Dimorphism in the P2Y1 ADP Receptor Gene Is Associated With Increased Platelet Activation Response to ADP
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《动脉硬化血栓血管生物学》
From the Department of Cardiovascular Sciences, University of Leicester, United Kingdom.
Correspondence to Professor N.J. Samani, Department of Cardiovascular Sciences, University of Leicester, Clinical Sciences Wing, Glenfield Hospital, Leicester, LE3 9QP, UK. E-mail njs@le.ac.uk
Abstract
Objective— The platelet ADP receptors P2Y1 and P2Y12 play a pivotal role in platelet aggregation. There is marked interindividual variation in platelet response to ADP. We studied whether genetic variants in the P2Y1 or P2Y12 genes affect platelet response to ADP.
Methods and Results— The P2Y1 and P2Y12 genes were screened for polymorphisms. Associations between selected polymorphisms and the platelet response to ADP (0.1, 1.0, and 10 μmol/L), assessed by whole blood flow cytometric measurement of fibrinogen binding to activated glycoprotein IIb-IIIa, were then determined in 200 subjects. Five polymorphisms were found in the P2Y1 gene and 11 in the P2Y12 gene. All polymorphisms were silent. A P2Y1 gene dimorphism, 1622AG, was associated with a significant (P=0.007) effect on platelet ADP response, with a greater response in carriers of the G allele (frequency 0.15). The effect was seen at all concentrations of ADP but greatest at 0.1 μmol/L ADP, where the response in GG homozygotes was on average 130% higher than that seen in AA homozygotes (P=0.006).
Conclusions— A common genetic variant at the P2Y1 locus is associated with platelet reactivity to ADP. This genotype effect partly explains the interindividual variation in platelet response to ADP and may have clinical implications with regard to thrombotic risk.
ADP is an important mediator of platelet aggregation. In this study, we show that a polymorphism (1622AG) in the P2Y1 ADP receptor gene is associated with a significant effect on platelet ADP response. This genotype effect explains some of the interindividual variability in platelet reactivity and may influence thrombotic risk.
Key Words: platelets ? thrombosis ? genes ? receptors ? adenosine diphosphate
Introduction
Platelet activation and thrombus formation play an integral role in the hemostatic mechanism after vascular injury. After disruption of atherosclerotic plaques, platelet aggregation also plays an important role in development of myocardial infarction and other acute coronary syndromes.1 Binding of platelets to von Willebrand factor and collagen is the initiating event in platelet activation. This leads to platelet degranulation and release of ADP. ADP, acting via specific receptors, causes further platelet activation and platelet aggregation. Therefore, ADP plays a key direct role in platelet activation. ADP-mediated activation also partly explains the platelet response to other agonists.2
Two platelet ADP receptors, P2Y1 and P2Y12, have been shown to initiate platelet activation when stimulated in concert.3 Both are heterotrimeric G-protein-coupled receptors: P2Y1 to Gq and P2Y12 to Gi. Stimulation at P2Y1 leads to intracellular calcium mobilization and platelet shape change,4 whereas stimulation at P2Y12 leads to inhibition of adenylyl cyclase5 and activation of phosphoinositide-3 kinase.6 The end effect is affinity modulation of the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor for fibrinogen, resulting in fibrinogen binding and platelet aggregation.7 It is well recognized that there is substantial interindividual variation in platelet response to ADP.8,9 The reasons for the interindividual variation are poorly defined, but the observation that the response is stable over time, in a given individual,9 raises the possibility that at least in part, it may be genetically controlled because of variation in the P2Y1 and/or P2Y12 genes.
P2Y1 and P2Y12 genes are located on chromosome 3. The P2Y1 gene spans 4 kb10 and is made up of a single exon of 3122 base pairs encoding a 372-aa protein.11 The P2Y12 gene spans 47 kb and is made up of 3 exons and 2 introns.12 There are 2 reference sequence mRNA variants listed in the National Center for Biotechnology Information (NCBI) Sequence Database for P2Y12, which differ in their 5'-untranslated region (UTR), long (1502 bp) and short (1474 bp).12 Hollopeter et al13 found platelets to express the shorter transcript variant, consisting of sequence from exons 2 and 3. The entire coding sequence for the 342-aa receptor is found in exon 3.13
Rare mutations within the P2Y12 gene have been shown to disrupt receptor function and lead to a bleeding diatheses.13,14 Whether there are more common variants in the 2 genes, which influence interindividual variation in platelet reactivity to ADP, has only recently come under investigation.15 The objectives of this study were to identify common P2Y1 and P2Y12 gene polymorphisms and investigate their possible functional effects on platelet reactivity.
Methods
Subjects and Sample Collection
A total of 200 white adult subjects of northern European origin were recruited. None had a history of coronary heart disease and none were taking any antiplatelet medication. Subjects were categorized as experiencing hypertension and diabetes on the basis of reported history. Smoking status was defined as current smoker, ex-smoker, or nonsmoker. The study was approved by the Leicestershire Research Ethics Committee, and all subjects gave written informed consent.
Individuals were seen in the morning and in a fasting state. Ten subjects attended on a second occasion, at least 6 weeks later, to permit analysis of intraindividual variability of the platelet ADP response. Smokers were asked to abstain from smoking for at least 24 hours before the visit, and this was confirmed by a carbon monoxide breath test. All subjects were rested supine for at least 20 minutes before venepuncture to minimize the effects of stress hormones, and blood was collected using a standardized phlebotomy technique designed to minimize platelet activation.16
Platelet Response Measurement
Blood samples were prepared for whole blood flow cytometric analysis within 10 minutes of collection essentially as described previously16,17 (see detailed Methods, available online at http://atvb.ahajournals.org).
