SHORT REPORT
Platelet glycoprotein Ilb polymorphism, traditional risk factors
and non-fatal myocardial infarction in young women
ALEXANDER P. REINER,] .2 3 STEPHEN M. SCHWARTZ,2 3 4
PRASANNA N. KUMAR,' FRITS R. ROSENDAAL/ RACHEL M. PEARCE,5 KAHLEN M. A R A M A K I , ' BRUCE M. PSATY' 2Λ AND DAVID S. SiscoviCK1'2'3 ]Department of Medicine, 2 Department of Epidemiology and 3 Cardiovascular Health Research Unit, University of Washington,
4Fred Hutchinson Cancer Research Center, Seattle, WA, USA, ' Department ofPathology, PSG Institute of Medical Sciences and Research, Tamilnadu, India, and 6 The Haemostasis and Thrombosis Research Centre and Department of Clinical Epidemiology, University Hospital Leiden, The Netherlands
Received 20 September 2000; accepted for publication 27 October 2000
Summary. Several platelet glycoprotein polymorphisms have been associated with an increased risk of myocardial infarction (MI) in studies that included predominantly men. In a population-based sample of 68 Caucasian women <45years old with non-fatal MI and 346 demographically similar control subjects, we found an increased risk of MI among women who possessed at least one copy of the glycoprotein Ilb Ser843 allele compared with those lacking the Ser843 allele (odds ratio 1-85; 95% confldence interval = 1-03-3-33). The increased risk was
present only in subgroups of women who smoked cigarettes. had hypercholesterolaemia or who had a family history of early onset MI. The Ser843 vanant of glycoprotein Ilb may be associated with an increased risk of MI in young women with other Cardiovascular risk factors. Additional studies involving larger numbers of subjects are needed to confirm this preliminary finding.
Keywords: myocardial infarction, glycoprotein Ilb, platelet, polymorphism.
Glycoprotein Ilb/IIIa is the major platelet receptor for fibrinogen and plays an important role in platelet-mediated thrombosis and the development of coronary heart disease. A number of studies have examined the role of the PLA2 variant of glycoprotein lila (Leu/Pro53) in the occurrence of myocardial infarction (MI), but the association remains controversial. A second common genetic glycoprotein Ilb/ lila variant, glycoprotein Ilb Ile/Ser843, has not been associated with acute coronary thrombotic events in several studies involving predominantly middle-aged to older men (Hato et al, 1997; Böttiger et al 1999, 2000). We previously
demonstrated the importance of the interactions of two inherited prothromboüc mutations, factor VArgSOGGIn and prothrombin G20210A, with other known Cardiovascular risk factors in women under the age of 45 years with MI (Rosendaal et al, 1997). In this report, we assessed the association of the two common platelet glycoprotein Ilb/IIIa polymorphisms, along with three other common platelet glycoprotein variants (glycoprotein Iba Thr/Met145,
glyco-protein la Glu/Lys50'' and glycoprotein la C807T), in young
Correspondence: Alexander P. Reiner, M.D., Harborview Medicai Center, 325 9th Avenue, Box 359756, Seattle, WA 98104, USA. E-mail: apremer@u.washington.edu
632
women with MI, and examined potential interactions with traditional Cardiovascular risk factors.
PATIENTS AND METHODS
Detailed methods of this population-based case-control study of acute MI in young women are described elsewhere (Rosendaal et al, 1997). Bnefly, the MI patients included women aged 18-44 years, residing in Western Washington State, who suffered a non-fatal acute MI between 1991 and
1995. Controls were women 18-44 years of age without a history of Cardiovascular disease from the same geographi-cal area frequency, matched to the age distribution oi tht cases.
Current smokers were defined äs subjects who reportccl smoking at least five cigarettes per week for at leasl si\ consecutive months. Subjects who were former smokers .is well äs those who had never or seldom smoked cigarettcs were classified together äs 'non-smokers'. Obesity ^vas defined äs a body mass index (BMI) > 30-0 kg/nr. A woman was classified äs hypertensive or diabetic il she reported ever receiving the diagnosis by a physician. Hypercholesterolaemia was defined äs a cholesterol measure-ment > 5-2 mmol/1. Family history of early onset MI was
M* ιPlatelct glycoprotein allele frequencies and genotypes in myocardial infarction cases and controls.
actorsj
ι.
