Author + information
- Received April 9, 2001
- Revision received July 20, 2001
- Accepted August 15, 2001
- Published online November 15, 2001.
- Khalid Barakat, MD, MRCP*,*,c (, )
- Simon Kennon, MB, MRCP*,
- Graham A Hitman, MD, FRCPc,
- Ebun Aganna, Bscc,
- Christopher P Price, PhD, FRCPathb,
- Peter G Mills, MA, FRCP*,
- Kulasegaram Ranjadayalan, Mphil, MRCP∥,
- Bernard North, PhDd,
- Heather Clarke, PhD, MRCPathb and
- Adam D Timmis, MD, FRCP*
- ↵*Reprint requests and correspondence:
Dr. Khalid Barakat, The London Chest Hospital, Bonner Road, London E2 9JX, United Kingdom
The goal of this study was to determine the interaction between smoking and the glycoprotein IIIa P1A2polymorphism in patients admitted with non–ST-elevation acute coronary syndromes (ACS).
An increased incidence of the P1A2polymorphism in smokers presenting with ST-elevation acute myocardial infarction (AMI) has recently been reported. We, therefore, postulated that, as a consequence of this interaction, fewer smokers with the P1A2polymorphism would present with non–ST-elevation ACS.
We performed a prospective cohort analysis of 220 white Caucasoid patients admitted with non–ST-elevation ACS fulfilling Braunwald class IIIb criteria for unstable angina who were stratified by smoking status.
There were twice as many nonsmokers as smokers. Nonsmokers compared with smokers were older (mean [SD]; 63.9 [11.2] vs. 57.6 [10.3]; p < 0.0001), more likely to have had a previous admission with unstable angina (24.3% vs. 13.2%; p = 0.051) and AMI (45.8% vs. 30.3%; p < 0.026), more likely to have undergone revascularization (24.3% vs. 1.8%; p = 0.028) and were more likely to be on aspirin on admission (60.4% vs. 44.7%; p = 0.026). The proportion of nonsmokers positive for the P1A2polymorphism was equivalent to that expected for this population but was significantly reduced in smokers (28.7% vs. 10%; Pearson chi-square = 9.09, p = 0.0026). In a logistic regression model, the odds ratio (OR) for being positive for the P1A2polymorphism was significantly reduced by smoking (OR [interquartile range]: 0.26 [0.11 to 0.62]; p = 0.0026).
There is a significant reduction in the P1A2polymorphism in smokers admitted with non–ST-elevation ACS compared with nonsmokers, which suggests an interaction between smoking and this polymorphism.
Platelet aggregation with subsequent coronary thrombosis is a key step in the development of acute coronary syndromes (ACS) (1–4). Activation of the platelet surface receptor glycoprotein IIb/IIIa (GP IIb/IIIa), which binds fibrinogen and von Willebrand factor, plays an important role in the development of such thrombus (5). Several point mutations have been described for the genes encoding the GP IIb/IIIa receptor, raising the possibility that they may be implicated in coronary thrombosis (6). One of these (C1565T) results in a substitution of proline for leucine at position 33 of the mature GP IIIa protein, which gives rise to two common alloantigens of GP IIIa (referred to as P1Aor Zw). P1A1is the more common allele, while P1A2is the presumed variant. Following the original study by Weiss (7)describing an association between P1A2and ACS, 23 more studies have been conducted on Caucasoids of North European extraction and have been subjected to a recent meta-analysis (8), which suggested that this polymorphism is not a marker for acute myocardial infarction (AMI). Some of the discrepancies between the studies have been attributed to differences in the risk factor profiles, particularly smoking, between the cases in the various studies (9). Environmental factors such as smoking are known to impinge heavily on risk; indeed, the risk of AMI is greatest in subjects with coagulation disorders who smoke (10). Previously, we have reported that smoking is predictive of ST-elevation in patients admitted with AMI (11). Recently, a case control study of young (<45 years) ST-elevation AMI (STAMI) survivors has reported an association between the P1A2polymorphism and STAMI but only in smokers (12). The interaction was such that smokers with the polymorphism had a 13-fold increased risk of AMI compared with nonsokers without the polymorphism (12). The inference from such an important interaction in STAMI is that this will be balanced by proportionally fewer smokers with the P1A2polymorphism presenting with milder ACS. In the current study we postulated that, after plaque rupture, the presence of smoking and the P1A2polymorphism would produce an exaggerated thrombotic response favoring STAMI such that there would be a decrease in the proportion of smokers with the P1A2polymorphism presenting with non–ST-elevation ACS. In order to investigate this possibility, we have prospectively analyzed an unselected cohort of patients admitted with non–ST-elevation ACS and compared the distribution of P1A2polymorphism in smokers with nonsmokers.