White cell count, platelet count, and mean platelet volume (MPV) were measured in EDTA-anticoagulated blood after 2 hours to allow stabilization of platelet volume using a Beckman Coulter counter. Fibrinogen was measured by the Clauss method on a Sysmex CA-1000 analyzer (Sysmex UK). Leukocyte DNA was extracted from a 10-mL blood sample using the PureGene DNA extraction kit (Gentra Systems).
P2Y1 and P2Y12 Gene Screening for Common Polymorphisms
On the basis of the response after stimulation of whole blood with 1.0 μmol/L ADP, 10 subjects from each end of the distribution of response were selected to undergo sequencing of the P2Y1 gene, including the exon and 500 bp of upstream sequence, and the region of the P2Y12 gene encompassing exons 2 and 3 and intron B. This strategy maximized the chances of identifying common polymorphisms that might affect function (further details of Methods available in online supplement).
Genotyping of Common P2Y1 and P2Y12 Polymorphisms
Polymorphisms to type in the full cohort were selected on the basis of their predicted allele frequencies and linkage disequilibrium data as described in Results. Four polymorphisms were typed in the P2Y12 gene and 1 in the P2Y1 gene (details of genotyping Methods available in online supplement).
Statistical Analysis
Unless stated otherwise, data are presented as mean±SD. Observed allele frequencies were compared with the Hardy-Weinberg equilibrium prediction using the 2 test. All response data were transformed before analysis using log (response [100-response]–1). The association between genotype and phenotype was tested by 1-way ANOVA. Associations at specific doses were further analyzed after adjustment for other parameters (age, resting level of bound fibrinogen, smoking status, and GPIIb-IIIa receptor expression) using multiple linear regression models. Analysis across all doses was performed using a random-effects model fitted by maximum likelihood that included a random effect for subject to adjust for within subject correlation. All analyses were performed using Stata 8.1 (Stata Corp.).
Results
Cohort Demographics
Mean age of the subjects was 47.3±6.0 years (range 25 to 56 years). A total of 87% were male, 18% were current smokers, and 32% were ex-smokers. Nine percent had a diagnosis of hypertension, and 1% were diabetic. Mean white cell count was 5.5±1.3x109 L, mean platelet count was 240.3±61.1x109 L, MPV was 9.2±1.2 fL, and mean plasma fibrinogen was 2.8±0.6 g/L. All of these variables were distributed normally.
Variability of Platelet Response to ADP
The distribution of subjects with respect to the percentage of platelets positive for bound fibrinogen at rest and after stimulation with the 3 doses of ADP are shown in Figure 1. Mean values (95% CIs) were as follows: resting level 2.6% (2.5 to 2.7%); 0.1 μmol/L ADP 13.5% (11.9 to 15.3%); 1.0 μmol/L ADP 69.9% (67.0 to 72.3%); and 10 μmol/L ADP 85.4% (83.9 to 86.9%). There were strong correlations between the different stimulating concentrations of ADP within subjects (0.1 versus 1.0 μmol/L, r=0.91; 0.1 versus 10 μmol/L, r=0.86; 1.0 versus 10 μmol/L, r=0.94) and a modest correlation between the resting level of bound fibrinogen to platelets and response to all doses of ADP (r0.38). In the 10 subjects studied on 2 occasions, a correlation between the ADP responses measured at the 2 visits was observed when comparing the response to all doses of ADP (0.1 μmol/L, r=0.56; 1.0 μmol/L, r=0.91; 10 μmol/L, r=0.93), indicating that the response to ADP is reproducible within an individual.
Figure 1. Percentage of platelets with fibrinogen bound to GPIIb-IIIa measured by whole blood flow cytometry in unstimulated (resting) samples and in samples stimulated with ADP at 0.1, 1.0, and 10 μmol/L. Data are shown for 200 subjects with medians.
Random effects analysis incorporating all 3 doses of ADP found that after adjusting for the resting level of bound fibrinogen, age (P<0.0001), smoking history (P=0.008), and GPIIb-IIIa expression (P=0.0005) were important independent regulators of the platelet response. GPIIb-IIIa expression showed 3-fold variation within the population studied (1.7 to 5.4 relative fluorescence units) and was distributed normally. The correlations between the level of GPIIb-IIIa expression and fibrinogen binding in response to ADP were 0.169 (P=0.016), 0.220 (P=0.002), and 0.266 (P<0.001) for the 0.1 μmol/L, 1.0 μmol/L, and 10 μmol/L of ADP, respectively. GPIIb-IIIa expression accounted for between 2.9% and 7.1% of the interindividual variation in response to ADP, dependent on stimulating concentration of ADP used. The proportions explained by age and smoking were 4.2% to 10.7% and 1.1% to 2.2%, respectively. Gender did not have an independent effect.
Identification of Common P2Y1 and P2Y12 Polymorphisms
The polymorphisms identified are shown in supplemental Table II (available online at http://atvb.ahajournals.org). All polymorphisms in the coding sequences of both genes were silent. In the P2Y1 gene, 2 polymorphisms were found with an allele frequency for the less common allele of >5%, 1 in the 5'-UTR (P2Y1 190GC), and 1 in the coding sequence (P2Y1 1622AG). These were partially linked, with P2Y1 190GC being slightly less common. From the P2Y1 gene, we therefore selected the 1622AG polymorphism for typing in the full cohort.
