European Journal of Vascular & Endovascular Surgery
Volume 36, Issue 2 , Pages 132-137, August 2008

Increased Platelet-monocyte Aggregation in Male Claudicants with the PlA1/A2 Polymorphism of Gp IIb/IIIa

  • J. McCaslin

      Affiliations

    • Queen Elizabeth Hospital, Gateshead, UK
    • ,
  • H. Ashour

      Affiliations

    • Queen Elizabeth Hospital, Gateshead, UK
    • ,
  • V. Bhattacharya

      Affiliations

    • Queen Elizabeth Hospital, Gateshead, UK
    • ,
  • M. Cleanthis

      Affiliations

    • Northern Vascular Unit, Freeman Hospital, Newcastle-upon-Tyne, UK
    • ,
  • A. Daly

      Affiliations

    • Newcastle University, Newcastle, UK
    • ,
  • G. Stansby

      Affiliations

    • Northern Vascular Unit, Freeman Hospital, Newcastle-upon-Tyne, UK
    • Corresponding Author InformationCorresponding author. Prof G. Stansby, Northern Vascular Unit, Freeman Hospital, Newcastle-upon-Tyne NE7 7DN, UK. Tel.: +191 2231125; fax: +191 2231489.

Received 31 October 2007; accepted 15 February 2008. published online 07 April 2008.

Article Outline

Abstract 

Objectives

To investigate the relationship between the PlA1/A2 polymorphism and platelet activation and aggregation in patients with Peripheral Arterial Disease (PAD).

Design

A prospective single-centre cohort study.

Methods

45 patients with PAD on aspirin 75mg were recruited and phenotyped/genotyped for the Gp IIb/IIIa PlA1/A2 polymorphism. Platelet-Monocyte Aggregation (PMAs) was evaluated using flow-cytometry.

Results

The formation of PMAs in the PlA2 group was higher but not statistically significant (p=0.17). However, when males were analysed separately, the formation of PMAs was significantly higher in the PlA2 group (p=0.0192). No difference was seen in the females.

Conclusions

In this study we show that the PlA1/A2 polymorphism primarily affects the aggregation of platelets to monocytes in males. The effect is not observed in females and understanding the mechanism behind this may help elucidate the way the polymorphism alters platelet function in the presence of aspirin.

Keywords: Peripheral arterial disease, Aspirin, Genetic polymorphism, Platelet

 

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Background 

Atheroma is a leading cause of morbidity and mortality throughout the world. Platelets contribute to the progression of atherosclerotic disease by adhering to the subendothelial matrix at sites of shear-induced mechanical injury to the vessel. Enhanced platelet activation and procoagulant activity may therefore contribute further to the pathophysiology of such events. The understanding that genetic factors play a role in the development of atheroma led to recent interest in polymorphisms of platelet receptors as a possible basis for this. Furthermore, there is a growing body of evidence to link these polymorphisms with resistance to antiplatelet agents,1, 2, 3 the implications of which are not yet fully appreciated.

Several genetic variants have been identified in the platelet receptor GPIIb/IIIa and attempts have been made to ascribe these to functional outcomes. The most studied sequence has been the PlA1/A2 (HPA-1) polymorphism, for which approximately 25% of Caucasians carry the variant A2 allele. The polymorphism results from a substitution of cytosine to thymidine at position 1565 on exon 2 of the GPIIIa gene. This point mutation results in the amino acid substitution of proline (Pro) to leucine (Leu) at position 33 in GPIIIa. The leucine-33 allele is known as PlA1 (HPA-1a) and has a frequency of 0.85. The proline-33 allele is known as PlA2 (HPA-1b) and has a frequency of 0.15. Genotypes can be PlA1/A1, PlA1/A2 or PlA2/A2 depending on simple Mendelian inheritance of the alleles. Controversy exists as to the exact clinical consequence of the polymorphism, with various previous studies resulting in conflicting results with regards to platelet activity.4, 5, 6, 7, 8 These population studies have shown that the polymorphism may worsen, have no effect or even improve clinical outcome from atheroma. This raises questions about how the polymorphism exerts its effect on patients, with aspirin resistance being one possible explanation for the wide variation in results. Understanding the underlying biochemical mechanisms behind the polymorphism effect is essential to explain the clinical differences seen in order to apply knowledge of the polymorphism in a clinical context.

The binding of platelets to leucocytes is an important part of their activation pathway. Detection of this interaction has been shown to be a sensitive marker of platelet activation.9 Flow cytometry can detect both platelets and leucocytes and determine if they are bound to one another using specific antibodies to unique surface proteins.

