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The [Pl.sup.A] Polymorphism of Glycoprotein IIIa Functions as a Modifier for the Effect of Estrogen on Platelet Aggregation.

The progression of coronary heart disease (CHD) accelerates markedly after menopause, and this increase is at least partially due to the drop of endogenous estrogen production by the ovaries.[1] Therefore, it was hypothesized that hormone replacement therapy (HRT), through the reduction of risk factors for CHD, may decrease the incidence and/or progression of CHD in postmenopausal women.[2] However, HRT, when tested in double-blind, randomized, prospective secondary prevention studies resulted in an early increase in coronary events (Heart and Estrogen/Progestin Replacement Study),[3] while late benefit from HRT has been deemed unlikely.[4] Our inability to account for the early increase in coronary events observed in the Heart and Estrogen/Progestin Replacement Study highlights the need for additional research on the effects of HRT on the cardiovascular and clotting systems.[4]

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.[6] 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,[10] and that this effect could vary according to the [Pl.sup.A] polymorphism.[11] 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.[12] Therefore, we used light transmission assay to test our hypothesis.

METHODS

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.[11] 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.[8] 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.[11] 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.[11]

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[13] 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.[6] 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.[14] 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.

RESULTS

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),[11] 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.

COMMENT

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.[15] One polymorphism of GPIIIa, [Pl.sup.A2],[16] has been shown to be associated with a higher incidence of intra-arterial thrombosis compared to [Pl.sup.A1/A1].[6] 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]).[18] 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.[16] 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.[16] 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.[13] 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.[19] 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.[3] 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.

References

[1.] Barrett-Connor E, Bush TL. Estrogen and coronary heart disease in women. JAMA. 1991;265:1861-1867.

[2.] The Writing Group for the PEPI Trial Effects of estrogen or estrogen/progestin regimens on heart disease risk factors in postmenopausal women: The Postmenopausal Estrogen/Progestin Interventions (PEPI) Trial. JAMA. 1995;273:199-208.

[3.] Hulley S, Grady D, Bush T, et al. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. JAMA. 1998;280:605-613.

[4.] Herrington DM, Reboussin DM, Brosnihan KB, et al. Effects of estrogen replacement on the progression of coronary-artery atherosclerosis. N Engl J Med. 2000;348:522-529.

[5.] Mendelsohn ME, Karas RH. Review articles: mechanisms of disease: the protective effects of estrogen on the cardiovascular system. N Engl J Med. 1999; 340:1801-1811.

[6.] Weiss EJ, Bray PF, Tayback M, et al. A polymorphism of a platelet glycoprotein receptor as an inherited risk factor for coronary thrombosis. N Engl J Med. 1996;334:1090-1094.

[7.] Goldschmidt-Clermont PJ, Roos CM, Cooke GE. Platelet PlA2 polymorphism and thromboembolic events: from inherited risk to pharmacogenetics. J Thromb Thrombolysis. 1999;8:89-103.

[8.] Feng D, Lindpaintner K, Larson MG, et al. Increased platelet aggregability associated with platelet GPIIla PlA2 polymorphism: the Framingham Offspring Study. Arterioscler Thromb Vasc Biol. 1999;19:1142-1147.

[9.] Michelson AD, Furman MI, Goldschmidt-Clermont P, et al. Plateiet GP IIIa Pl(A) polymorphisms display different sensitivities to agonists. Circulation. 2000; 101:1013-1018.

[10.] Nakano Y, Oshima T, Matsuura H, Kajiyama G, Kambe M. Effect of 17betaestradiol on inhibition of platelet aggregation in vitro is mediated by an increase in NO synthesis. Arterioscler Thromb Vasc Biol. 1998;18:961-967.

[11.] Cooke GE, Bray PF, Hamlington J, Pham DM, Goldschmidt-Clermont PJ. [Pl.sup.A2] polymorphism and efficacy of aspirin. Lancet. 1998;351:1253.

[12.] Goldschmidt-Clermont PJ, Schulman SP, Bray PF, et al. Refining the treatment of women with unstable angina: a randomized, double-blind, comparative safety and efficacy evaluation of Integrelin versus aspirin in the management of unstable angina. Clin Cardiol. 1996;19:869-874.

[13.] Long BJ, Tilghman SL, Yue W, Thiantanawat A, Grigoryev DN, Brodie AM. The steroidal antiestrogen ICI 182,780 is an inhibitor of cellular aromatase activity. J Steroid Biochern Mol Biol. 1998;67:293-304.

[14.] Faraday N, Goldschmidt-Clermont PJ, Bray PF. Gender differences in platelet GPIIb-IIIa activation. Thromb Haemost. 1997;77:748-754.

[15.] Nurden AT. Polymorphisms of human platelet membrane glycoproteins: structure and clinical significance. Thromb Haemost. 1995;74:345-351.

[16.] Newman PJ, Derbes RS, Aster RH. The human platelet alloantigens, [Pl.sup.A1] and [Pl.sup.A2], are associated with a leucine33/proline33 amino acid polymorphism in membrane glycoprotein Ilia, and are distinguishable by DNA typing. J Clin Invest. 1989;83:1778-1781.

[17.] Ridker PM, Hennekens CH, Schmitz C, Stampfer MJ, Lindpaintner K. PlA1/ A2 polymorphism of platelet glycoprotein IIIa and risks of myocardial infarction, stroke, and venous thrombosis. Lancet. 1997;349:385-388.

[18.] Kim HO, Jin Y, Kickler TS, Blakemore K, Kwon OH, Bray PF. Gene frequencies of the five major human platelet antigens in Afro Caribbean, white, and Korean populations. Transfusion. 1995;35:863-867.

[19.] Jun SS, Chen Z, Pace MC, Shaul PW. Estrogen upregulates cyclooxygenase-1 gene expression in ovine fetal pulmonary artery endothelium. J Clin Invest. 1998;102:176-183.

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
Geographic Code:1USA
Date:Jan 1, 2001
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