Consecutive white Caucasoid patients admitted with non–ST-elevation ACS were recruited if they fulfilled the criteria for Braunwald class 3B unstable angina (13). Electrocardiographic changes (ST depression, T-wave inversion) were not required for inclusion, but patients who developed Q waves were excluded as were patients with a myocardial infarction in the previous 21 days. Other exclusion criteria included percutaneous coronary intervention in the previous six months and cardiac failure (New York Heart Association grade 3 or 4). The study was approved by the local ethics committee (East London and City Health Authority).
Baseline characteristics including demographic, clinical, smoking status and biochemical data as well as details of the presenting electrocardiogram (ECG) were collected prospectively and stored electronically. Medication being taken before admission was documented. Current smokers were classed as smokers, while ex-smokers and nonsmokers were grouped as nonsmokers. Ex-smokers were defined as patients who had stopped smoking at least one month before the index admission.
Blood sampling and biochemical analysis
In addition to samples taken for routine laboratory analysis according to hospital protocols, samples were also taken on admission (before antithrombotic therapy) for troponin I (Tn I) assay and GP IIIa genotyping and at 12, 24 and 48 h after admission for Tn I assays only. Whole blood and, after separation, serum samples were stored at −80°C for later analysis in batches. Troponin I concentrations were measured using a one-step sandwich immunoassay with magnetic separation (Bayer Immuno 1 Analyser: Bayer Plc., Newbury, United Kingdom). The minimum detection limit was 0.1 μg/l. The coefficient of variation was 3.2% at 2.5 μg/l and 10% at 0.3 μg/l (manufacturers datasheet). As recommended by the manufacturers, the cutoff point used was 0.1 μg/l.
Determination of GP IIIa genotypes
Genomic DNA wa extracted from 200 μl of whole blood with QIAmp (96) blood kit (QIAGEN, Crawley, United Kingdom). Glycoprotein IIIa genotyping was determined as previously described. Briefly, a 266 base pair (bp) fragment in exon 2 was amplified using previously described oligonucleotide primers (7), with subsequent restriction digest of the polymerase chain reaction product with Msp 1. The C1565T substitution results in an extra restriction site with Msp 1 such that Msp 1 restriction digest of P1A2individuals yields three fragments (171 bp, 50 bp and 45 bp) compared with two fragments for P1A1individuals (221 bp and 45 bp). These fragments were then separated on 2.5% agarose gels.
Results for continuous variables are presented as means and SDs. Two sample ttests were used to compare smokers with nonsmokers. Variables not normally distributed are presented as medians and interquartile ranges and compared using the Mann-Whitney Utest. Variables clinically or statistically significant (p < 0.05) on univariate analysis were considered for entry into a multiple logistic regression model. We then looked at the effect of adding variables insignificant on univariate analysis to test if any of these became important after adjustment. Results from the logistic regression were expressed as the odds of an individual having one or more copies of the P1A2allele to a reference category for categorical variables and as the relative odds associated with a 1 SD increase in continuous variables. Pearson chi-square test was used to compare GP IIIa genotype in smokers and nonsmokers.
