Hormone therapy and platelet function

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Vol. 2, No. 1 2005

Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Tamas Bartfai – Harold L. Dorris Neurological Research Center and The Scripps Research Institute, USA

DISEASE Cardiovascular diseases MECHANISMS

Hormone therapy and platelet function Xing-Hong Leng, Paul F. Bray* Thrombosis Research, Department of Medicine and Pediatrics, Baylor College of Medicine, One Baylor Plaza, BCM 286, N1319, Houston, TX 77030, USA

There is evidence that platelets from women are hyperreactive compared to platelets from men, that

Section Editor: Cam Patterson – Farmal Biomedicines, LLC, USA

endogenous estrogen regulates platelet reactivity, that estrogen therapies modulate platelets response to agonists, and that estrogen acts directly on platelets

Pathophysiology of CHD: sites of estrogen action

in a non-genomic manner. A greater understanding of

CHD is the most common cause of morbidity and mortality among American men and women. MI and other acute coronary syndromes develop when a platelet thrombus forms at the site of a ruptured or eroded unstable coronary atherosclerotic plaque. The pathophysiology of MI is summarized in Fig. 1; atherosclerosis involves the vessel wall and endothelium, lipid metabolism, inflammation; thrombosis is regulated by platelet reactivity, coagulation, fibrinolysis and inflammation. As indicated in Fig. 1, estrogen affects virtually all components of this pathophysiology, but its effects on platelets are the least well studied. There is a substantial body of older evidence indicating that HT is cardioprotective [1]. These studies were consistent with the ability of HT to reduce LDL cholesterol and raise HDL cholesterol, but all were observational in design, such that it is not possible to determine if hormones make women healthy, or healthy women take hormones. Over the past seven years, the results of a series of large randomized clinical trials with HT have consistently shown no benefit, and perhaps even harm, for cardiovascular outcomes, the most prominent studies being the Heart and Estrogen/Progestin Replacement Study (HERS) and the Women’s Health Initiative (WHI) [2–4]. These adverse CHD outcomes occurred in spite of improvements in LDL and HDL cholesterol, suggesting HT may have an adverse effect on thrombosis or inflammation. Furthermore, the risk of HT for CHD events became apparent soon after randomization, suggesting insufficient time for an effect of HT to induce atherosclerosis [2]. Substantial prior research has focused on the relationship between HT use and the development of atherosclerosis. This review focuses on

the complex physiology and pharmacogenetic effects of sex hormones and their receptors on hemostasis will be important for the most rational management of patients using hormone therapy. Introduction The use of postmenopausal hormone therapy (HT) has undergone dramatic and controversial changes in the past three years. Previously, HT had risen to the status of standard of care for postmenopausal women, in some cases as preventative therapy rather than symptom management. But the ‘pendulum of use’ has largely swung in the opposite direction, based on results from randomized clinical trials that showed no benefit or harm for outcomes of breast cancer, stroke and myocardial infarction (MI). Estrogen has a plethora of physiologic effects, such that one might expect a wide distribution of responses to its use. Considering this complexity as well as the small absolute effects of HT for coronary heart disease (CHD) outcomes and some basic research and animal studies suggesting vascular benefit of HT, it is plausible that some women are now being denied these medications even though they are not at risk of harm or might even benefit from HT. In order to know which patients are at risk of HT side effects and which are not, it is desirable to understand better the effects of HT on individual components of disease pathophysiology. *Corresponding author: P.F. Bray ([email protected]) 1740-6765/$ ß 2005 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.ddmec.2005.05.028

