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Trans Am Clin Climatol Assoc. 2006; 117: 103–112.
PMCID: PMC1500939

Platelet Reactivity and Genetics down on the Pharm

Abstract

It is widely accepted that there is marked inter-individual variation in platelet function, although this has not been formally studied in large numbers of subjects. We have recruited and studied three large populations: one to characterize platelet variation and the factors responsible for it; a second to estimate heritabilities of the variation in platelet function; and a third to test for interactions between platelet genes and hormone therapy in postmenopausal women. We find that platelet variability is substantial, inherited and can be reproducibly detected. We genotyped 2145 women in the Heart and Estrogen/progestin Replacement Study followed for coronary heart disease (CHD) events for 6 years and found that common combinations of polymorphisms in the genes for GPIbα and GPVI were associated with hormone therapy harm, benefit or no effect. If corroborated in future studies, platelet genotyping could be used to predict postmenopausal hormone safety.

Introduction

Human blood platelets are two-faced. On the one hand, they are critical for preventing excessive blood loss after vascular injury. Blood is not normally supposed to come into contact with the subendothelium. Elements present in the extracellular matrix, such as collagen, induce platelet plug formation to stop bleeding and begin the repair process. But sometimes platelets do their job too well. In certain settings, such as atherosclerosis, vascular stenoses or other pathologic shear conditions, platelets can become activated and bind adhesive plasma proteins. Platelet plug formation in such settings can result in undesirable vessel occlusion with resultant ischemia (1).

The steps of platelet thrombus formation are shown in Figure 1. Platelets travel through the blood vessel at rapid rates. Initially, platelets attach and roll along the exposed subendothelium. This process requires immobilized von Willebrand factor (VWF) and the VWF receptor on platelets, the glycoprotein (GP) Ib-V-IX complex (2). This interaction has a very fast “on” rate and results in a much slower movement of the platelets and a longer period of platelet contact with the vessel wall. Platelet collagen receptors, with slower “on” rates, are then able to interact with collagen and effect firm adhesion. Figure 2 depicts some of the major receptors and organelles involved in platelet activation and aggregation. Upon attachment and rolling along collagen, GP Ibα and GP VI transmit activation signals inside the platelet. A series of as-yet-incompletely characterized intracellular signaling events induces granule secretion. ADP is released from dense granules and in a positive feedback loop, amplifies platelet activation. Alpha granules fuse with the plasma membrane, increasing P-selectin availability and releasing fibrinogen into the platelet microenvironment.

Fig. 1
Platelet deposition to subendothelium of injured vessel. The three phases of platelet response to an injured vessel are described above. The key platelet adhesive and signaling receptors are shown, as well as their extracellular matrix ligand.
Fig. 2
Molecular basis of platelet activation-aggregation. Shown are the von Willebrand factor receptor, GP Ib-V-IX; the signaling collagen receptor, GP VI; a generic G-protein coupled receptor, GPRC, and the G protein β3 subunit, GNβ3; P-selectin, ...

Simultaneous to many of the previously described reactions, platelet integrin α2β1 is activated, enabling firm adhesion to collagen. Platelet activation also results in the conversion of the GP IIb–IIIa (integrin αIIbβ3) receptor from a low affinity to a high affinity state. High affinity GP IIb–IIIa binds fibrinogen and VWF, which act as bridges to other activated platelets, thus enabling the platelet thrombus to grow and expand. The latter process is often referred to as the “final common pathway” for platelet aggregation and its importance is underscored by the clinical utility of GP IIb–IIIa blockers in the setting of percutaneous coronary interventions (3).