In the P2Y12 gene, there were 9 common polymorphisms, 5 of which (P2Y12 145CT, IntB137CT, IntB742TC, IntB798delA, and 252GT) were in complete linkage disequilibrium. A further polymorphism (P2Y12 2014CT) was in partial linkage with these but more common. The following alleles were carried together: 145C, IntB137C, IntB742T, IntB798del, 252G or 145T, IntB137T, IntB742C, IntB798A, and 252T. From the P2Y12 polymorphisms, we therefore typed IntB742TC (to represent the 5 linked polymorphisms), 234CT, 1622CT, and 2014CT. Although the P2Y12 2087delA deletion was also common, this was not typed because the nature of the sequence around the deletion (a run of 7 or 8 As) prevented an efficient method for genotyping being developed.
Effect of Common P2Y1 and P2Y12 Gene Polymorphisms on Platelet ADP and TRAP Responses
All polymorphisms were in Hardy-Weinberg equilibrium in the cohort (Table). Carrying 1 G allele at the P2Y1 1622 position was found to be significantly associated with increased platelet response to ADP. This was apparent across the whole range of stimulating concentrations, when analyzed separately or in aggregate (Table; Figure 2). The major difference was between the AA and the GG genotype. For example, at the 0.1 μmol/L ADP concentration, there was a 130% greater response in GG homozygotes when compared with AA homozygotes (P=0.006 after adjustment for age, resting level of bound fibrinogen, smoking, and GPIIb-IIIa receptor expression; Table; Figure 2). The response of the AG genotype was also statistically significantly different from that of AA genotype at all doses and about one third of the effect seen with the GG genotype. On average, across all doses of ADP, 3.4% of the interindividual variation in response was explained by the P2Y1 1622 genotype.
Platelet Activation in Response to ADP by P2Y1 and P2Y12 Genotypes
Figure 2. Fibrinogen binding to platelets (expressed as percent positive) in unstimulated (resting) samples and after stimulation with 0.1, 1.0, and 10 μmol/L ADP in relation to the P2Y1 1622 genotypes (AA, n=147; AG, n=46; GG, n=7). Data are shown as box plots demonstrating medians, 25th and 75th percentiles, and range for each genotype.
In the P2Y12 gene, none of the polymorphisms were found to have a significant effect on platelet response to ADP after adjustment for other covariates (Table). In addition, no effect was observed of any genotype on resting platelet activation state (data not shown).
The interindividual response to thrombin receptor activity peptide (TRAP) at all 3 concentrations also showed a wide variation (data not shown). There was significant correlation between the responses to the 3 concentrations of TRAP and ADP (r=0.51 to 0.84). The association of the P2Y1 A1622G polymorphism with the responses to TRAP is shown in Table III (available online at http://atvb.ahajournals.org). There was a trend toward a higher response at all doses for GG homozygotes, and the aggregate effect after adjustment for age, resting level of bound fibrinogen, and GPIIb-IIIa receptor expression was significant (P=0.037). The magnitude of the genotype-related effect was not as great as with ADP at corresponding doses, and after adjustment for the ADP response, the aggregate effect was no longer significant (P=0.678).
Discussion
The role of platelets in normal hemostasis is incontrovertible, and they are also implicated in arterial thrombosis, as evidenced by the effectiveness of antiplatelet therapy in reducing mortality and morbidity in atherothrombotic disease states.18 There is a high degree of interindividual variability in the platelet response to all agonists, and in particular to ADP.8,9 This is further supported by data in the present study. More important, this variation is reproducible over time in any given individual,9 which points to a potential genetic influence on the platelet response to ADP. To examine this possibility, we used a strategy to identify common sequence variation within the 2 platelet ADP receptor genes that may affect function. An AG polymorphism was identified at position 1622 in the P2Y1 gene that was found to have a significant association with platelet response to ADP, as defined by the binding of fibrinogen to activated GPIIb-IIIa. This is the first report of genetic variation at the P2Y1 locus that is associated with the platelet response to ADP.
Five polymorphisms were identified in the P2Y1 ADP receptor gene and 11 polymorphisms in the P2Y12 gene. Of these, a number have been detailed previously in the NCBI single-nucleotide polymorphism (SNP) database (Table I, available online at http://atvb.ahajournals.org), although several are novel. All coding sequence polymorphisms identified in this study were silent, indicating that there are no common polymorphisms that directly contribute to the variation observed in platelet response to ADP by affecting the amino acid structure of either the P2Y1 or the P2Y12 receptor.
However, a synonymous polymorphism in the P2Y1 gene was associated with variation in platelet response to ADP. The fact that the effect was seen at all doses of ADP strongly indicates a true genotype effect. A 130% increase in platelet response to the lowest stimulating dose of ADP (0.1 μmol/L) was observed when comparing those subjects homozygous for the G allele at the P2Y1 1622 position with those homozygous for the A allele. The effect of the polymorphism on platelet response to ADP at an individual level is further underscored by the fact that 6 of the 7 individuals with the GG genotype had a response in the top 50% of the range at all doses.
In addition to its association with the platelet response to ADP, the P2Y1 1622AG polymorphism also showed an association with the response to TRAP but to a much lesser extent than was observed with the ADP response. The effect was abolished after adjustment for the ADP response. These findings are important for several reasons. First, up to 50% of the response to TRAP is attributable to the secondary effect of ADP released from the platelet-dense granules, acting through the P2Y receptors.2 Reproduction of the genotype effect with a second agonist that is heavily reliant on secondary ADP release indicates that the observed effect with ADP is genuine. Second, the fact that the magnitude of the genotype-related effect with TRAP was less than with ADP and was abolished after adjustment for the ADP response strongly suggests that the genotype-related effect is indeed mediated via the ADP receptor and not a result of some generalized hyper-reactivity of the cell.