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Glossary CEE: conjugated equine estrogen. CHD: coronary heart disease. COX-1: cyclooxygenase-1. The enzyme that converts arachidonic acid to the chemical messengers called prostaglandins. ER: estrogen receptor. GP: glycoprotein. HERS: Heart and Estrogen/Progestin Replacement Study. HT: hormone therapy. Megakaryocyte: the bone marrow precursor cell that generates platelets. MI: myocardial infarction. OCP: oral contraceptive pill. Shear stress: the force generated between two adjacent planes of laminar flow in a viscous solution. VWF: Von Willebrand factor. VWF is an adhesive glycoprotein that mediates platelet adhesion to the exposed subendothelial collagen surface. Quantitative or qualitative defects of VWF results the most common inherited bleeding disorder, von Willebrand disease. Washed platelets: Refers to platelets that have been separated from the plasma by centrifugation and washing in buffer, so the intrinsic platelet response can be studied without the interference of plasma factors. WHI: Women’s Health Initiative. Wiskott–Aldrich syndrome: A rare, X-linked syndrome characterized by reduced numbers of small platelets, eczema and recurrent pyogenic infection.

the influence of HT on thrombosis and especially the effects of estrogen on platelet function.

Platelets and coronary artery disease Platelets are important mediators of both inflammation and thrombosis. Physiologic, pathologic and clinical studies have established a critical role for platelets in acute coronary syndromes [5]. Platelet deposition onto the subendothelium is proportional to the SHEAR STRESS (see Glossary) [6], such that platelets play a particularly important role in arterial thrombosis. Upon arterial plaque rupture, von Willebrand factor (VWF) molecules are rapidly localized to the subendothelium and the initial platelet contact with the wound is a tethering of platelets to this insoluble form of VWF. The platelet receptor mediating this tethering to VWF is the glycoprotein (GP) Iba subunit of the GPIb-IX-V complex [6]. Slowing of the platelets also allows GPVI crosslinking by collagen, which amplifies the signaling to and activation of integrins a2b1 and aIIbb3 (also called GPIIb-IIIa). Subsequent firm platelet adhesion is through integrin a2b1 binding to exposed collagen and aIIbb3 binding to VWF and fibrinogen. An expanding thrombus ensues when platelets aggregate via the intercellular bridging of fibrinogen and VWF binding to the activated conformation of aIIbb3. Recruitment and exposure of tissue factor initiates coagulation, which results in fibrin deposition upon and stabilization of the newly formed platelet thrombus. Antithrombin, the Protein C anticoagulant 86

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pathway and the fibrinolytic system regulate fibrin generation. Blood flow ceases when an occlusive platelet–fibrin plug forms.

Gender effects on platelet reactivity Several studies have shown female platelets are hyperreactive compared to male platelets [7,8]. We have shown that WASHED PLATELETS from healthy women [8] and from female siblings of patients with premature CHD [9] bind significantly more exogenous fibrinogen than do their male counterparts. We have also shown that washed platelets (see Glossary) from female mice are hyperreactive to low concentrations of agonists compared with platelets from their male siblings [10]. These differences are independent of cyclooxygenase-1 (COX1, see Glossary) and secreted ADP, platelet size, surface expression of aIIbb3 and GPIb-IX-V, and were not blocked by apyrase or aspirin. Thus, in both these human and animal studies the platelets of females were more reactive than those of males and the sex differences we observed were intrinsic to platelets.

Rationale for considering hormonal effects on platelets The basis for the gender difference in platelet function is likely to be very complex. If platelet reactivity were the sole cause of coronary ischemic events, premenopausal women would be expected to experience more, not fewer, MIs than men of comparable age. However, high estrogen states like menses and childbirth give women a unique need for exceptional hemostasis. Although some portion of these gender differences in platelet function may be non-hormonal – rather a difference between XX and XY platelets (e.g. the genes encoding the androgen receptor and the WISKOTT– ALDRICH SYNDROME (see Glossary) protein are on the X chromosome) – there are many lines of evidence supporting a hormonal effect on platelets. Fig. 2 summarizes possible pathways and mediators by which estrogen could modify platelet function.

