The [Pl.sup.A] Polymorphism of Glycoprotein IIIa Functions as a Modifier for the Effect of Estrogen on Platelet Aggregation.
Platelet aggregation, via the platelet receptor glycoprotein (GP) IIb-IIIa, is instrumental to the progression of CHD and to the development of coronary thrombosis responsible for acute ischemic coronary syndromes. A polymorphism of GPIIIa (integrin [[Beta].sub.3]), [Pl.sup.A2], has been found to be associated with an increased incidence of intravascular thrombosis. However, the status of [Pl.sup.A2] as a risk factor for CHD remains a controversial issue (for review, see reference 7). Nevertheless, this polymorphism was shown to increase platelet responsiveness.[8,9]
Based on the results of previous studies, we hypothesized that estrogen inhibits platelet aggregation, and that this effect could vary according to the [Pl.sup.A] polymorphism. Furthermore, since aspirin is widely used as an antiplatelet agent in patients with CHD, the combination of aspirin and estrogen was studied on either [Pl.sup.A1/A1] or [Pl.sup.A1/A2] platelets. Standard light transmission ("turbidimetric") platelet aggregometry is designed to detect platelet hypofunction in the evaluation of hemorrhagic conditions. Platelet aggregation studied in vitro with this assay has also been established as a reliable way to titrate specific platelet antagonists applied to patients with CHD. Therefore, we used light transmission assay to test our hypothesis.
Studies were performed on platelets obtained from 20 healthy men (10 [Pl.sup.A1/A1] and 10 [Pl.sup.A1/A2]) and 10 premenopausal healthy women (5 [Pl.sup.A1/A1] and 5 [Pl.sup.A1/A2]). The [Pl.sup.A1/A1] and [Pl.sup.A1/A2] individuals were matched for age and race. After establishing each individual's [Pl.sup.A] genotype, repeat phlebotomy was performed for functional studies. Blood was drawn through a 19-gauge needle into 3.2% sodium citrate between 7 AM and 9 AM and in the fasting state. Volunteers were healthy and had not taken medications for at least the preceding 10 days. The first 4 mL of drawn blood was discarded; platelet-rich plasma was prepared as described[9,12] and was incubated at 37[degrees]C for 30 minutes, with or without inhibitors, prior to the addition of agonists and stirring.[11,12]
The platelet counts were adjusted to a range of 3 X [10.sup.5] to 4 X [10.sup.5] platelets per microliter. Epinephrine and adenosine diphosphate (ADP) have been shown to be [Pl.sup.A2]-sensitive agonists and consequently were selected as the agonists used for this study. Response of [Pl.sup.A1/A1] and [Pl.sup.A1/A2] platelets to agonists can also vary independently of the [Pl.sup.A2] polymorphism. Therefore, to normalize differences in responsiveness to agonists between [Pl.sup.A1/A1] and [Pl.sup.A1/A2] platelets, a titrated dose of agonist was selected to generate a uniform aggregation response, as required for studies with inhibitors. Hence, epinephrine and ADP dose-response curves were established for each individual, and the lowest dose for each agonist that provided greater than 60% aggregation was selected for further experiments with inhibitors.
We tested the effect of 17[Beta]-estradiol ([E.sub.2]: [10.sup.-11], [10.sup.-10], [10.sup.-9], and [10.sup.-8] mol/L), aspirin (0.056, 0.56, 5.6, and 56 [micro]mol/L) alone, and aspirin (0.056 to 56 [micro]mol/L) plus [E.sub.2] ([10.sup.-10] mol/L) on platelet aggregation using the turbidimetric assay.[11,12] The effect of ICI 182780 (ICI), an antiestrogen molecule that competes for the estrogen receptor on platelet aggregation in relation to estrogen inhibition and the [Pl.sup.A] polymorphism was studied in a subset of the population. The genotyping of the subjects was performed as reported elsewhere. Experiments studying platelet aggregation were performed without knowledge of the [Pl.sup.A] genotype. Informed consent was obtained from all subjects, and the study protocol was approved by the Human Subject Review Committee of The Ohio State University (Columbus, Ohio).
Because of the known effect of gender on platelet responsiveness, as well as the difference in blood concentration of endogenous estrogen, data on platelets from women and men were analyzed separately. Two-way analysis of variance was used to define the significance of overall differences between the effect of [E.sub.2] alone, [E.sub.2] plus aspirin, and [E.sub.2] plus ICI on aggregation of platelets from [Pl.sup.A1/A1]- and [Pl.sup.A1/A2]-positive individuals. When overall differences were statistically significant, a t test was used to define the significance of differences between specific points. A P value of less than .05 was considered statistically significant.
The average dose of epinephrine required to induce more than 60% aggregation was 4.9 [+ or -] 0.8 [micro]mol/L for [Pl.sup.A1/A1] and 4.2 [+ or -] 1.1 for [Pl.sup.A1/A2] individuals (P = .67), and the average close of ADP was 4.6 [+ or -] 0.6 [micro]mol/L for [Pl.sup.A1/A1] and 3.8 [+ or -] 0.6 for [Pl.sup.A1/A2] individuals (P = .34).