We chose to study fibrinogen binding as the preferred assay for studying platelet activation by the ADP receptors rather than platelet aggregation because it is more directly linked to receptor activation, and unlike aggregation, is unaffected by any thromboxane generation related to extracellular calcium concentration.19,20 Nonetheless, one would anticipate the increased fibrinogen binding in response to ADP in carriers of the 1622G allele to be translated into increased aggregation. However, the exact magnitude of the effect on aggregation needs to be determined in future studies.
The mechanism by which the P2Y1 1622 polymorphism is associated with increased platelet response to ADP remains to be elucidated. Given that the polymorphism is itself silent and our study has not revealed any other common polymorphism affecting the primary structure of P2Y1, the likelihood is that this is through an effect on P2Y1 receptor expression. Associations between receptor expression and silent polymorphisms have been observed in studies on other platelet receptors.21,22 In addition, a recent study by Hechler et al23 found that overexpression of the P2Y1 receptor in a transgenic mouse model resulted in platelet hyper-reactivity to ADP. We have sequenced the region 1000-bp upstream of the P2Y1 gene in 13 of the subjects and identified an additional 10 SNPs, all of which have a degree of linkage to the 1622AG polymorphism (data not shown). One of these, a CT substitution at –545, is in complete linkage disequilibrium with the 1622AG site. These findings suggest a potential link to regulation of expression within the promoter region of the gene and provide a target for further investigation. The effects of carrying the P2Y1 1622 polymorphism on downstream signal transduction pathways, and in particular calcium flux, also need to be investigated. It also needs to be borne in mind that what we report is an association of the P2Y1 1622 polymorphism with increased platelet response to ADP, and that until the functional basis of this is established, it remains possible that this reflects the effect of an adjacent gene rather than P2Y1 itself.
Age, smoking status, and platelet GPIIb-IIIa expression were all found to be important independent regulators of the platelet response to ADP. A positive correlation was found between platelet response and increasing age and also with the density of GPIIb-IIIa on platelets, whereas a negative correlation was found with current smokers. Increasing age has been recognized to be associated with increased platelet response to ADP by others,24 although the underlying mechanism for this is poorly understood. As would be expected, fibrinogen binding was correlated with the expression levels of GPIIb-IIIa on each platelet. However, it is worth noting that there was no effect of the human platelet antigen (HPA)-1a/b polymorphism on GPIIb-IIIa expression or fibrinogen binding (data not shown). A number of studies have attempted to characterize the effect of acute and chronic smoking on platelet reactivity and have produced conflicting results.24–26 Current smokers in this study had abstained from smoking for 24 hours before platelet function testing. Nonetheless, an effect of smoking status was still observed. Importantly, adjusting for each of these variables (GPIIb-IIIa expression, age, and smoking status) did not diminish the significance of the association between fibrinogen binding and the P2Y1 1622 dimorphism. The proportion of the total interindividual variation in response explained by the P2Y1 1622 polymorphism and the other variables combined is modest, suggesting that other variables affecting the response remain to be identified. Although the overall proportion of the interindividual variation in response to ADP explained by the P2Y1 A1622G polymorphism is small (2.7% to 3.9% across the 3 concentrations of ADP), mainly because the frequency of the GG genotype is low, it should be emphasized that at an individual level, those homozygous for the G allele had a markedly exaggerated response to ADP, particularly at the lower doses.
Screening of the P2Y12 gene identified several polymorphisms, including 5 in complete linkage disequilibrium. However, none were found to affect platelet reactivity. Four of the linked polymorphisms, equivalent to IntB137CT, IntB742TC, IntB798delA, and 252GT in our study, have been described recently by Fontana et al.15 Interestingly, in their study, there was an association between the 2 haplotypes defined by these linked polymorphisms and platelet response to ADP, as measured by maximal platelet aggregation. Carriage of the minor haplotype was associated with higher maximal aggregation and greater inhibition of iloprost-stimulated cAMP accumulation.15 Despite a larger number of heterozygotes and homozygotes for the minor haplotype in our study, we did not detect any effect of this variant on fibrinogen binding to activated GPIIb-IIIa. Indeed, the response in heterozygous subjects was somewhat lower than that in the subjects homozygous for the more common allele (Table). The reason for the discrepancy between the 2 studies is unclear. Different methodologies were used in the 2 studies: flow cytometry here compared with aggregometry by Fontana et al,15 and as discussed earlier, the precise relationship between the results from the 2 assays remains to be established. An alternative explanation, especially given our finding of a significant effect of age on fibrinogen binding in response to ADP and the known effect of age on aggregometry responses,24 is the difference in the demographics of the subjects studied. In contrast to our cohort, the subjects of Fontana et al were all males and significantly younger (18 to 35 years of age).
Whether the increased platelet reactivity associated with the P2Y1 1622G allele increases the risk of thrombotic events remains to be determined. There is also emerging evidence that the variability in platelet response to ADP may be implicated in the variation in response observed with certain antiplatelet agents.27,28 Those patients with a high pretreatment response to ADP may not achieve the desired degree of platelet inhibition at conventional doses of those agents, and this may be a factor influencing treatment failure in the setting of unstable atherothrombotic disease. Whether the P2Y1 1622 polymorphism identified here plays a role in this phenomenon will be interesting to investigate.
In summary, we report an association between a silent polymorphism at position 1622 in the coding sequence of the P2Y1 ADP receptor gene and platelet reactivity. Carrying 1 G allele at this position is associated with an increased platelet activation response to ADP. Identification of this genotype effect partly explains the interindividual variation in platelet response to ADP and may have clinical implications with regard to risk of arterial thrombosis and individual response to certain antiplatelet agents.
Acknowledgments
This work was supported by grants from the British Heart Foundation and the British Cardiac Society. We would like to thank Laurence Hall for technical advice on sequencing.