Endogenous sex hormones and human platelet function (see Glossary) use 3-hydroxysteroid dehydrogenase to synthesize 17b-estradiol (E2), which regulates platelet formation [11]. There is intriguing evidence that the platelet content of E2 is higher than the plasma concentration in both males and females [12]. In this case, it is possible that platelet localization at the sites of vessel injury could result in supra-physiologic local concentrations of E2. Several physiologic properties of platelets from women have been shown to vary with the phase of the menstrual cycle, with pregnancy and with hormone use, strongly suggesting that estrogen, progesterone and/or other hormones regulate platelet activation and function. In our studies of platelets from women during different phases of their menstrual cycles, we found enhanced platelet fibrinogen binding MEGAKARYOCYTES

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Figure 1. Schematic of sites of estrogen action in arterial occlusion. Estrogen acts on various tissues and modulates pathologic processes that contribute to the pathophysiology of coronary heart disease. Some critical mediators in each category are (1) in endothelium: estrogen receptors, endothelin-1, cytokines, growth factors, adhesion molecules, prostaglandins (e.g. prostacyclin), nitric oxide (NO), NO synthase and signaling molecules (e.g. PI3K, Akt, MAPK); (2) inflammation: estrogen receptors, cell adhesion molecules (e.g. ICAM-1, VCAM-1, E-selectin), monocyte chemoattractant protein-1, C-reactive protein, tumor necrosis factor-a and interleukin-6; (3) atherosclerosis: estrogen receptors, NO, HDL-cholesterol, antioxidants, oxidized LDL, lipoprotein(a), tissue factor, cytokines, reactive oxygen species and metalloproteinases. The mediators of estrogen effects on fibrin formation/lysis and platelets are largely unknown. The consequences of estrogen on these components of CHD pathophysiology are discussed in the table and text.

in the luteal phase [8]. Platelet adhesion to collagen and a2adrenergic receptor expression vary by phase of the menstrual cycle [13,14]. Platelet aggregation is enhanced during human pregnancy [15] and pregnant mice lacking the estrogenmetabolizing gene, estrogen sulfotransferase, have elevated

blood levels of estrogen, increased platelet reactivity and spontaneous fetal loss due to placental thrombosis [16]. Numerous human studies have suggested some benefit to antiplatelet therapy in recurrent spontaneous abortion complicated by placental thrombosis.

Figure 2. Possible pathways by which estrogen affects platelets. Estrogen can act on platelets directly (e.g. non-genomic means) or via the megakaryocyte precursor. Estrogen can also affect megakaryocyte/platelet function indirectly through other systems; for example, by either changing the threshold of platelet activation or modifying the production of endogenous platelet agonists or inhibitors. HSD, 3b-hydroxysteroid dehydrogenase; EST, estrogen sulfotransferase.

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Exogenous sex hormones and clinical thrombosis outcomes There is little controversy regarding the relationship between hormone use and the risk of venous thrombosis. An association between oral contraceptive pill (OCP) use and arterial thrombosis has been noted for nearly 30 years and a WHO multicenter study showed a fivefold increased risk of MI in OCP users [17]. OCPs appear to enhance platelet reactivity, adhesiveness and aggregation [18,19]. Our own studies found increased platelet fibrinogen binding to washed platelets from women taking OCPs [8]. As mentioned above, recent randomized clinical trials have indicated HT is a risk for stroke and MI [2–4]. Estrogen acts directly upon hematopoietic cells [20], and oral HT and high-dose subcutaneous E2 increases the number of bone marrow megakaryocytes [21]. The only large study (the Framingham Study with 768 women) found that HT use was associated with increased ADP-induced platelet aggregation (P = 0.03) [22]. Smaller studies (less than 20 subjects) have been inconsistent regarding the effects of HT on platelet function, finding platelet reactivity to be increased, decreased or not changed, and most did not consider confounding variables that affect platelet function (e.g. cigarette use, plasma factors, etc.). The few available data on the effect of progestins on platelet effect function are controversial, with reports that

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progesterone can enhance [23] or inhibit [23] platelet activity. Our own studies suggest that progesterone does not activate platelets in vivo or in vitro (unpublished data).