Previous studies on patients with atheroma have shown increased platelet activation in the form of fibrinogen binding, direct platelet aggregation and surface markers of platelet activation (e.g. PAC-1, P-selectin).5, 10, 11, 12 Other studies, however, have shown that platelets are unaffected and even protected from activation by the PlA2 allele.13, 14 These studies have been performed in the context of healthy volunteers however, and as such extrapolation of the results into patients with vascular disease is difficult. This study uses platelet-monocyte binding as a marker of platelet activation. This has been shown to be a more sensitive marker than fibrinogen binding or surface markers and perhaps gives an indication of chronic platelet activation rather than simply the acute activation seen well with other tests.9

The aim of this study was to investigate the link between the PlA1/A2 polymorphism and platelet aggregability/activation in a population of patients with peripheral arterial disease (PAD).

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Methods 

Ethical approval was granted from the local ethics committee. 45 Patients with PAD were recruited from patients attending the vascular unit at the Queen Elizabeth Hospital, Gateshead, UK. All patients had a history suggestive of stable Intermittent Claudication (IC) and an Ankle Brachial Pressure Index (ABPI) of less than 0.9 or radiologically proven vascular disease on angiography or duplex ultrasound. No patients had rest pain or critical ischaemia. All patients gave written consent to participate in the study. Patients with concomitant inflammatory processes or with conditions known to affect platelet function were excluded. Specific exclusions were patients with diabetes mellitus, critical ischaemia, active ulceration or gangrene, malignancy or infection. Patients were all taking 75mg of aspirin daily and were on no other antiplatelet agents or anticoagulants for the duration of the study.

Phlebotomy 

A standardised phlebotomy technique was used which has been previously advocated for platelet work.15, 16, 17 Venepuncture was performed with patients sitting upright after at least 10 minutes of rest. No tourniquet was used where-ever possible, but on occasions was used to find a suitable vein, then released before any sample was drawn. The first 4ml of blood was discarded, or used for unrelated tests (e.g. Cholesterol level). Vacutainers (Becton-Dickinson, Cambridgeshire, UK) were employed to standardise the rate of phlebotomy and minimise unnecessary handling of the samples. For polymorphism phenotyping, platelet flow cytometry and platelet aggregation tests, samples were collected in 0.109M trisodium citrate in a 1:9 dilution (citrate:blood). For DNA analysis, blood was collected in plastic K2 EDTA tubes and for soluble P-selectin, blood was collected in gold top BD tubes with a clot activator gel (Becton-Dickinson, Cambridgeshire, UK). Samples used for phenotyping were stored at room temperature and used within 24 hours as directed by the manufacturer. Samples for platelet flow cytometry were prepared within 3 minutes as described below. Samples for DNA analysis were frozen within 24 hours at −20°C and thawed once required.

Phenotyping 

Detection of the PlA phenotype by flow cytometry has been described previously,18 although no formal validation of the commercially available kit (American Diagnostica, USA) exists in the literature. We used this single-colour flow cytometric technique to elicit patient PlA status and then validated our own results using the gold-standard PCR.

RFLP-PCR 

Restriction-Fragment Length Polymorphism (RFLP-PCR) was used to ascertain genotype as previously described.4, 5, 10, 19, 20 Samples were stored at −20°C in K2 EDTA tubes. After thawing, aliquots of blood underwent DNA extraction using the automated BioRobot M48 workstation (Qiagen Ltd, UK). Essentially cells were lysed, then MagAttract Suspension (Qiagen Ltd, UK) was added to bind nucleic acids; MagAttract particles were efficiently separated by applying a magnet to the pipette tips and washing to remove contaminants; elution reagent was added to the washed magnetic particles and the mixture was heated to optimize the yield of nucleic acid.

Primers were manufactured as previously described20 (Invitrogen, Paisley, UK) and diluted in purified water to 25μM. DNA was diluted in purified water to give a final concentration of 25–50ng/μL. Amplification of the sequence to give a 338bp product was performed using HotstarTaq DNA polymerase (Qiagen Ltd, UK). Several ‘blank’ samples were amplified as controls for contamination. The restriction endonuclease used for the digest was ScrFI which digests CC'NGG sequence. With this PCR product, the enzyme digested the PLA1 allele into 3 fragments; 214bp, 46bp and 78bp. The PLA2 allele was digested further due to the nucleotide base substitution into fragments of 77bp, 137bp, 46bp and 78bp. The fragments were separated and identified by 2.2% agarose gel electrophoresis with ethidium bromide staining.

Platelet monocyte aggregation 

The technique used here is based on that first developed by Li et al.9 It was modified for our purposes to focus on monocytes rather than all leucocytes with the addition of the monocyte specific CD14 antibody. It is a three-colour flow-cytometric technique which uses whole blood with minimal sample manipulation.