Using men's platelets, we found that the inhibitory effect of [E.sub.2] on aggregation induced with epinephrine was more pronounced in [Pl.sup.A1/A2] than [Pl.sup.A1/A1] platelets (Figure 1). For [Pl.sup.A1/A2] platelets, aggregation was significantly inhibited with all 4 (physiologic) concentrations of [E.sub.2], while in [Pl.sup.A1/A1] platelets, aggregation decreased significantly only with the highest concentration of [E.sub.2] ([10.sup.-8] mol/L). The concentration of [E.sub.2] required to inhibit [Pl.sup.A1/A1] platelet aggregation to the extent of [Pl.sup.A1/A2] platelets was greater by at least 3 orders of magnitude ([is greater than]1000-fold). The inhibitory effect of [E.sub.2] on platelet aggregation induced with ADP was also significantly greater in [Pl.sup.A1/A2] compared to [Pl.sup.A1/A1] platelets (P [is less than] .01), although overall, inhibition of platelet aggregation by [E.sub.2] was less pronounced when ADP was used as the agonist, relative to platelets activated with epinephrine (Table). The addition of ICI [10.sup.-9] mol/L significantly reduced (P [is less than] .01) the inhibition of platelet aggregation provided by [E.sub.2] ([10.sup.-10] mol/L). Hence, in contrast with experiments performed in the absence of ICI, there was no significant difference in platelet aggregation between [Pl.sup.A1/A1] and [Pl.sup.A1/A2] platelets when exposed to [E.sub.2] and ICI (Figure 2).
[Figures 1-2 ILLUSTRATIONS OMITTED]
We also studied the inhibition of epinephrine-induced platelet aggregation by [E.sub.2] alone ([10.sup.-10] mol/L), aspirin alone (0.056 to 56 [micro]mol/L, covering the spectrum of therapeutic concentrations for CHD), or [E.sub.2] ([10.sup.-10] mol/L) plus aspirin (0.056 to 56 [micro]mol/L). As we reported previously,[9,11] low concentrations of aspirin decreased platelet aggregation induced by epinephrine more efficiently with [Pl.sup.A1/A2] than with [Pl.sup.A1/A1] platelets. Estrogen had no additional inhibitory effect on aggregation once platelets were already exposed to aspirin prior to the addition of [E.sub.2]. Thus, the combined effect of [E.sub.2] [10.sup.-10] mol/L plus aspirin was similar to that observed with aspirin alone.
The effect of exogenous [E.sup.2] was also studied on aggregation of platelets obtained from premenopausal women, using epinephrine and ADP as agonists (Figure 4 and Table). As with men, the addition of [E.sub.2] was found to inhibit the platelets of [Pl.sup.A1/A2]-positive women more efficiently than their [Pl.sup.A1/A1]-positive counterparts.
[Figures 3-4 ILLUSTRATION OMITTED]
Inhibition by Estrogen of Adenosine Diphosphate-Induced Aggregation(*)
[E.sub.2], [Pl.sup.A1/A1] [Pl.sup.A1/A2] P Value mol/L Men (n = 6) 0 76.4 [+ or -] 3.9 77.5 [+ or -] 2.7 NS [10.sup.-11] 76.2 [+ or -] 1.9 67.7 [+ or -] 2.2 0.017 [10.sup.-10] 78.0 [+ or -] 2.6 64.5 [+ or -] 5.0 0.038 [10.sup.-9] 74.5 [+ or -] 3.0 61.0 [+ or -] 2.6 0.007 [10.sup.-8] 57.7 [+ or -] 2.4 46.6 [+ or -] 4.3 0.047 Women (n = 5) 0 81.5 [+ or -] 1.5 74.4 [+ or -] 4.1 NS [10.sup.-11] 82.5 [+ or -] 2.0 72.6 [+ or -] 6.2 NS [10.sup.-10] 73.3 [+ or -] 6.7 64.0 [+ or -] 13.8 NS [10.sup.-9] 82.0 [+ or -] 4.0 67.8 [+ or -] 13.2 NS [10.sup.-8] 60.3 [+ or -] 2.6 44.2 [+ or -] 9.5 NS
(*) Data show percent aggregation - SEM. NS indicates not significant.
Our results demonstrate that the effect of estrogen on platelet aggregation is greater for [Pl.sup.A2]-positive individuals and therefore is genetically determined. According to extensive studies on the predictive value of the in vitro aggregation assay for the titration of specific platelet blockers, one might speculate that the clinical beneficial effect of estrogen on platelet thrombosis would be greater for [Pl.sup.A1/A2]-positive individuals than for [Pl.sup.A1/A1]-positive individuals.