Baseline characteristics according to smoking status
A total of 220 white Caucasoid patients formed the study group. There were twice as many nonsmokers as smokers (Table 1). Smokers tended to be younger, but there were no differences with regard to gender nor in other recognized clinical risk factors for coronary disease, namely, hypertension, diabetes and family history. Nonsmokers were more likely to have had a previous ACS and prior revascularization. Although more nonsmokers were taking aspirin on admission, there were no significant differences in other antianginal therapies. Nonsmokers had higher serum creatinine levels on admission with a tendency to having higher cholesterol. No significant differences were noted in the serum glucose on admission. Troponin I was elevated in similar proportions in smokers and nonsmokers.
Genotypic distributions and allelic frequencies of P1A1/A2polymorphism among smokers and nonsmokers
Examining the study group as a whole, the P1A1/A2allelic frequencies were similar to those expected for this population (Table 2). Examining the group stratified by smoking status revealed that the frequency of the P1A2allele in nonsmokers was very similar to that expected for North European Caucasoids. However, a marked reduction in the proportion of smokers positive for this allele was noted (28.7% vs. 10%; p = 0.0026).
Multivariate predictors of subjects positive for the P1A2polymorphism
Logistic regression analysis showed that smoking reduced the odds of subjects positive for the P1A2polymorphism by 74% (p = 0.0026) (Table 3). No other factors including previous cardiac history and treatment differences predicted P1A2carriage.
Interaction between smoking and the P1A2polymorphism
This study has shown that the frequency of the P1A2allele is reduced in smokers as compared with nonsmokers in consecutive white Caucasoid patients admitted with non–ST-elevation ACS, an observation that has not previously been reported. Thus, in smokers the odds of carrying one or more copies of the P1A2allele was reduced by 74%. The allelic frequency of this polymorphism in nonsmokers and for the group overall was equivalent to the percentage predicted for Caucasoids of North European extraction from a recent meta-analysis of 5,799 controls (8). These results support an important interaction between smoking and the P1A2polymorphism in ACS.
Determinants of ST elevation
ST-elevation is a marker of coronary occlusion (14)and is predicted by smoking (11). The current concept that STAMI and non–ST-elevation ACS differ in the degree of coronary occlusion that they produce is now being challenged (15). Recent evidence would suggest that the spectrum of syndromes that make up ACS with non–ST-elevation ACS at the milder end of the spectrum and STAMI and death at the other reflect the stability of thrombus after plaque rupture (15). Fibrin-rich thrombus is the hallmark of STAMI, which is slow to lyse through endogenous fibrinolytic mechanisms and, thus, presents as AMI. Although fibrin produces a stable clot, it is most amenable to thrombolytic therapy, resulting in the excellent response to fibrinolytic therapy (16). In this paradigm of ACS, the factors determining stability of thrombus at the time of plaque rupture will be the “hemostatic milieu” to which the contents of the plaque are exposed. This “milieu” is itself a mulifactorial phenomenon, which in the simplest model is due to the interaction between environmental and genetic factors. Smokers have a heightened thrombotic response and so are more prone to STAMI. Paradoxically, therefore, proportionally fewer smokers will present with non–ST-elevation ACS as we have previously demonstrated (17).