6 artmein l, 'dical •lent ofJ } f t t — . igaretle·* 1 üstorv o, -j * ' p b may hi womeii | ' Studie* l Allele frequency .· -imitrphism ,1, protcin llb ,-vr"4 1 ^•prolem UJa i { f U l Γ" iicprotem Ia , ^()T ,'α ΙΛΊ „ui'prolein la ,νοΓΓ '<!H'"Pr"te)n Iba Π r Met1"" Allele Iles4? Ser813 Leu" Pro" Glu505 Lys50? 807C 807T Thr145 Met145 Cases 0-551 0449 0-824 0-176 0-897 0-103 0-559 0-441 0-949 0-051 Controls 0-630 0-370 0-838 0-162 0-909 0-091 0-627 0-373 0-916 0-084 Genotype IIe843/IIe843 Ile843/Ser84i Ser843/Ser843 Leu "/Leu" Leu "/Pro" Pro"/Pro" Glu5"5/«^05 GIus05/Lys505 Lysro5/Lys505 807(C/C) 807(C/T) 807(T/T) Thr145/Thr145 Thr'45/Met14S Met'45/Met145 Genotype Cases (n = Number 18 39 1] 46 20 2 56 10 2 22 32 14 61 7 0 frequency = 68) (%) (26-5) (57-3) (16-2) (67-6) (29-4) (2-9) (82-4) (147) (2-9) (32-4) (47-0) (20-6) (89-7) (10-3) (0-0) Controls (n Number 140 156 50 241 98 7 286 57 3 135 164 47 290 54 2 = 346) (%) (40-5) (45 1) (14-4) (69-7) (28-3) (2-0) (82-7) (16-4) (0-9) (39-0) (47-4) (13-6) (83-8) (15-6) (0-6) OR' (CI95) ] 85 (1 03-3 33) 1-14 (0-65-2-01) 1-06 (0-53-2-12) 1-26 (0-72-2-20) 0 58 (0-25-1-33) "Odds ratlos were calculated for individuals who are either homozygous or heterozygous for the less common allele compared with individuals »hu are homozygous for the more common allele.fiR. nHds ratio: (7Γ95. 95% confidenc:e interval.
olatelel
s Willi
defined äs having at least one first-degree relative who
jullered an acute MI before the age of 55 years. Genotype analysis for five common platelet glycoprotein morphisms (included in Table I) was performed by poly-merase chain reaction (PCR) amplification of genomic DNA, followed by restriction enzyme digestion (PCR-RFLP) (Reiner
Λ αϊ, 2000).
The association of platelet glycoprotein genotypes with MI was exammed using unconditional logistic regression adjusted for age, and expressed äs odds ratios (OR) and
95% confidence intervals (CI95). We assessed the extent to »"hich associations with glycoprotein polymorphisms were modified by other cardiovascular risk factors through flratified analyses. We then used logistic regression models to estimate the OR for persons possessing both the genotype of interest and the risk factor, persons possessing only Ihe genotype of interest but not the risk factor, and persons Possessing only the risk factor but not the genotype, for each combination of platelet glycoprotein genotype and cardio-vascular risk factor, all relative to persons lacking both the genotype and risk factor. We measured the strength of «le effect modiflcation by calculating the relative excess risk due to interaction (RERI), which takes on the null value of
Π when there is no difference in the risk of MI associated *lth the genotype according to the level of the cardio-v«scular risk factor. The statistical precision of the ""agnitude of interaction was assessed by calculating 95% »nfidence intervals on the RERI (Hosmer & Lemeshow,
1992).
RESULTS
Of the initial 112 MI patients and 525 control women that were interviewed for the case-control study, DNA samples for platelet glycoprotein genotyping were available for 68 Caucasian MI patients and 346 Caucasian controls. The 68 MI patients had a mean age of 39-7 years (ränge
23-44 years) and the 346 control women had a mean age of 37-8 years (ränge 19-44 years). The majority of both MI cases (90%) and control subjects (96%) were premeno-pausal. Only 4% of MI cases and 11% of control subjects were current users of oral contraceptives. Traditional cardiovascular risk factors were more common in MI patients than in controls: current smoking (71% vs. 21%), obesity (44% vs. 16%), hypercholesterolaemia (75% vs. 36%), hypertension (35% vs. 9%) and diabetes (15% vs. 3%). There were no significant differences in demographic characteristics or cardiovascular risk factors between the women who had DNA samples available for analysis and those who did not (data not shown).
The distribution of glycoprotein genotypes among the controls subjects was in Hardy-Weinberg equilibrium. As shown in Table I, MI cases (73-5%) were more likely to carry at least one copy of the less common Ser84 ? allele of
glycoprotein llb than control subjects (59-5%), although the frequencies of Ser843 homozygotes were similar (16-2% vs.