Variables contributing to controversial effects of HT Intrinsic effects Almost all studies have used platelets either in whole blood or in plasma, such that it is not possible to know whether the effect of hormone is directly on the megakaryocyte/platelet or affects other components of the assay, such as plasma proteins. Estrogens affect plasma levels of coagulation factors, anticoagulants and proteins in the fibrinolytic pathways (Table 1). Overall, the impression is that HT favors thrombosis with elevations of coagulation factors and depressions of anticoagulants and fibrinolytic factors. Studying platelet function in the presence of plasma does not allow evaluation of the hormone effect on platelets. For this reason we have focused our studies on washed platelets in order to assess the intrinsic nature of hormone effects on platelets. Our studies with both human and mouse washed platelets have shown enhanced reactivity in female platelets compared with male platelets [8–10]. Preliminary studies in ovariectomized mice suggest that HT modulates platelet sensitivity to agonists, with conjugated equine estrogen (CEE) (see Glossary)-treated mice displaying enhanced sensitivity to collagen peptides (unpublished observations).

Table 1. Summary of postmenopausal hormone therapy-induced changes in hemostasis biomarkers No change Coagulation Fibrinogen Factor VIII Factor VII activity Factor VII antigen Factor IX Activated protein C ratio D-dimer Prothrombin F1.2 Von Willebrand factor Anticoagulants Antithrombin Protein C Protein S Tissue factor pathway inhibitor Fibrinolysis Plasminogen activator inhibitor-1 Tissue plasminogen activator Plasminogen Thrombin activatable fibrinolysis inhibitor Miscellaneous Viscosity Homocysteine C-reactive protein

Increase

Decrease

3+ 3+ ns

3+ ns 1+ ns 1+ 2+

ns

3+

ns 1+

ns

4+ ns 4+

1+ ns

None None Thrombotic ns ns ns Thrombotic Thrombotic None Thrombotic None None Thrombotic

4+ 4+

Antithrombotic Thrombotic None None

1+ 1+

Antithrombotic Antithrombotic Thrombotic

ns

4+

Predicted effect on hemostasis

1+ = weak evidence; 4+ = very strong evidence; ns: not sufficient evidence data to draw conclusions. Strength of evidence based on the number of studies, the number of subjects in each study and whether it was prospective. The C-reactive protein association is with atherothrombotic events.

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Route Oral administration can be especially important considering the ‘first-pass effect’ of oral estrogen that results in hormone concentrations in hepatic sinusoidal blood that are four to five times higher than those in peripheral blood [24]. This issue is important because the effect of hormones on platelet function could be indirect; for example, hormones could directly affect the liver, which synthesizes a factor that acts on megakaryocytes and/or platelets. Furthermore, many genes are up or downregulated within 4 h of exposure to estradiol [25], so that there can be physiologic consequences of continuous stimulation/exposure (as in transdermal administration) versus the ‘peaks and valleys’ obtained with the oral route. For these reasons, we believe that different routes of delivery will produce different platelet responses depending upon the time blood is obtained relative to hormone administration.

Formulation Interpretation of various studies of hormone effects on platelet function is complicated by the use of different hormone preparations with different timing of administration, with or without progesterone. In clinical studies in America, HT most commonly refers to oral CEE without or with medroxyprogesterone acetate. CEE is extracted from the urine of pregnant mares and contains at least 10 estrogens, including estrone, 17b-estradiol (E2), 17a-estradiol, equilin, 17b-dihydroequilin, 17a-dihydroequilin, equilenin, 17b-dihydroequilenin, 17a-dihydroequilenin and Delta (8)-estrone. The three most common are estrone (50%), equilin (25%) and 17a-dihydroequilin (15%) [26]. E2 is the most potent and most plentiful estrogen produced by the ovary, but all of the CEEs in their unconjugated form are biologically active and can interact with recombinant human estrogen receptor (ER) a and ER b. The affinity for the two ERs can vary by twofold among the different estrogens, with E2 and 17b-dihydroequilin having the highest affinity for both receptors [27]. In addition to CEE, there are other commonly used estrogen preparations, including oral E2. Progestin formulation can also confound the issue; Thomas et al. found that synthetic progestin caused platelet activation and thrombosis, whereas natural progesterone did not [28]. Just as different estrogen formulations have different effects on triglyceride levels [29], we believe that CEE and estradiol have different effects on platelet reactivity.

