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Ann Thorac Surg 2007;83:134-138
© 2007 The Society of Thoracic Surgeons
a Department of Cardiothoracic Surgery, Papworth Hospital, Cambridge, United Kingdom
b Department of Clinical Pharmacology, Papworth Hospital, Cambridge, United Kingdom
c Department of Cardiovascular Medicine, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom
Accepted for publication August 1, 2006.
* Address correspondence to Dr Lim, Department of Cardiothoracic Surgery, Papworth Hospital, Papworth Everard, Cambridge CB3 8RE, United Kingdom. (Email: eric.lim{at}cvsnet.org).
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Abstract |
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 Top Abstract IntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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METHODS: Blood was collected from consenting patients and DNA extracted. Polymerase chain reaction and restriction fragment length polymorphism analysis was performed to detect PlA2, C807T, and A842/C50T polymorphisms. Aspirin efficacy was assessed using light transmission platelet aggregometry, and reported as percentage aggregation and EC50 concentrations using the technique of Born.
RESULTS: Of 90 patients, 80 consented to further genetic testing, of whom 63 patients were randomly assigned to medium- (325 mg) or low-dose (100 mg) aspirin. The PlA2, C807T, and A842/C50T gene frequencies were 30%, 66%, and 21%, respectively, with no identifiable differences in the baseline platelet aggregation. Postoperatively, after 5 days of aspirin, platelet aggregation was consistently but not significantly impaired with PlA2 and A842/C50T carriers and consistently but not significantly improved with C50T carriers. An interaction term was identified on percentage aggregation and EC50 using epinephrine. The interaction coefficient describes a higher aggregation of 19% (95% confidence interval: 2 to 36; p = 0.03) and less inhibition with an EC50 of –2.07 (–4.19 to 0.04; p = 0.06) in patients who were both PlA2 positive and receiving low-dose aspirin.
CONCLUSIONS: Genetic polymorphisms that affect the response to aspirin are common. The impaired response of persons with the PlA2 polymorphism to aspirin may be dose related, with significant improvement observed in patients using medium- rather than low-dose aspirin.
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Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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In the dawn of pharmacogenomics, little is known about how genes affect the individual response to aspirin after cardiopulmonary bypass (itself a potent stimulator of platelets). To evaluate the feasibility of genetic profiling for targeted postoperative antiplatelet therapy, we performed a subanalysis of the results from a randomized trial to evaluate the impact of the candidate polymorphisms on baseline aggregation and the response to different aspirin doses after cardiopulmonary bypass.
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Material and Methods |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Genotyping of Polymorphisms
Blood was collected from each patient, and DNA was extracted using standard techniques. Informed consent for molecular genetic testing was obtained in each case.
Genotyping
Identification of the respective alleles in each gene was carried out using polymerase chain reaction and restriction fragment length polymorphism analysis (PCR-RFLP). Oligonucleotides were designed from flanking intronic and exonic sequence of the respective genes (Table 1). Digestion of the 105 bp ITGB3 fragment with MspI produced fragments of 67 and 38 bp only in the presence of the P1A2 allele due to the T to C variation in exon 3. Digestion of the 149 bp ITGA2 fragment with Hpy188I produced fragments of 124 and 25 bp only in the presence of the 807C allele in exon 7. Digestion of the 156 bp PTGS1 C50T fragment with FauI produced fragments of 85 and 71 bp only in the presence of the 50C allele in exon 2. Digestion of the 198 bp PTGS1 A-842G fragment with BseSI produced fragments of 162 and 36 bp only in the presence of the –842 allele in the intronic sequence. Fragments obtained after digestion with the specific restriction endonucleases were separated on a 3% agarose gel and visualized using ethidium bromide, allowing identification of heterozygous or homozygous status for each allele.
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Statistical Methods
Platelet aggregation and EC50 concentrations of heterozygous or homozygous carriers of the candidate gene were compared against the normal. The primary outcome was compared using regression (analysis of covariance [ANCOVA]) with robust standard errors to adjust for baseline values of platelet aggregation and EC50 concentrations. Formal tests of interaction [5] were applied to determine any differences in the response between carriers of the candidate polymorphisms and aspirin dosage. Analyses were performed on Stata 9.0 (StataCorp, College Station, Texas).
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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PlA2 Polymorphism
Carriers of PlA2 polymorphism had consistently higher baseline adjusted difference in percentage aggregation for all three agonist, albeit without reaching statistical significance. The results of EC50 concentrations were uniform, with lower amounts of agonist required to induce 50% aggregation, consistent with less effective platelet inhibition by aspirin (Table 4).
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C807T Polymorphism
There was no evidence that heterozygote carriers of C807T polymorphism had any important differences in baseline adjusted difference in percentage aggregation for all three agonist and EC50 concentrations. However, participants who were homozygous for polymorphism had consistently higher baseline adjusted difference in percentage aggregation for all three agonist, albeit without reaching statistical significance. The results of EC50 concentrations were uniform, with lower amounts of agonist required to induce 50% aggregation, consistent with less effective platelet inhibition by aspirin (Table 3). There was no evidence of an interaction with aspirin dosage.