Received May 28, 2004; accepted September 29, 2004.
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Correspondence to Professor N.J. Samani, Department of Cardiovascular Sciences, University of Leicester, Clinical Sciences Wing, Glenfield Hospital, Leicester, LE3 9QP, UK. E-mail njs@le.ac.uk
Abstract
Objective— The platelet ADP receptors P2Y1 and P2Y12 play a pivotal role in platelet aggregation. There is marked interindividual variation in platelet response to ADP. We studied whether genetic variants in the P2Y1 or P2Y12 genes affect platelet response to ADP.
Methods and Results— The P2Y1 and P2Y12 genes were screened for polymorphisms. Associations between selected polymorphisms and the platelet response to ADP (0.1, 1.0, and 10 μmol/L), assessed by whole blood flow cytometric measurement of fibrinogen binding to activated glycoprotein IIb-IIIa, were then determined in 200 subjects. Five polymorphisms were found in the P2Y1 gene and 11 in the P2Y12 gene. All polymorphisms were silent. A P2Y1 gene dimorphism, 1622AG, was associated with a significant (P=0.007) effect on platelet ADP response, with a greater response in carriers of the G allele (frequency 0.15). The effect was seen at all concentrations of ADP but greatest at 0.1 μmol/L ADP, where the response in GG homozygotes was on average 130% higher than that seen in AA homozygotes (P=0.006).
Conclusions— A common genetic variant at the P2Y1 locus is associated with platelet reactivity to ADP. This genotype effect partly explains the interindividual variation in platelet response to ADP and may have clinical implications with regard to thrombotic risk.
ADP is an important mediator of platelet aggregation. In this study, we show that a polymorphism (1622AG) in the P2Y1 ADP receptor gene is associated with a significant effect on platelet ADP response. This genotype effect explains some of the interindividual variability in platelet reactivity and may influence thrombotic risk.
Key Words: platelets ? thrombosis ? genes ? receptors ? adenosine diphosphate
Introduction
Platelet activation and thrombus formation play an integral role in the hemostatic mechanism after vascular injury. After disruption of atherosclerotic plaques, platelet aggregation also plays an important role in development of myocardial infarction and other acute coronary syndromes.1 Binding of platelets to von Willebrand factor and collagen is the initiating event in platelet activation. This leads to platelet degranulation and release of ADP. ADP, acting via specific receptors, causes further platelet activation and platelet aggregation. Therefore, ADP plays a key direct role in platelet activation. ADP-mediated activation also partly explains the platelet response to other agonists.2
Two platelet ADP receptors, P2Y1 and P2Y12, have been shown to initiate platelet activation when stimulated in concert.3 Both are heterotrimeric G-protein-coupled receptors: P2Y1 to Gq and P2Y12 to Gi. Stimulation at P2Y1 leads to intracellular calcium mobilization and platelet shape change,4 whereas stimulation at P2Y12 leads to inhibition of adenylyl cyclase5 and activation of phosphoinositide-3 kinase.6 The end effect is affinity modulation of the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor for fibrinogen, resulting in fibrinogen binding and platelet aggregation.7 It is well recognized that there is substantial interindividual variation in platelet response to ADP.8,9 The reasons for the interindividual variation are poorly defined, but the observation that the response is stable over time, in a given individual,9 raises the possibility that at least in part, it may be genetically controlled because of variation in the P2Y1 and/or P2Y12 genes.
P2Y1 and P2Y12 genes are located on chromosome 3. The P2Y1 gene spans 4 kb10 and is made up of a single exon of 3122 base pairs encoding a 372-aa protein.11 The P2Y12 gene spans 47 kb and is made up of 3 exons and 2 introns.12 There are 2 reference sequence mRNA variants listed in the National Center for Biotechnology Information (NCBI) Sequence Database for P2Y12, which differ in their 5'-untranslated region (UTR), long (1502 bp) and short (1474 bp).12 Hollopeter et al13 found platelets to express the shorter transcript variant, consisting of sequence from exons 2 and 3. The entire coding sequence for the 342-aa receptor is found in exon 3.13
Rare mutations within the P2Y12 gene have been shown to disrupt receptor function and lead to a bleeding diatheses.13,14 Whether there are more common variants in the 2 genes, which influence interindividual variation in platelet reactivity to ADP, has only recently come under investigation.15 The objectives of this study were to identify common P2Y1 and P2Y12 gene polymorphisms and investigate their possible functional effects on platelet reactivity.
Methods
Subjects and Sample Collection
A total of 200 white adult subjects of northern European origin were recruited. None had a history of coronary heart disease and none were taking any antiplatelet medication. Subjects were categorized as experiencing hypertension and diabetes on the basis of reported history. Smoking status was defined as current smoker, ex-smoker, or nonsmoker. The study was approved by the Leicestershire Research Ethics Committee, and all subjects gave written informed consent.
Individuals were seen in the morning and in a fasting state. Ten subjects attended on a second occasion, at least 6 weeks later, to permit analysis of intraindividual variability of the platelet ADP response. Smokers were asked to abstain from smoking for at least 24 hours before the visit, and this was confirmed by a carbon monoxide breath test. All subjects were rested supine for at least 20 minutes before venepuncture to minimize the effects of stress hormones, and blood was collected using a standardized phlebotomy technique designed to minimize platelet activation.16
Platelet Response Measurement
Blood samples were prepared for whole blood flow cytometric analysis within 10 minutes of collection essentially as described previously16,17 (see detailed Methods, available online at http://atvb.ahajournals.org).