10μl of blood was added to a pre-mixed tube of antibodies containing; 20μl CD61-FITC (Becton Dickinson, Oxford, UK), 5μl CD14-PE (DakoCytomation, Cambridgeshire, UK), 7μl CD45-PerCP (DakoCytomation, Cambridgeshire, UK) and 60μl PBS. A second tube was used as an isotype control and was mixed in the same way except for substituting the CD61-FITC for 20μl IgG1-FITC (Becton Dickinson, Oxford, UK). The tubes were then incubated in the dark for 20 minutes before the addition of 1ml of fixative.

Flow cytometry was performed within 2 hours of fixation using a Becton Dickinson FACScan Flow Cytometer. 500 monocytes were collected for analysis on WinMDI 2.8.

A dotplot of CD14 versus CD45 was used to identify the monocyte population. This was then gated and a histogram of CD61 binding calculated. This is a measurement of platelet aggregation to the monocytes and was calculated as percent positive cells (PPC).

Application of the platelet-monocyte binding data for determination of aspirin resistance was done using a cut-off value above which platelets were considered to be inadequately ‘blocked’ by aspirin. This has been used in the past by several groups to define aspirin resistance in-vitro,21, 22, 23 although standardisation between methods makes it difficult to apply these values directly. Previous attempts to define a cut-off have shown approximately 5–60% of the population to be inadequately treated by aspirin.24 Using this method, a cut-off was defined as the mean binding (12.5% platelet to monocyte aggregates).

Some studies have identified a link between oestrogens and platelet function in regard to the PlA polymorphism.25, 26 The authors of these papers concluded that oestrogens may protect against the effect of the polymorphism. Analysis of the sexes individually was therefore performed.

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Results 

45 patients were recruited into the study and divided into 2 groups; PlA1 homozygotes and PlA2 homozygotes/heterozygotes. The two groups were well matched in terms of demographics (see Table 1), although significantly more PlA2 carriers had had a previous stroke or TIA than the PlA1 homozygotes. 11 patients (24.4%) carried the PlA2 allele with the remaining 34 patients belonging to the PlA1 homozygote group in concordance with previously published figures.4 2 patients were homozygous for the PlA2 allele (1 male, 1 female) and were included with the heterozygotes for the purpose of analysis.

Table 1. Patient demographics
PlA1PlA2PlA1 v PlA2
Mean age68.865.9p=0.421*
Male (%)26 (76.5)6 (54.5)p=0.163**
Active smokers (%)9 (26.5)5 (45.5)p=0.237**
Mean cholesterol4.844.77p=0.841*
Ischaemic heart disease (%)14 (41.1)4 (36.4)p=0.777**
Previous cerebral event (%)0 (0)3 (27.3)p=0.002**

Percentage of total patients shown in brackets.

∗Mann-Whitney test.

∗∗Chi-Square test.

Overall Platelet Monocyte Aggregates (PMA) showed a non-significant (p=0.17, Mann-Whitney Test) trend towards a difference between the genotypes (Fig. 1). Further analysis of the PMA was carried out by defining a cut-off value at which the platelet was not effectively inhibited from aggregation by aspirin (Mean aggregation, 12.5%). 38% of the population lay above this value and would be considered aspirin resistant. A chi-squared analysis reveals significantly more PlA2 carriers in the aspirin resistant group (p=0.04) and is shown in Fig. 2.

  • View full-size image.
  • Figure 1 

    Platelet Monocyte Aggregation in PlA1 versus PlA2. Mann-Whitney test shows a trend towards higher binding in PlA2 genotype but is not significant (p=0.17) in both sexes combined (left). PMA formation by genotype in males only is significant (p=0.019) (right).

  • View full-size image.
  • Figure 2 

    Aspirin resistance as defined by PMA formation above the mean aggregation of 12.5%. Significantly more patients with the PlA2 allele are found in the aspirin resistant group (p=0.0418, Chi-squared test). This association is stronger in the males (p=0.0101) but not evident in the females (p=0.9282).

Sex differences 

The females in the study showed no significant difference between the genotypes (p=0.62, Mann-Whitney test), but the males showed stronger differences than in mixed sex, achieving significance in the PMA test directly (10.9% PlA1 versus 18.4% PlA2, p=0.02, Mann-Whitney test) and using an aspirin resistance cut-off value of the mean PMA as previous (p=0.01, Chi-squared test). These results are shown in Figure 1, Figure 2.