14-5%). There was a significantly increased risk of MI for women who carried the glycoprotein llb Ser's43
allele (Ser843/Ser84i or IIe84VSer843) compared with the
* 2001 Blackwell Science Ltd, Bntish Journal of Haematology 112: 632-636
Table IIA. Risk of myocardial infarction among young women associated with the GPIIb Sers4 3 allele, by cardiovascular risk factors.
OR, odds ratio; CI95, 95% confidence interval; RERI, relative excess risk due to interaction. Ile843/Ile843 genotype (OR 1-85; CI95 = 1-03-3-33).
The OR comparing the Ile845/Ser843 genotype with the
Ile845/Ile843 genotype was 1-90 (CI95 = 1-03-3-50).
The OR comparing the Ser843/Ser843 genotype with the
Ile84i/Ile's43 genotype was 1-69 (CI95 = 0-74-3-85).
There was no association between glycoprotein Ilb genotype and smoking, obesity, hypertension, diabetes or hyper-cholesterolaemia among either cases or controls (data not shown). Of the four other platelet glycoprotein poly-morphisms analysed, the genotype distributions were similar among cases and controls (Table I).
In analyses stratified according to the presence or absence of other cardiovascular risk factors, the glycoprotein Ilb Sers4i allele was associated with a ^three- to fourfold
increased risk of MI in the subgroup of women who smoked cigarettes, who had a serum cholesterol level > 5-2 mmol/l and who had a family history of early onset MI in first-degree relatives (Table IIA). In contrast, the glycoprotein
Ser843 allele was not associated with increased risk of MI
in women who were non-smokers, who had a serum cholesterol level s 5-2 mmol/l and who had a negative family history. No significant effect modifications were observed when the risk of MI associated with the glycoprotein Ilb Ser843 allele was stratified according to
obesity, hypertension, diabetes, or the presence or absence of the factor V Leiden and prothrombin G20210A mutations (data not shown). 4
Analysis of the separate and combined effects of thf glycoprotein Ilb Ser843 allele with smoking (Table IIB l
indicates that smoking by itself was associated with a threefold increased risk of MI compared with non-smokers without the glycoprotein Ilb Ser843 allele, while <hf
glycoprotein Ilb Ser843 allele was not associated with λδ
in the absence of smoking. However, the combination « smoking and the glycoprotein Eb Ser843 allele was
associated with a 10-fold increase in risk compared «fttn 2001 Blackwell Science Ltd, Britisfi Journal of Haematology 112: 632-»'
Smoking Current non-smokers Current smokers Cholesterol level £ 5-2 mmol/l > 5-2 mmol/l
Family history of early MI
No Yes
Table IIB. Glycoprotein Ilb Glycoprotein Ilb genotype Ser's4 '-negative Ser84 '-positive Ser84 '-negative Ser84 '-positive Ser84 '-negative Ser84 '-positive Ser84 '-negative Ser84 '-positive Ser84 '-negative Ser84 '-positive Ser843-negative Ser84 '-positive Patients (n = 68) Ser84'+ Ser84 '-Number (%) '-Number (%) 10 (50-0) 10 (50-0) 40(83-3) 8(16-7) 10(58-8) 7(41-2) 40(78-4) 11(21-6) 29(69-0) 13(31-0) 19 (79-2) 5 (20-8)
Ser843 allele and other cardiovascular
Risk factor Smoking Not current Not current Current Current Cholesterol < 5-2 mmol/l s 5-2 mmol/l > 5-2 mmol/l > 5-2 mmol/l Family History of MI No No Yes Yes Controls (n = Ser84' + Number (%) 162 (59-1) 44 (61-1) 133 (61-0) 69 (57-0) 182 (61-9) 20 (44-4)
risk factors: separate
OR 1 0-70 3-39 9-86 1 0-91 2-43 6-33 1 1-37 1-78 7-95 346) Ser84 '-Number (%) 112 (40-9) 28 (38-9) 85 (39-0) 52 (43-0) 112 (38-1) 25 (55-6)
women who did not smoke and did not carry the
glycoprotein Ilb Ser843 allele; the effect of smoking on
the association of the glycoprotein Ilb allele with non-fatal MI was greater than predicted by the addition of the risks associated with smoking alone and the
glyco-prctem Hb Ser843 allele alone (RERI = 6-77, CI95 = 0-43,
13-11). Qualitatively similar but less pronounced effect modiflcations were observed for the combinations of
the glycoprotein üb Ser843 allele and plasma cholesterol
or family history of MI (Table IIB). The results of this analysis were altared only slightly by additional adjustment for the confounding variables of smoking and hypercholesterolaemia.