For many years we have been interested in how inherited variations in platelet genes affect platelet function, and hence the risk of bleeding or thrombosis. Genetic studies in the late 1980’s characterized mutations in the genes for GP IIb and GP IIIa that result in quantitative or qualitative defects in the protein product (4,5). This inherited bleeding disorder is called Glanzmann thrombasthenia. In last decade we have tackled a more challenging problem: the identification and characterization of platelet risk factors for arterial thrombosis, specifically myocardial infarction (MI), stroke and peripheral arterial disease (PAD). Platelet risk factors presumably induce the hyperreactivity that has been associated with these clinical disorders of arterial thrombosis (6,7). The primary goals of the research presented herein are to identify genetic and environmental predictors of platelet hyperreactivity and to test for genetic predictors of drug benefit or risk.

Results And Discussion

The current clinical evaluation of platelet reactivity is performed in the setting of bleeding. For this reason, platelets are maximally stimulated in order to diagnose platelet dysfunction or hyporeactivity. Therefore, we designed a series of assays to detect hyperreactivity using standard aggregometry, flow cytometry and shear-dependent assays of platelet function. Early morning phlebotomies were performed on relaxed, supine healthy donors after an overnight fast. Figure 3 shows the marked variability of platelet reactivity in a normal population. Note that at submaximal concentrations of agonist, the extent of aggregation varies from 0% to 100% Similar data were obtained when epinephrine, ADP and ristocetin were used as agonists. Using 0.4 μM epinephrine, a small population was identified with hyperreactive platelets (Figure 4A) (8). Compared to most subjects, platelets from these individuals had a clear hyperreactive phenotype. Such an “outlier” response could be intrinsic to the platelets, or due to technical issues or day-to-day variation. Substantial effort was made to exclude technical issues: samples were processed and assays run by the same technician, with the same reagents and same equipment. Considering the variability in inter-individual platelet responsiveness, it was imperative to determine whether such platelet hyperreactivity was reproducible in the same subject over time. Figure 4B shows the results of our reproducibility study. Twenty-seven different subjects were studied four times each, with each set of studies being performed one week apart. Figure 4B shows the raw data for each visit from all subjects arbitrarily ordered from the lowest to the highest responder. We observed very good reproducibility. Only 7 subjects demonstrated greater than 60% aggregation more than once and 5 of 7 subjects who showed 60% aggregation on at least 2 occasions showed hyperreactivity on each visit. Thus, we conclude that 0.4 μM epinephrine can easily and efficiently identify a reproducible ex vivo platelet hyperreactive phenotype. Based on these findings, the classification of hyperreactivity should be restricted to those individuals who demonstrate more than 60% aggregation to epinephrine on at least two occasions.

Fig. 3
Variation in platelet reactivity. The results of collagen-induced platelet aggregation in citrated plasma using platelets from 280 healthy donors are shown.
Fig. 4
Identification of reproducible platelet hyperreactivity. Panel A, A histogram is shown with the numbers of subjects displayed on the y-axis and percent aggregation to 0.4 μM epinephrine on the x-axis. A total of 359 subjects were studied. Panel ...

Having identified individuals with a hyperreactive platelet phenotype, we considered whether they were distinguished by any demographic variables. Subjects with hyperreactive platelets were more likely to be female (p = 0.015 by chi-squared test) and have higher plasma fibrinogen levels (p = 0.007 by Student’s t test). Platelets from these individuals also displayed increased aggregation to other agonists, excluding an artifact specific to epinephrine and indicating hyperreactivity is typically “global.” We also found a number of factors intrinsic to platelets that were associated with enhanced reactivity (9). At lower levels of stimulation, these platelets more readily secreted their granules. In the unstimulated state, these platelets have a significantly higher expression of GP IIb–IIIa. Finally, there was an association between a hyperreactive platelet phenotype and a common polymorphism in the gene for the β3 subunit of heterotrimeric G proteins. This latter finding dovetails with new data from an on-going study we are conducting at Johns Hopkins University School of Medicine, in which we found significant heritability for platelet aggregation, using both low and high concentrations of epinephrine and ADP (10). Demonstrating that platelet hyperreactivity is reproducible and heritable supports the use of large and unbiased genetic approaches to identify new genes and genetic variations responsible for this phenotype. Such studies are underway.