If ACS are truly a spectrum determined by the stability and perhaps extent of clot then any risk factors (environmental or genetic) accumulating at one end of the spectrum should be balanced by a similar reduction in those same factors at the opposing end of the spectrum. This seems to hold true for smoking, and it would also seem to hold true for the interaction between smoking and the P1A2polymorphism. The results of Ardissino et al. (12), which found an association between AMI and the P1A2polymorphism in young smokers who had survived STAMI, support our hypothesis and the results of this study. In the aforementioned study, smokers with AMI were 50% more likely to have the polymorphism as are nonsmokers with AMI. Our results indicate that smokers admitted with non–ST-elevation ACS have a 74% reduction in the odds of carrying one or more copies of this allele. Combining the results of these two studies, there is a shortfall of smokers with the polymorphism, which is presumably accounted for by a proportion of these patients presenting with sudden death. This is supported by a Finnish autopsy series that has reported an excess of the P1A2polymorphism in sudden death victims who died of AMI but only if there was evidence of coronary thrombus (18). They also reported an association of the P1A2polymorphism with more vulnerable plaques but with less severely stenosed coronary arteries. As many as 75% of sudden death victims may have no evidence of coronary thrombosis, and, in a proportion of these patients, death may be secondary to AMI caused by progressive atherosclerotic narrowing of the culprit vessel (19,20). In essence, the pathophysiology of AMI may fall into two distinct bands: those secondary to thrombus on a vulnerable plaque and those secondary to progressive arterial narrowing. Furthermore, smoking favors the development of vulnerable plaques and coronary thrombosis with subsequent AMI (21,22).
The inconsistencies of the many studies that have tested for associations between the P1A2polymorphism and AMI have been attributed to differences in case and control selections (8). As described in the preceding text, it can be seen that any association studies testing for associations between thrombotic genetic markers and coronary syndromes need well-defined study groups, stratified not only on the basis of risk factors but also on the basis of the underlying pathophysiology of specific ACS. At present it is only crudely possible with the use of the 12 lead ECG, creatinine kinase and troponin levels to subdivide ACS into three increasingly more severe categories, namely: troponin negative non–ST-elevation ACS, troponin positive non–ST-elevation ACS and STAMI. The role of environmental and genetic factors, although overlapping in these three categories, may influence the development of these syndromes to different degrees. Thus, an important interaction in any one of these categories may be overlooked if all categories are grouped as one. As an example, in the Physicians Health study (9), which reported no association between P1A2and AMI, the prevalence of smoking was very low (15%) in this presumably health-conscious group. Our findings, together with those of Ardissino et al. (12), would suggest that this negative result is entirely consistent with this group of very few smokers. Furthermore, the low frequency of the P1A2polymorphism (21%) for our cohort as a whole (smokers and nonsmokers) (Table 2)once again reinforces the need for accurate phenotyping in the definition of AMI if the subtle molecular, genetic and pathophysiologic differences in the spectrum of ACS are to be resolved.
This is an observational study, and the influence of the differences between smokers and nonsmokers with regard to age and aspirin on admission as potential confounding variables should be kept in mind. Nevertheless, the interaction between smoking and the P1A2polymorphism remains highly significant despite introducing age and aspirin into the multiple logistic regression model and suggests that our results are strong.
We have found a lower than expected P1A2allelic frequency in smokers admitted with non–ST-elevation ACS. This suggests an important interaction between smoking and the P1A2polymorphism in patients admitted with ACS.
☆ Dr. Barakat is supported by an MRC Clinical Training Fellowship. Biochemical assays were funded by Bayer Pharmaceutical, Plc., Newbury, United Kingdom.
- acute coronary syndromes
- acute myocardial infarction
- ST-elevation acute myocardial infarction
- Tn I
- troponin I
- Received April 9, 2001.
- Revision received July 20, 2001.
- Accepted August 15, 2001.
- American College of Cardiology
- Kennon S,
- Barakat K,
- Suliman A,
- et al.
- Ardissino D,
- Mannucci P.M,
- Merlini P.A,
- et al.
- A report of the American College of Cardiology and American Heart Association task force on practice guidelines (committee on the management of patients with unstable angina)
- Rentrop K.P
- Kennon S,
- Suliman A,
- MacCallum P.K,
- et al.
- Mikkelson J,
- Perola M,
- Laippala P,
- et al.
- Leach I.H,
- Blundell J.W,
- Rowley J.M,
- et al.
- Feng D,
- Lindpainter K,
- Larson M.G,
- et al.