Platelet aggregation after rupture or ulceration of an atherosclerotic plaque is mediated by GPIIb-IIIa, the platelet receptor for fibrinogen and von Willebrand factor. Glycoprotein IIb-IIIa, particularly the GPIIIa subunit, has been found to be highly polymorphic. One polymorphism of GPIIIa, [Pl.sup.A2], has been shown to be associated with a higher incidence of intra-arterial thrombosis compared to [Pl.sup.A1/A1]. Although the status of [Pl.sup.A2] as a risk factor for CHD has remained controversial,[7,17] the proaggregatory effect of this polymorphism is not[8,9] and has been attributed to a lower threshold for platelet aggregation on exposure to agonists. It is estimated that 25% to 30% of whites and 15% to 20% of Afro Caribbeans carry at least 1 [Pl.sup.A2] allele ([Pl.sup.A1/A2] or [Pl.sup.A2/A2]). The [Pl.sup.A2] variant results from the presence of a proline instead of a leucine at amino acid 33 of the GPIIIa subunit, due to a missense mutation with a cytosine substituting for a thymidine at position 1565 in exon II of the GPIIIa gene. The [Pl.sup.A1] allele codes for a leucine at position 33 of GPIIIa, while the [Pl.sup.A2] allele codes for a proline residue. We show here that this leucine to proline substitution dramatically increases platelet sensitivity to the inhibitory effect of estrogen on platelet aggregation.
The effect of estrogen on platelet aggregation is mediated via an estrogen receptor (ER[Alpha] or ER[Beta]), as demonstrated by the inhibitory effect of ICI, a specific estrogen receptor inhibitor. The mechanism(s) by which estrogen provides its inhibitory effect is not yet fully characterized, but 2 aspects of the pathway involved are clear: (1) it involves the [Pl.sup.A] polymorphism of the GPIIIa subunit of the fibrinogen receptor, and (2) it could not be mediated by a classic genomic effect of the estrogen receptor, as platelets are anucleated fragments derived from megakaryocytes. It was also instructive to learn that the effect of aspirin and that of estrogen on platelet aggregation are overlapping. Over a large spectrum of aspirin concentrations, the addition of estrogen to aspirin had no additional inhibitory effect on platelet aggregation. Estrogen has been shown in endothelial cells to up-regulate cyclooxygenase-1 (COX-1) expression and activity, resulting in increased prostacyclin (PGI2) production. Aspirin inhibits COX-1 activity and thromboxane A2 production. Our data suggest that the nongenomic effect of the estrogen receptor detected in this study might involve prostaglandins and the GPIIIa subunit of the fibrinogen receptor.
Moreover, our findings suggest that the beneficial inhibitory effect of estrogen on platelet aggregation may be lost in patients already treated with aspirin and vice versa. Such findings should be further investigated in the context of studies in which patients with CHD are treated concurrently with aspirin and HRT. Finally, our data on estrogen inhibition of platelet aggregation provide another potential beneficial effect of HRT for the prevention of cardiovascular events, in addition to other effects reported previously. It was unexpected that the effect of [E.sub.2] on aggregation would be readily detectable on the platelet-rich plasma of premenopausal women. In spite of the known higher concentrations of endogenous [E.sub.2] present in the blood of premenopausal women relative to men, the inhibitory effect of exogenous [E.sub.2] on aggregation of women's platelets was readily detectable. Our findings suggest that the ICI-inhibitable receptor for estrogen on the surface of platelets may bind preferentially to free [E.sub.2] relative to protein-bound [E.sub.2] found in the blood, and that addition of exogenous [E.sub.2] increases the fraction of the unbound hormone.
Mr Boudoulas was a recipient of The Samuel J. Roessler Research Scholarship of The Ohio State University College of Medicine and Public Health, Columbus, Ohio. Dr Goldschmidt-Clermont is the recipient of an Established Investigator Award of The American Heart Association (Dallas, Tex), of National Institutes of Health (Bethesda, Md) grants GM-53236 and HL-52315, and of a grant from the Bremer Foundation. The technical support of Youm M. Pham and Yiwen Liu-Stratton is greatly appreciated.
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Accepted for publication August 30, 2000.
From the Heart and Lung Institute and Division of Cardiology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio (Mr Boudoulas and Drs Cooke and Roos); Thrombosis Research Section, Baylor University, Houston, Tex (Dr Bray); and Division of Cardiology, Department of Medicine, Duke University, Durham, NC (Dr Goldschmidt-Clermont).
Presented at the Ninth Annual William Beaumont Hospital DNA Technology Symposium, DNA Technology in the Clinical Laboratory, Royal Oak, Mich, April 13-15, 2000.
Reprints: Pascal J. Goldschmidt-Clermont, MD, Division of Cardiology, Duke University Medical Center, DUMC 3845, Durham, NC 27710.
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|Author:||Boudoulas, Konstantinos D.; Cooke, Glen E.; Roos, Christine M.; Bray, Paul F.; Goldschmidt-Clermont,|
|Publication:||Archives of Pathology & Laboratory Medicine|
|Date:||Jan 1, 2001|
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