A842G/C50T Polymorphism
Carriers of A842G/C50T polymorphism had consistently lower baseline adjusted difference in percentage aggregation for all three agonist, albeit without reaching statistical significance. The results of EC50 concentrations were consistent. Higher amounts of agonist were required to induce 50% aggregation, suggesting more effective inhibition by aspirin (Table 4). There was no evidence of any interaction with aspirin dosage.
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Comment |
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PlA2 Polymorphism
The functional significance of the PlA2 polymorphism on the platelet IIb/IIIa receptor function is not yet completely elucidated, but it is one of the most common and well known polymorphisms associated with genetic aspirin resistance [6] and coronary thrombosis [7]. These findings have not been entirely consistent. The Physicians Health Study (a large prospective observational study) reported no association between PlA2 and the risk of myocardial infarction, stroke, or venous thrombosis; neither was there any evidence of a protective effect from aspirin administration [8]. The dose of aspirin used in the Physicians Health Study was 325 mg. The results of our study suggest that carriers of PlA2 polymorphism had a uniformly more impaired response to aspirin after coronary surgery compared with homozygotes with the PlA1 genotype. Although the findings did not reach statistical significance, the results were consistent on the six different aggregation tests. On (formal) interaction testing, we discovered that the response of carriers of the PlA2 polymorphism to aspirin is potentially modifiable. Participants with PlA2 polymorphism had a better response with lower aggregation and higher EC50s when receiving a medium dose (325 mg) of aspirin compared with a low dose (100 mg) on epinephrine-induced aggregation.
We are unsure as to why these responses were preferentially detectable on epinephrine-induced aggregation, a finding consistent with the Framingham Offspring Study, where incremental aggregation was observed using epinephrine as an agonist on samples obtained from subjects with PlA2 polymorphism, with similar nonsignificant findings with ADP [2].
C807T Polymorphism
The C807T polymorphism is associated with an increased expression of the platelet GP Ia/IIa receptor [9]. This was a common polymorphism in our sample, with 38% being heterozygous and a further 28% homozygous for this polymorphism. Analysis comparing heterozygous and homozygous carriers with the norm did not reveal any discernable pattern despite heterozygous individuals expressing intermediate levels of receptor density [9]. The findings of increased platelet aggregation [10] were largely confined to participants homozygous for this polymorphism (Table 3). It is difficult to determine the clinical significance of this finding. The differences in the point estimates were small (2% to 5%), and the upper bound of the 95% confidence interval was a maximum 13% difference in aggregation on collagen, which is unlikely to contain an important difference.
A842G/C50T Polymorphism
The 842G/C50T alleles exhibit complete linkage dysequilibrium (nonrandom association between 842G/C50T such that it more likely to occur together on a chromosome than other combinations of alleles). The 842G variant is located in the promoter of COX-1 and persons heterozygous for 842G/C50T that is associated with increased aspirin effectiveness. Unlike the other two polymorphisms, carriers of this genotype have a greater sensitivity to aspirin, possibly due to reduced levels of COX-1 [11]. Our results confirmed complete linkage dysequilibrium, as both the A842G and the C50T polymorphisms were sequenced. In our study, all patients who were heterozygous or homozygous carriers of A842G polymorphisms also had identical C50T polymorphism. The results of aggregation were also consistent with a greater response to aspirin, with lower aggregation and a greater platelet inhibition with higher EC50 levels. The confidence intervals were wide, and the lower bound contained up to an 18% reduction in aggregation; it is difficult to determine whether this represents an important difference that our study was not powered to detect.
Potential Limitations
Our original study was powered to detect a 30% difference in aggregation between any two arms. In this subanalysis, we were unable to determine whether the absence of statistical significance implied no true difference. The findings of increased aggregation with C807T and reduced aggregation with A842G/C50T polymorphisms were consistent with previous reports, but our findings did not reach statistical significance.
Clinical Implications
Our main findings suggest that it may be possible to improve the response of PlA2 carriers to aspirin by using a higher dose. A 20% improvement in platelet aggregation is considerable, bearing in mind the overall reduction of 30% in percentage aggregation after 5 days of aspirin therapy. If facilities for the screening and detection of PlA2 polymorphism are not available, it may be prudent to consider medium-dose aspirin as the dosage of choice. Approximately 1 in 3 patients carry this silent polymorphism.
Certainly, further work is required to determine the clinical utility of preoperative screening for PlA2 polymorphism, and to determine whether optimum platelet inhibition leads to substantial clinical results. Although our study was confined to postoperative patients, the results are likely to reflective the gene-drug interaction, rather than any confounding effects of surgery.
In conclusion, genetic polymorphisms of PlA2, C807T, and C50T are common. There did not appear to be any identifiable differences in the baseline platelet aggregation. The responses to aspirin were consistently but not significantly impaired with PlA2 and C807T carriers and consistently but not significantly improved with C50T carriers. The response of persons with the PlA2 polymorphism to aspirin may be dose related, with significant improvement observed in patients using medium- rather than low-dose aspirin.
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Acknowledgments |
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References |
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 H.-S. Lee Invited commentary Ann. Thorac. Surg., January 1, 2007; 83(1): 138 - 139. [Full Text] [PDF]
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