What is the clinical significance of identifying “functional” genetic variations in platelet genes? Genetic risk factors themselves cannot be modified. Most likely the greatest value is to discover variants that interact with drugs or the environment, since these factors can be modified. Because we have previously shown gender and hormonal effects on platelet function, we have been particularly interested in the possibility of identifying genetic predictors of hormone therapy harm or benefit in postmenopausal women. Perhaps the most publicized clinical trial in history—the Women’s Health Initiative (WHI) study of hormone therapy in postmenopausal women—found that compared to women randomized to placebo, women randomized to estrogen with or without progesterone experienced either more adverse coronary heart disease (CHD) events or no benefit (11–13). Perhaps less publicized was the first ever clinical trial addressing the heart benefits of hormone therapy in women with established CHD. This study, known as the Heart and Estrogen/progestin Replacement Study (HERS), was published in 1996 and found early harm for women receiving estrogen plus progesterone, but an overall lack of benefit after four and nearly seven years of follow-up (14,15). We established a collaboration with Dr. David Herrington at Wake Forest University, one of the principle Investigators in HERS, in order to assess whether genetic variations in platelet genes might affect CHD outcomes in women using hormone therapy. Our analyses focused on genes encoding platelet adhesion/activation receptors involved in the earliest stages of platelet activation, GP Ibα and GP VI (see Figure 2). The positions and approximate allele frequencies of the single nucleotide polymorphisms we studied are illustrated in Figure 5.

Fig. 5
Polymorphisms of platelet genes involved in early activation events.

The −5T/C polymorphism of GP1BA.

We found that there was a higher CHD event rate in carriers of the C allele at both 1 and 6 years in women randomized to placebo. Although this is interesting in and of itself, we were mostly interested in the interaction between genotypes and hormone use. We found that hormone therapy in subjects with the TT genotype was associated with harm, with the greatest risk occurring early after initiation of therapy (16). Intriguingly, hormone therapy was associated with benefit in the 25% of women who carry the C allele of GP1BA.

The −5T/C polymorphism of GP6.

No excess risk for recurrent CHD events was observed with carriage of either allele of the 13254T/C polymorphism in women randomized to placebo. However, we found that hormone therapy in subjects with the TT genotype was associated with slight benefit. But there was evidence for harm for the 30% of women carrying the C allele, with the greatest risk again occurring early after initiation of therapy.

We next determined whether CHD risk assessment could be further refined by considering combinations of these genotypes. There was no significant effect of hormone therapy on CHD risk for women with the most common and least common combinations of genotypes; i.e., TT for each gene and TC/CC for each gene, respectively. This accounts for 62.2% of the 2,090 women in this study. However, for 21.2% of women with the TT GP1BA and TC/CC GP6 genotypes, hormone therapy conferred significant harm. Equally important was the finding that hormone therapy conferred significant benefit for the 16.6% of women with the TC/CC GP1BA and TT GP6 genotypes. The differences in CHD events between the hormone and placebo groups became apparent early and persisted throughout follow-up.

In summary, we have shown that ex vivo platelet hyperreactivity can be defined in a simple and reproducible manner. Platelet hyperreactivity appears global and the molecular mechanisms responsible for this phenotype will likely involve multiple pathways. Prospective studies are needed to correlate ex vivo platelet hyperreactivity with clinical events. This intermediate phenotype will be useful for identifying novel genes and other risk factors for arterial thrombosis. We have also shown that polymorphisms in platelet genes interacted with HT to modify the risk for CHD events for postmenopausal women with established coronary artery disease. Replication of these findings in other cohorts may permit a more rational approach for HT use.

ACKNOWLEDGMENTS

I would like to acknowledge my collaborators in these studies: Donald Yee, Jing-fei Dong and K. Vinod Vijayan (Baylor College of Medicine); David Herrington (Wake Forest University); Diane Becker, Lewis Becker and Nauder Faraday (Johns Hopkins University); and Rasika Mathias (National Institutes of Health). This research has been supported by grants HL65229, HL68829, HL072518, HL074729 from the National Institutes of Health.

