develop ST segment elevation myocardial infarction instead of non-ST segment elevation infarction or unstable angina. We could assume that after erosion or rupture of the atherosclerotic plaque, patients homozygous for the HPA-3b allele tend to form a larger occlusive thrombus leading to ST elevation myocardial infarction. Additionally, patients with ST segment elevation myocardial infarction had a higher frequency of HPA-3b allele homozygosity compared to healthy controls. Platelet function has been shown to be associated with acute coronary syndromes. Enhanced platelet activation has been observed in patients with myocardial infarction; this activation is more intense in patients with ST elevation compared to non-ST elevation myocardial infarction [12]. Although there is considerable interest in the association between GPIIb/IIIa receptor polymorphisms and coronary acute ischemic syndromes different studies provided contradictory results [4–6]. The HPA-1b polymorphism may represent an inherited risk factor that promotes the thromboembolic complications of coronary artery disease. Individuals carrying the HPA-1b allele displayed a lower threshold for platelet activation [13] and increased platelet aggregability [14]. Many studies support the hypothesis that the presence of HPA-1b polymorphic allele could be a genetic prothrombotic factor associated with the risk of developing myocardial infarction especially at young age [4,15]. Other studies do question the association between the HPA-1b allele and coronary thrombosis [16]. A recent meta-analysis on the relation of the HPA-1b polymorphism and myocardial infarction including 5298 patients with myocardial infarction and 5285 control subjects from 19 independent studies, suggested that there is no evidence for a definite relation between the HPA-1b polymorphism and myocardial infarction [2]. The two GPIIb/IIIa subunits are encoded by separate but closely associated genes located on the long arm of chromosome 17. HPA-3a/HPA-3b is a common polymorphism of the GPIIb subunit of the GPIIb/IIIa platelet receptor that like HPA-1 could play a role in the development of posttransfusion purpura and neonatal alloimmune thrombo-

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cytopenic purpura. Contrary to HPA-1 polymorphism there is little information about the significance of the GPIIb polymorphism on cardiovascular disease. There have been only a few studies on the association between the HPA-3 polymorphism and arterial thrombotic disorders. In Japanese people, the HPA-3b allele frequency was 65% in 100 controls and 59% in 88 patients with a first myocardial infarction [17], whereas no measurable impact of the HPA-3 polymorphism on coronary artery disease or myocardial infarction was detected in angiographically evaluated patients by Bottiger et al. [6]. Moreover, in another study no significant relation was found between the HPA-3 genotypes and the risk for thrombosis and restenosis after stent placement [8]. On the contrary, a small study on young women from the US found an association between HPA-3b and myocardial infarction in the presence of other risk factors [9]; women having the HPA-3b allele and being current smokers had a 10-fold increased risk for myocardial infarction. Finally, Reiner et al. [18] observed an association between HPA-3b homozygosity and ischemic stroke in young women with risk factors for atherosclerosis. In our study patients homozygous for the HPA-3b allele showed a higher probability of presenting myocardial infarction with ST segment elevation instead of NSTEMIUA acute coronary syndrome . To our knowledge, this is the first time that this polymorphism in the GPIIb subunit of the GPIIb/IIIa is shown to be indirectly related to the amount and characteristics of coronary thrombus in ACS, since ST elevation myocardial infarction is caused by a larger, occlusive thrombus compared to NSTEMI. The differences observed among the various studies examining the significance of HPA-3 polymorphism could be explained either by the different ethnicity and/or by the synergism of various environmental factors and genetic variants in developing a certain phenotype; it is well known that increased platelet aggregability is associated with many of the known risk factors for atherothrombosis [19]. It has to be noted that the association of the HPA-3b homozygosity and ST elevation infarction in our study remained unchanged after adjustment for age and traditional cardiovascular risk factors. There was no association between the HPA-3 genotypes and occurrence of cardiovascular events during follow up. This may be attributed to the small number of cardiovascular events during follow up or may reflect the similar rate of adverse events between STEMI and non-STEMI as also confirmed in our study. A large population study suggested that heritable factors play a major role in determining platelet aggregation [19]; of the overall variance in platelet aggregation, 21% to 30% was due to heritable factors . However, the HPA-1b allele contributed only 0.8% to the overall variation in platelet aggregability to epinephrine and b 0.5% to aggregation to other agonists. Therefore, given the substantial role of the heritable factors, further studies are needed to identify genetic variants regulating platelet function. A previous study indicated that the HPA-3b variant is associated with

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increased platelet aggregation and decreased clot retraction [20], a finding which could explain at least in part the findings of our study. Further studies are required to fully elucidate the significance of HPA-3 polymorphism in the increase of thrombotic risk and its potential role in the pharmacogenetic approach of the treatment of coronary artery disease. 4.1. Study limitations The association of the HPA-3b homozygosity and ST elevation infarction in our study remained unchanged after adjustment for age and traditional cardiovascular risk factors. However, the relatively small number of patients in our study may explain the high odds ratio for HPA-3 b/b, which is higher than the expected for the effect of a single nucleotide polymorphism. Acknowledgement This work was supported by the Greek General Secretariat for Research and Technology (programme EPAN YB/88). References [1] Shah PK. Pathophysiology of coronary thrombosis: role of plaque rupture and erosion. Prog Cardiovasc Dis 2002;44:357–68. [2] Meisel C, Lopez JA, Stangl K. Role of platelet glycoprotein polymorphisms in cardiovascular diseases. Arch Pharmacol 2004;369:38–54. [3] Newman PJ. Alloimune thrombocytopenias. In: Loscalzo JS, Chafer AI, editors. Thrombosis and hemorrhage. Boston: Blackwell Scientific; 1994. p. 529–43. [4] Weiss EJ, Bray PF, Tayback M, et al. A polymorphism of a platelet glycoprotein receptor as an inherited risk factor for coronary thrombosis. N Engl J Med 1996;334:1090–4. [5] Goodall AH, Curzen N, Panesar M, et al. Increased binding of fibrinogen to glycoprotein IIIa-Proline33(HPA-1b, PlA2, Zwb) positive platelets in patients with cardiovascular disease. Eur Heart J 1999;20:742–7. [6] Bottiger C, Kastrati A, Koch W, et al. HPA-1 and HPA-3 polymorphisms of the platelet fibrinogen receptor and coronary artery disease and myocardial infarction. Thromb Haemost 2000;83:559–62. [7] Carter AM, Catto AJ, Bamford JM, Grant PJ. Association of the platelet glycoprotein IIb HPA-3 polymorphism with survival after acute ischemic stroke. Stroke 1999;30:2606–11.

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