White cell count, platelet count, and mean platelet volume (MPV) were measured in EDTA-anticoagulated blood after 2 hours to allow stabilization of platelet volume using a Beckman Coulter counter. Fibrinogen was measured by the Clauss method on a Sysmex CA-1000 analyzer (Sysmex UK). Leukocyte DNA was extracted from a 10-mL blood sample using the PureGene DNA extraction kit (Gentra Systems).
P2Y1 and P2Y12 Gene Screening for Common Polymorphisms
On the basis of the response after stimulation of whole blood with 1.0 μmol/L ADP, 10 subjects from each end of the distribution of response were selected to undergo sequencing of the P2Y1 gene, including the exon and 500 bp of upstream sequence, and the region of the P2Y12 gene encompassing exons 2 and 3 and intron B. This strategy maximized the chances of identifying common polymorphisms that might affect function (further details of Methods available in online supplement).
Genotyping of Common P2Y1 and P2Y12 Polymorphisms
Polymorphisms to type in the full cohort were selected on the basis of their predicted allele frequencies and linkage disequilibrium data as described in Results. Four polymorphisms were typed in the P2Y12 gene and 1 in the P2Y1 gene (details of genotyping Methods available in online supplement).
Statistical Analysis
Unless stated otherwise, data are presented as mean±SD. Observed allele frequencies were compared with the Hardy-Weinberg equilibrium prediction using the 2 test. All response data were transformed before analysis using log (response [100-response]–1). The association between genotype and phenotype was tested by 1-way ANOVA. Associations at specific doses were further analyzed after adjustment for other parameters (age, resting level of bound fibrinogen, smoking status, and GPIIb-IIIa receptor expression) using multiple linear regression models. Analysis across all doses was performed using a random-effects model fitted by maximum likelihood that included a random effect for subject to adjust for within subject correlation. All analyses were performed using Stata 8.1 (Stata Corp.).
Results
Cohort Demographics
Mean age of the subjects was 47.3±6.0 years (range 25 to 56 years). A total of 87% were male, 18% were current smokers, and 32% were ex-smokers. Nine percent had a diagnosis of hypertension, and 1% were diabetic. Mean white cell count was 5.5±1.3x109 L, mean platelet count was 240.3±61.1x109 L, MPV was 9.2±1.2 fL, and mean plasma fibrinogen was 2.8±0.6 g/L. All of these variables were distributed normally.
Variability of Platelet Response to ADP
The distribution of subjects with respect to the percentage of platelets positive for bound fibrinogen at rest and after stimulation with the 3 doses of ADP are shown in Figure 1. Mean values (95% CIs) were as follows: resting level 2.6% (2.5 to 2.7%); 0.1 μmol/L ADP 13.5% (11.9 to 15.3%); 1.0 μmol/L ADP 69.9% (67.0 to 72.3%); and 10 μmol/L ADP 85.4% (83.9 to 86.9%). There were strong correlations between the different stimulating concentrations of ADP within subjects (0.1 versus 1.0 μmol/L, r=0.91; 0.1 versus 10 μmol/L, r=0.86; 1.0 versus 10 μmol/L, r=0.94) and a modest correlation between the resting level of bound fibrinogen to platelets and response to all doses of ADP (r0.38). In the 10 subjects studied on 2 occasions, a correlation between the ADP responses measured at the 2 visits was observed when comparing the response to all doses of ADP (0.1 μmol/L, r=0.56; 1.0 μmol/L, r=0.91; 10 μmol/L, r=0.93), indicating that the response to ADP is reproducible within an individual.
Figure 1. Percentage of platelets with fibrinogen bound to GPIIb-IIIa measured by whole blood flow cytometry in unstimulated (resting) samples and in samples stimulated with ADP at 0.1, 1.0, and 10 μmol/L. Data are shown for 200 subjects with medians.
Random effects analysis incorporating all 3 doses of ADP found that after adjusting for the resting level of bound fibrinogen, age (P<0.0001), smoking history (P=0.008), and GPIIb-IIIa expression (P=0.0005) were important independent regulators of the platelet response. GPIIb-IIIa expression showed 3-fold variation within the population studied (1.7 to 5.4 relative fluorescence units) and was distributed normally. The correlations between the level of GPIIb-IIIa expression and fibrinogen binding in response to ADP were 0.169 (P=0.016), 0.220 (P=0.002), and 0.266 (P<0.001) for the 0.1 μmol/L, 1.0 μmol/L, and 10 μmol/L of ADP, respectively. GPIIb-IIIa expression accounted for between 2.9% and 7.1% of the interindividual variation in response to ADP, dependent on stimulating concentration of ADP used. The proportions explained by age and smoking were 4.2% to 10.7% and 1.1% to 2.2%, respectively. Gender did not have an independent effect.
Identification of Common P2Y1 and P2Y12 Polymorphisms
The polymorphisms identified are shown in supplemental Table II (available online at http://atvb.ahajournals.org). All polymorphisms in the coding sequences of both genes were silent. In the P2Y1 gene, 2 polymorphisms were found with an allele frequency for the less common allele of >5%, 1 in the 5'-UTR (P2Y1 190GC), and 1 in the coding sequence (P2Y1 1622AG). These were partially linked, with P2Y1 190GC being slightly less common. From the P2Y1 gene, we therefore selected the 1622AG polymorphism for typing in the full cohort.