DISCUSSION

Wolf, Boston: As you go from TT to TC to CC Mis there a dose effect?

Bray, Houston: Yes.

Colwell, Charleston: I enjoyed it very much. I’d like to return to the increased sensitivity in the normal population that you showed so very nicely with some correlation with fibrogen in normal women. There’s a large literature on platelet hyperreactivity among people with diabetes and it extends down to those with impaired glucose tolerance as well. And I just wonder if in the group of normals you might have had some diabetic individuals?

Bray: We have that data, and there were no diabetics in our population. We didn’t allow anybody taking any kind of prescription medication to be included.

Schafer, Philadelphia: A very nice lecture Paul, as usual. In correlating your in vitro hyper-reactivity data with platelets, as you know, platelets in vivo are exposed to more complex forces such as hemodynamic stimuli and interactions with other cells. Have you had a chance to look to see if the same individuals who are hyper-reactive to epinephrine in vitro are also hyper-reactive in vivo to endogenous catecholamines? For example, following standardized exercise?

Bray: No we haven’t done that. I thought you were going to ask about shear. Some of our assays did include a shear component, and while they were less reproducible than the epinephrine data, there was a correlation between hyper-reactivity to 0.4 micomolar epinephrine and shear-induced platelet aggregation.

Sacher, Cincinnati: I also enjoyed that very much. As a surrogate for platelet turnover, one could sort of look at the mean platelet volume, and since platelets are more reactive when they are younger. Have you looked at any stratification on that basis?

Bray: That’s a great question. I’m very interested in mean platelet volume for quite a few reasons. In fact, there are reports that hormone therapy increases your mean platelet volume. And of all the platelet parameters ever examined Mand there have been very few that have been correlated prospectively with clinical outcomes Mthere are two studies where a higher mean platelet volume was correlated with a clinical outcome. So, we have looked, but we have found no correlation with hyper-reactivity defined by 0.4 micromolar epinephrine.

Stevenson, Stanford: Have you looked into the relevance of your findings to pregnancy? Is there anything that might help us to understand miscarriage and things of that sort? I am curious because pregnancy has hormonal changes and relative glucose intolerance.

Bray: Another good question that we’re quite interested in. In fact we submitted a grant a year ago in which we proposed to examine these issues in pre-eclampsia. There is a literature on platelet reactivity in pregnancy Mnot a lot of literature Mand platelets do appear to be more reactive during pregnancy. The blood of pregnant women contains more platelet micro-particles than the blood of non-pregnant women. And there are even more micro-particles in the blood of patients with pre-eclampsia.

Billings, Baton Rouge: Paul, have you looked at aspirin and plavix in your studies?

Bray: We have, but in the data that I showed from our hyper-reactivity study, subjects were excluded who were using aspirin or non-steroidals because the goal was to develop a database of platelet function. We have banked DNA, and intend to perform additional genetic studies on this material. We are collaborating with Diane and Lewis Becker and Nauder Faraday at Johns Hopkins to identify genes involved in aspirin responsiveness. But to assess the clinical usefulness of our platelet hyper-reactivity definition, we need to collaborate with cardiologists. But we will not be able to use epinephrine as the agonist in such a study if patients are already taking aspirin, since aspirin blocks epinephrine-induced platelet aggregation.

Abboud, Iowa City: Is the hyper-reactivity related in any way to adrenergic receptor density-alpha or beta receptors on platelets?

Bray: We have not measured adrenergic receptor levels. There’s no antibody to the alpha-2 adrenergic receptor, which is the epinephrine receptor on platelets. There is a polymorphism in the alpha-2 adrenergic receptor that we have looked at, but it did not correlate with platelet hyperreactivity.

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