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Discussion 

In this study we have shown that the PlA1/A2 polymorphism primarily affects the aggregation of platelets to monocytes in male claudicants. The effect is not observed in females and understanding the mechanism behind this may be key to understanding the way the polymorphism alters platelet function in the presence of aspirin. Platelet-monocyte interaction has been shown previously in clinical situations such as during cardiopulmonary bypass,27 angioplasty,28, 29, 30 stroke31 and ischaemic heart disease.32, 33 The role of these aggregates is not fully understood, but it is known that leukocytes are activated by such an interaction with platelets.34, 35 Platelet adhesion to monocytes is mediated by P-selectin (CD62P), the counter receptor on monocytes being PSGL-1.36 There is also evidence to show that GP IIb/IIIa has a role to play in platelet-leukocyte interactions,37, 38 monocytes binding fibrinogen via MAC-133, 39 to this integrin. May et al. showed the binding of platelets to monocytes appeared to be blocked by antiplatelet agents,33 although they used a combination of agents in their study, possibly negating the effects of resistance to the agents. A plausible explanation of the results seen here is that the polymorphism directly affects either the GP IIb/IIIa receptor, or the aspirin effect on that receptor, and hence alters the interaction of the platelet and monocyte. This modified reaction has the potential to worsen atheroma via inflammatory and other pathways, leading to poorer clinical outcomes seen in various studies of the PlA1/A2 polymorphism. This complex chain of events may also go some way to explaining the heterogeneous results of other studies, as different methods of assessing platelet function may not be as sensitive to the effects shown here. Indeed, platelet-monocyte aggregates have been shown to be one of the most sensitive methods of detecting platelet activation available to researchers.40

In our study, we were unable to draw any conclusions about the difference between heterozygote carriers of the PlA2 allele and homozygote carriers due to the small number of homozygotes in our study. However, it is interesting to note that both homozygotes were in the aspirin resistant group, and moreover, the male homozygote had the highest PMA binding in the study at 32.13% (compared to a mean of 12.5%). Larger studies would enable the separate analysis of the 3 genotypes to further delineate any cumulative effect of the PlA2 allele.

The sex difference seen in this study may simply reflect the background difference in platelet activation between men and women. However, there is some evidence to show that oestrogens may play a role in the pathophysiology of the polymorphism25, 26 and as such may lead to the pronounced effect in men as seen here. As all our patients are post-menopausal, oestrogen levels alone cannot fully explain the effect seen here.

The interaction is a complex one between oestrogen levels and the levels/number/types of receptors for these oestrogens on the platelet surface. It is more likely that it is this interaction that modulates the effect rather than the amount of oestrogen per se. Studies have shown that the platelet-monocyte interaction varies with the menstrual cycle41 and also that after the menopause, there is a decreased platelet activation status compared with premenopausal women.42 Clearly further work needs to be done to investigate this important link between sex and platelet activation and the effect of this link on genetic polymorphisms. To date no study has shown such a marked difference between men and women with the polymorphism in-vivo.

The issue of compliance with medication is a possible factor in the resistance to aspirin. However, we have no reason to believe that there should be a difference in compliance due to the polymorphism studied here.

The future of antiplatelet therapy is likely to involve ‘tailoring’ a regimen to individual patients. Large studies have shown unequivocally the benefit of aspirin to the population,43, 44, 45, 46 however this effect is not universal to the individual and the reasons behind this are only just becoming clear. Studies such as CAPRIE47 reveal an improvement in outcome when using Clopidogrel compared to aspirin and a more pronounced benefit is seen when combined agents are used, as in the CURE trial.48 These studies suggest that certain patients do better on agents other than aspirin and by identifying these patients they can be treated with higher dose, alternative or combined antiplatelet therapies. Identifying the patients at high-risk of vascular events despite taking aspirin is now the subject of vigorous research. It is anticipated that surrogate tests such as genetic screening for recognised polymorphisms such as PlA1/A2 will form the basis of the individualisation of antiplatelet therapy. Clearly much more work is needed before we can fully understand the basis of aspirin resistance, along with work to look at resistance to other agents and whether there is a benefit to swapping agents in-vitro. After this, larger clinical trials will be needed to determine if those results seen in the laboratory setting transfer to genuine clinical benefit. This study adds further weight to the growing body of evidence to show that patients with the PlA2 allele have higher levels of platelet activation than those without, even in the presence of aspirin, and that this may be one of a host of genes soon to be screened in the routine treatment of patients with atheroma.

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Acknowledgements 

The authors would like to acknowledge the help from staff at the School of Surgical and Reproductive Sciences flow cytometry facility, Newcastle University for their technical help with the project.

Funding for this project was by Gateshead Health NHS Foundation Trust research and development fund. There was no direct involvement of the sponsors in the design, running or write-up of the study.

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Conflict of Interests 

None declared.

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PII: S1078-5884(08)00155-X

doi:10.1016/j.ejvs.2008.02.016

European Journal of Vascular & Endovascular Surgery
Volume 36, Issue 2 , Pages 132-137, August 2008

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