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From Medicine/Thrombosis Research (X.-H.L., W.Z., P.F.B.), Baylor College of Medicine, Houston, Tex; and Vascular Biology (B.N.), Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Germany.
Correspondence to Paul F. Bray, MD, Thrombosis Research Section, Baylor College of Medicine, One Baylor Plaza, BCM 286, N1319, Houston, TX 77030. E-mail pbray{at}bcm.tmc.edu
| Abstract |
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Key Words: estrogen glycoprotein VI hormone replacement therapy platelets
| Introduction |
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| Materials and Methods |
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Assessment of Estrogen Activity
The biologic effect of estrogen was assessed by changes in mouse uteri,5 because plasma E2 levels do not represent the biologic effect of estrogen activity in the oral CEE study and because the oral delivery follows an absorption metabolic decay curve. The doses used in this study produced an effect on the uterus similar to that observed with human ERT dosing. For completeness, plasma E2 was measured (Diagnostic Systems Laboratories) and was below the lower limit of detection (<1.5 pg/mL) after oral administration of CEE and E2 (probably attributable to the timing of plasma collection, which was
18 hours after last gavage) and was 49.1±33.1 pg/mL after SQ administration (normal E2 is 31±13 to 58±27 pg/mL6).
Platelet Preparation and Analysis
Preparation of washed platelets, flow cytometric analysis of platelet size and fibrinogen binding, and platelet aggregation were performed as previously described.7 To maximize quality control, only 1 pair of mice (1 hormone-treated and 1 placebo-treated) were studied per day. COL-RP was synthesized and crosslinked with glutaraldehyde. Submaximal concentrations of COL-RP were used to assess platelet reactivity: 0.02 µg/mL for flow cytometry and 0.01 µg/mL for aggregation. Surface expression of platelet adhesive receptor glycoproteins (GPs) were quantified using PE-conjugated anti-GPIb
(Xia.G5-PE, Emfret Analytics, Würzburg, Germany), FITC-conjugated anti-
IIb (anti-CD41, BD Pharmingen), and anti-GPVI antibodies.8
Effects of ERT are presented as mean±SE percent change from the placebo value and analyzed with a 1-sample t test using StatView software (SAS Institute).
| Results and Discussion |
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In an effort to address the possible mechanisms by which estrogens might affect platelet function, we measured levels of adhesive membrane GPs. No difference between placebo and any estrogen therapy was observed for GPIb
and GPIIb-IIIa (integrin
IIbß3). However, the 3 ERT regimens altered platelet surface GPVI levels in a pattern similar to COL-RPinduced platelet activation (Figure 2A). A strong correlation between COL-RPinduced platelet activation and surface GPVI level was observed in these studies (Figure 2B), suggesting the changes in COL-RPinduced platelet reactivity caused by ERT are mediated in part via GPVI or its downstream signaling pathways.
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This is the first report of ERT regulating GPVI expression and the lack of an ERT effect on GPIb
or GPIIb-IIIa expression (Figure 2A) suggests hormone-specific effects on GPVI expression. The pivotal role of the platelet GPVI collagen receptor in platelet activation and thrombus formation has been demonstrated using selective agonists and GPVI-deficient platelets.8 Modest reductions in GPVI density (
12% less than normal platelet levels) can have a major effect on cell adhesion under high shear conditions.10 Estrogens could act directly on megakaryocytes to regulate GPVI expression (eg, GPVI transcription or altering the expression of a metalloproteinase that induces platelet GPVI shedding). Because our assays assessed the in vivo effects of estrogens, we cannot exclude an indirect effect whereby estrogen could induce nonmegakaryocyte cells to secrete a factor that could regulate platelet GPVI expression.