In the P2Y12 gene, there were 9 common polymorphisms, 5 of which (P2Y12 145CT, IntB137CT, IntB742TC, IntB798delA, and 252GT) were in complete linkage disequilibrium. A further polymorphism (P2Y12 2014CT) was in partial linkage with these but more common. The following alleles were carried together: 145C, IntB137C, IntB742T, IntB798del, 252G or 145T, IntB137T, IntB742C, IntB798A, and 252T. From the P2Y12 polymorphisms, we therefore typed IntB742TC (to represent the 5 linked polymorphisms), 234CT, 1622CT, and 2014CT. Although the P2Y12 2087delA deletion was also common, this was not typed because the nature of the sequence around the deletion (a run of 7 or 8 As) prevented an efficient method for genotyping being developed.
Effect of Common P2Y1 and P2Y12 Gene Polymorphisms on Platelet ADP and TRAP Responses
All polymorphisms were in Hardy-Weinberg equilibrium in the cohort (Table). Carrying 1 G allele at the P2Y1 1622 position was found to be significantly associated with increased platelet response to ADP. This was apparent across the whole range of stimulating concentrations, when analyzed separately or in aggregate (Table; Figure 2). The major difference was between the AA and the GG genotype. For example, at the 0.1 μmol/L ADP concentration, there was a 130% greater response in GG homozygotes when compared with AA homozygotes (P=0.006 after adjustment for age, resting level of bound fibrinogen, smoking, and GPIIb-IIIa receptor expression; Table; Figure 2). The response of the AG genotype was also statistically significantly different from that of AA genotype at all doses and about one third of the effect seen with the GG genotype. On average, across all doses of ADP, 3.4% of the interindividual variation in response was explained by the P2Y1 1622 genotype.
Platelet Activation in Response to ADP by P2Y1 and P2Y12 Genotypes
Figure 2. Fibrinogen binding to platelets (expressed as percent positive) in unstimulated (resting) samples and after stimulation with 0.1, 1.0, and 10 μmol/L ADP in relation to the P2Y1 1622 genotypes (AA, n=147; AG, n=46; GG, n=7). Data are shown as box plots demonstrating medians, 25th and 75th percentiles, and range for each genotype.
In the P2Y12 gene, none of the polymorphisms were found to have a significant effect on platelet response to ADP after adjustment for other covariates (Table). In addition, no effect was observed of any genotype on resting platelet activation state (data not shown).
The interindividual response to thrombin receptor activity peptide (TRAP) at all 3 concentrations also showed a wide variation (data not shown). There was significant correlation between the responses to the 3 concentrations of TRAP and ADP (r=0.51 to 0.84). The association of the P2Y1 A1622G polymorphism with the responses to TRAP is shown in Table III (available online at http://atvb.ahajournals.org). There was a trend toward a higher response at all doses for GG homozygotes, and the aggregate effect after adjustment for age, resting level of bound fibrinogen, and GPIIb-IIIa receptor expression was significant (P=0.037). The magnitude of the genotype-related effect was not as great as with ADP at corresponding doses, and after adjustment for the ADP response, the aggregate effect was no longer significant (P=0.678).
Discussion
The role of platelets in normal hemostasis is incontrovertible, and they are also implicated in arterial thrombosis, as evidenced by the effectiveness of antiplatelet therapy in reducing mortality and morbidity in atherothrombotic disease states.18 There is a high degree of interindividual variability in the platelet response to all agonists, and in particular to ADP.8,9 This is further supported by data in the present study. More important, this variation is reproducible over time in any given individual,9 which points to a potential genetic influence on the platelet response to ADP. To examine this possibility, we used a strategy to identify common sequence variation within the 2 platelet ADP receptor genes that may affect function. An AG polymorphism was identified at position 1622 in the P2Y1 gene that was found to have a significant association with platelet response to ADP, as defined by the binding of fibrinogen to activated GPIIb-IIIa. This is the first report of genetic variation at the P2Y1 locus that is associated with the platelet response to ADP.
Five polymorphisms were identified in the P2Y1 ADP receptor gene and 11 polymorphisms in the P2Y12 gene. Of these, a number have been detailed previously in the NCBI single-nucleotide polymorphism (SNP) database (Table I, available online at http://atvb.ahajournals.org), although several are novel. All coding sequence polymorphisms identified in this study were silent, indicating that there are no common polymorphisms that directly contribute to the variation observed in platelet response to ADP by affecting the amino acid structure of either the P2Y1 or the P2Y12 receptor.
However, a synonymous polymorphism in the P2Y1 gene was associated with variation in platelet response to ADP. The fact that the effect was seen at all doses of ADP strongly indicates a true genotype effect. A 130% increase in platelet response to the lowest stimulating dose of ADP (0.1 μmol/L) was observed when comparing those subjects homozygous for the G allele at the P2Y1 1622 position with those homozygous for the A allele. The effect of the polymorphism on platelet response to ADP at an individual level is further underscored by the fact that 6 of the 7 individuals with the GG genotype had a response in the top 50% of the range at all doses.
In addition to its association with the platelet response to ADP, the P2Y1 1622AG polymorphism also showed an association with the response to TRAP but to a much lesser extent than was observed with the ADP response. The effect was abolished after adjustment for the ADP response. These findings are important for several reasons. First, up to 50% of the response to TRAP is attributable to the secondary effect of ADP released from the platelet-dense granules, acting through the P2Y receptors.2 Reproduction of the genotype effect with a second agonist that is heavily reliant on secondary ADP release indicates that the observed effect with ADP is genuine. Second, the fact that the magnitude of the genotype-related effect with TRAP was less than with ADP and was abolished after adjustment for the ADP response strongly suggests that the genotype-related effect is indeed mediated via the ADP receptor and not a result of some generalized hyper-reactivity of the cell.