DISCUSSION
Our results suggest a possible association between the
Ser843 variant of platelet glycoprotein Ilb and the risk of MI
in young women. The increased risk of MI associated with
glycoprotein Ilb Ser843 was only present among women who
smoked cigarettes, had elevated serum cholesterol levels or had a positive family history of MI, and the interaction with smoking appeared strong. Women who possessed the
glycoprotein Ilb Ser843 allele and who were current smokers
had an «lO-fold increased risk of MI, whereas there was no
increased risk associated with the glycoprotein Ilb Ser843
allele in women who were non-smokers.
A recent in vitro study indicated that the glycoprotein Ilb
Ser843 variant is associated with both increased platelet
aggregation and decreased clot retraction compared with
thelle843 variant (Bray et cd, 1999). Whether the Ile/Ser843
Substitution is causally responsible for these effects or whether the dimorphism is genetically linked to another causative locus in the glycoprotein Ilb gene or elsewhere remains to be determined. Further functional
charac-terization of the glycoprotein üb Ile/Ser843 polymorphism
may contribute to our understanding of the role of the platelet glycoprotein Ilb/IIIa receptor in the patho-genesis of arterial thrombosis and potentially allow risk stratification of MI patients (e.g. the identification of individuals particularly likely to benefit from glycoprotein üb/IIIa inhibitor therapy). Cigarette smoking is associated with increased glycoprotein Ilb/IIIa receptor activation (Markovitz et cd, 1999), äs well äs with increased circulating levels of fibrinogen (Folsom, 1999), the ligand that binds to glycoprotein Ilb/IIIa. These findings provide possible mechanisms underlying the synergism we observed between the genetic variant of platelet glycoprotein Ilb and smoking in the pathogenesis of acute MI.
In contrast to our results, several larger studies involving predominantly male patients of a wider age ränge have not
observed an association between the Ser843 variant of
glycoprotein Ilb and the risk of MI (Hato et al, 1997; Böttiger «t a/, 2000) or coronary stent occlusion (Böttiger et al, 2000). However, the genetic influence on risk of MI may be stronger in younger women than in older women or fflen (Marenberg et al, 1994). Furthermore, premenopausal women exhibit increased platelet glycoprotein Ilb/IIIa activation in response to platelet agonists compared with
young men (Faraday et al, 1997). Of note, we recently
observed an association between glycoprotein Ilb Ser843
homozygosity and ischaemic stroke in young women with other cardiovascular risk factors (Reiner et al, 2000). In contrast, Carter et al (1999) found no relationship between the glycoprotein Ilb polymorphism and the occurrence of ischaemic stroke in elderly men and women, but did
report an association between the Ser84i variant and a
decreased risk of stroke-related death.
The major limitation of our study is the relatively small number of cases (MI is a rare event in women < 45 years of age), which may increase the likelihood of a false-positive association. The performance of multiple subgroup analyses may also increase the probability that an observed association may have arisen by chance. Furthermore, while our results do not support an association between several other platelet glycoprotein polymorphisms and MI in young women, our sample size may not have sufficient power to detect a small effect, particularly for the less frequent polymorphisms. Thus, analysis of a larger number of patients will be required to exclude these other platelet glycoprotein polymorphisms äs risk factors for MI in young women. In addition, äs our study included only women who survived an acute MI, it is possible that an association may have been missed if a particular genotype is also associated with early post-MI mortality. The lack of an increased risk of MI in women who were homozygous for
the glycoprotein Ilb Ser843 variant in our study may suggest
the possibility of a spurious association. Alternatively, the
lack of association in Ser843 homozygous women could be
äs a result of either the relatively small number of cases or
an over-representation of the Ser843/Ser843 genotype among
the fatal MI cases. Thus, confirmation of our preliminary association between the glycoprotein Ilb polymorphism and the risk of MI in young women will require further study involving a larger number of subjects, äs well äs patients who suffer fatal MI.
ACKNOWLEDGMENTS
We thank Fran Chard, Karen Graham, Carol Handley-Dahl, Judy Kaiser, Marlene Bengeult, Carol Ostergard, Denize Koriander, Barb Twaddell, Sandy Tronsdal and Jill Ashman for assistance with the study, Benjamin Siscovick for providing technical assistance and Ida McCormick for assistance in preparation of the manuscript.
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