Additional platelet activation in vivo likely occurs after platelet adhesion, and we also examined the effect of thrombin on platelets from the ERT-treated mice. Neither oral regimen had a significant influence on fibrinogen binding (Figure 3), whereas SQ E2 increased platelet sensitivity to thrombin. Other investigators have also found that transdermal E2 was more active than oral E2 in increasing platelet activation to thrombin.11 Interestingly, the responses to COL-RP and thrombin were not concordant, indicating estrogens influence platelet function in an agonist specific fashion.
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It is important to note that any mechanism whereby estrogen affects platelet function could be influenced by at least 3 variables: non-E2 estrogens in CEE, higher plasma levels of E2 after SQ E2, or the differences attributable to peaks and troughs of oral estrogens compared with the steady levels of SQ estrogens. In addition, because oral estrogen results in high portal vein estrogen levels, at least some of the estrogen effects could be mediated by first-pass metabolism in the liver and altered hepatic gene expression.
In summary, we have shown that in vivo estrogens affect platelet reactivity in an agonist-specific fashion and depend on the hormone formulation and mode of delivery. Altered GPVI expression likely mediated some of these effects. Cautiously extrapolating to postmenopausal women using ERT, these estrogen-induced platelet functional changes could contribute to cardiovascular complications. More human studies are needed to assess specific aspects of vascular complications of hormone therapy; specifically, does oral E2 lack the reported risks of CEE? And is there risks related to daily fluctuations of estrogen levels?
| Acknowledgments |
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| Footnotes |
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| References |
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2. Wilcox JG, Hwang J, Hodis HN, Sevanian A, Stanczyk FZ, Lobo RA. Cardioprotective effects of individual conjugated equine estrogens through their possible modulation of insulin resistance and oxidation of low-density lipoprotein. Fertil Steril. 1997; 67: 5762.[CrossRef][Medline] [Order article via Infotrieve]
3. Gawaz M. Role of platelets in coronary thrombosis and reperfusion of ischemic myocardium. Cardiovasc Res. 2004; 61: 498511.
4. Sit AS, Modugno F, Hill LM, Martin J, Weissfeld JL. Transvaginal ultrasound measurement of endometrial thickness as a biomarker for estrogen exposure. Cancer Epidemiol Biomarkers Prev. 2004; 13: 14591465.
5. Evans JS, Varney RF, Koch FC. The mouse uterine weight method for the assay of estrogens. Endocrinology. 1941; 28: 747752.
6. Gong M, Wilson M, Kelly T, Su W, Dressman J, Kincer J, Matveev SV, Guo L, Guerin T, Li XA, Zhu W, Uittenbogaard A, Smart EJ. HDL-associated estradiol stimulates endothelial NO synthase and vasodilation in an SR-BI-dependent manner. J Clin Invest. 2003; 111: 15791587.[CrossRef][Medline] [Order article via Infotrieve]
7. Leng XH, Hong SY, Larrucea S, Zhang W, Li TT, Lopez JA, Bray PF. Platelets of female mice are intrinsically more sensitive to agonists than are platelets of males. Arterioscler Thromb Vasc Biol. 2004; 24: 376381.
8. Nieswandt B, Watson SP. Platelet-collagen interaction:is GPVI the central receptor? Blood. 2003; 102: 449461.
9. Godsland IF. Effects of postmenopausal hormone replacement therapy on lipid, lipoprotein, and apolipoprotein (a) concentrations: analysis of studies published from 19742000. Fertil Steril. 2001; 75: 898915.[CrossRef][Medline] [Order article via Infotrieve]
10. Sarratt KL, Chen H, Kahn ML, Hammer DA. Platelet receptor glycoprotein VI-mediated adhesion to type I collagen under hydrodynamic flow. Ann Biomed Eng. 2004; 32: 970976.[CrossRef][Medline] [Order article via Infotrieve]
11. Gu M, Xi X, Englund GD, Berndt MC, Du X. Analysis of the roles of 143-3 in the platelet glycoprotein Ib-IX- mediated activation of integrin
IIbß3 using a reconstituted mammalian cell expression model. J Cell Biol. 1999; 147: 10851096.
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