We chose to study fibrinogen binding as the preferred assay for studying platelet activation by the ADP receptors rather than platelet aggregation because it is more directly linked to receptor activation, and unlike aggregation, is unaffected by any thromboxane generation related to extracellular calcium concentration.19,20 Nonetheless, one would anticipate the increased fibrinogen binding in response to ADP in carriers of the 1622G allele to be translated into increased aggregation. However, the exact magnitude of the effect on aggregation needs to be determined in future studies.
The mechanism by which the P2Y1 1622 polymorphism is associated with increased platelet response to ADP remains to be elucidated. Given that the polymorphism is itself silent and our study has not revealed any other common polymorphism affecting the primary structure of P2Y1, the likelihood is that this is through an effect on P2Y1 receptor expression. Associations between receptor expression and silent polymorphisms have been observed in studies on other platelet receptors.21,22 In addition, a recent study by Hechler et al23 found that overexpression of the P2Y1 receptor in a transgenic mouse model resulted in platelet hyper-reactivity to ADP. We have sequenced the region 1000-bp upstream of the P2Y1 gene in 13 of the subjects and identified an additional 10 SNPs, all of which have a degree of linkage to the 1622AG polymorphism (data not shown). One of these, a CT substitution at –545, is in complete linkage disequilibrium with the 1622AG site. These findings suggest a potential link to regulation of expression within the promoter region of the gene and provide a target for further investigation. The effects of carrying the P2Y1 1622 polymorphism on downstream signal transduction pathways, and in particular calcium flux, also need to be investigated. It also needs to be borne in mind that what we report is an association of the P2Y1 1622 polymorphism with increased platelet response to ADP, and that until the functional basis of this is established, it remains possible that this reflects the effect of an adjacent gene rather than P2Y1 itself.
Age, smoking status, and platelet GPIIb-IIIa expression were all found to be important independent regulators of the platelet response to ADP. A positive correlation was found between platelet response and increasing age and also with the density of GPIIb-IIIa on platelets, whereas a negative correlation was found with current smokers. Increasing age has been recognized to be associated with increased platelet response to ADP by others,24 although the underlying mechanism for this is poorly understood. As would be expected, fibrinogen binding was correlated with the expression levels of GPIIb-IIIa on each platelet. However, it is worth noting that there was no effect of the human platelet antigen (HPA)-1a/b polymorphism on GPIIb-IIIa expression or fibrinogen binding (data not shown). A number of studies have attempted to characterize the effect of acute and chronic smoking on platelet reactivity and have produced conflicting results.24–26 Current smokers in this study had abstained from smoking for 24 hours before platelet function testing. Nonetheless, an effect of smoking status was still observed. Importantly, adjusting for each of these variables (GPIIb-IIIa expression, age, and smoking status) did not diminish the significance of the association between fibrinogen binding and the P2Y1 1622 dimorphism. The proportion of the total interindividual variation in response explained by the P2Y1 1622 polymorphism and the other variables combined is modest, suggesting that other variables affecting the response remain to be identified. Although the overall proportion of the interindividual variation in response to ADP explained by the P2Y1 A1622G polymorphism is small (2.7% to 3.9% across the 3 concentrations of ADP), mainly because the frequency of the GG genotype is low, it should be emphasized that at an individual level, those homozygous for the G allele had a markedly exaggerated response to ADP, particularly at the lower doses.
Screening of the P2Y12 gene identified several polymorphisms, including 5 in complete linkage disequilibrium. However, none were found to affect platelet reactivity. Four of the linked polymorphisms, equivalent to IntB137CT, IntB742TC, IntB798delA, and 252GT in our study, have been described recently by Fontana et al.15 Interestingly, in their study, there was an association between the 2 haplotypes defined by these linked polymorphisms and platelet response to ADP, as measured by maximal platelet aggregation. Carriage of the minor haplotype was associated with higher maximal aggregation and greater inhibition of iloprost-stimulated cAMP accumulation.15 Despite a larger number of heterozygotes and homozygotes for the minor haplotype in our study, we did not detect any effect of this variant on fibrinogen binding to activated GPIIb-IIIa. Indeed, the response in heterozygous subjects was somewhat lower than that in the subjects homozygous for the more common allele (Table). The reason for the discrepancy between the 2 studies is unclear. Different methodologies were used in the 2 studies: flow cytometry here compared with aggregometry by Fontana et al,15 and as discussed earlier, the precise relationship between the results from the 2 assays remains to be established. An alternative explanation, especially given our finding of a significant effect of age on fibrinogen binding in response to ADP and the known effect of age on aggregometry responses,24 is the difference in the demographics of the subjects studied. In contrast to our cohort, the subjects of Fontana et al were all males and significantly younger (18 to 35 years of age).
Whether the increased platelet reactivity associated with the P2Y1 1622G allele increases the risk of thrombotic events remains to be determined. There is also emerging evidence that the variability in platelet response to ADP may be implicated in the variation in response observed with certain antiplatelet agents.27,28 Those patients with a high pretreatment response to ADP may not achieve the desired degree of platelet inhibition at conventional doses of those agents, and this may be a factor influencing treatment failure in the setting of unstable atherothrombotic disease. Whether the P2Y1 1622 polymorphism identified here plays a role in this phenomenon will be interesting to investigate.
In summary, we report an association between a silent polymorphism at position 1622 in the coding sequence of the P2Y1 ADP receptor gene and platelet reactivity. Carrying 1 G allele at this position is associated with an increased platelet activation response to ADP. Identification of this genotype effect partly explains the interindividual variation in platelet response to ADP and may have clinical implications with regard to risk of arterial thrombosis and individual response to certain antiplatelet agents.
Acknowledgments
This work was supported by grants from the British Heart Foundation and the British Cardiac Society. We would like to thank Laurence Hall for technical advice on sequencing.
Received May 28, 2004; accepted September 29, 2004.
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