Technical There are numerous approaches for assessing various aspects of platelet function, each with advantages and disadvantages and each with challenges of quantification and standardization. Different investigators have used different assays (e.g. aggregation in whole blood, plasma or

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buffer; flow cytometry detection of platelet activation; calcium flux) with different conditions (e.g. different agonists, different concentrations of agonists, static versus shear) making comparisons between studies difficult. Some tantalizing information may be teased out of these studies; Dores et al. observed that the effects of HT on platelet adhesion differed according to the substrate (fibronectin versus collagen I or III) [30]. Chen et al. found that the effect of HT on platelet function was maximal at one and three months but was reduced or lost at six months [31]. The studies of Miller et al. are consistent with the hypothesis that the effects of oral HT on platelet function are prothrombotic, but the in vitro effect of E2 on platelets is not [32].

Non-genomic effects of HT on platelet function Estrogen acts via a non-genomic mechanism in a variety of tissues [33], utilizing the classic ERs as well as novel receptors [34]. Very little molecular or cell biology of platelet steroid hormone receptors is known. We have shown that human megakaryocytes and platelets express ER b, raising the possibility that there is a direct hormonal effect on this cell lineage [35]. We have also shown that platelet ER b protein is expressed and is glycosylated, but the consequences of this unusual post-translational modification are not understood [36]. ER a and ER b have at least 20 and 10 mRNA splice variants, respectively [37,38]. We have identified an alternate splice form of ER b in human platelets [36], but the full repertoire of ER transcripts in platelets is unknown. We have also detected ER a protein in both human and mouse platelets (Fig. 3) but suspect that this form lacks either exon 4 or exon 6 [35]. In vitro studies indicate 17b-estradiol has non-genomic effects on platelets [39–42]. At concentrations 1000– 100,000 times physiologic levels, E2 inhibits aggregation [39,40]. However, physiologic concentrations of E2 increase platelet intracellular calcium concentration, have profound effects on platelet signaling and lower the threshold for integrin aIIbb3 activation in an ER-dependent manner [12]. This effect may be lost after prolonged exposure of platelets to physiologic concentrations of estrogen [12,40] (and our own unpublished data).

Interaction of genetics with hormone therapy Genetic susceptibility has been proposed as a potential mediator of increased risk of CHD in women using HT [43]. Genetic polymorphisms in platelet genes, such as adhesive receptors or ERs, might enhance the risk. We have found that platelet genetic variations modify the in vitro effects of 17b-estradiol on platelet function [44], and our preliminary studies suggest that certain genetic variations in platelet glycoproteins interact with estrogen to increase the risk of MI, whereas other gene variations reduce the risk [45]. www.drugdiscoverytoday.com

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prescriptions are filled per year [46], such that an enormous number of women remain at risk of MI, stroke and venous thromboembolism. Thus, it becomes an important health issue to understand the mechanisms by which HT is prothrombotic. The pharmacogenetic effects of estrogen on platelet behavior can have great potential to improve the safety and efficacy of individualized HT in postmenopausal women. A greater understanding of the physiology, and molecular and cell biology of the effects of sex hormones and their receptors on hemostasis will be important for developing more effective and safe forms of HT. A corollary is that women might require different or, in some situations, more intensive thromboprophylaxis than men require.

Acknowledgements Supported by American Heart Association Scientist Development Award 0335057N (Leng), National Institutes of Health grant HL65229 (Bray) and Baylor College of Medicine.

References Figure 3. Analysis of ER a and ER b in platelets and megakaryocytes. (a) 250 ng of total RNA from the indicated tissues was reversetranscribed and amplified by PCR with the use of primers specific for ER a (upper) or ER b (lower). NT, no-template control; MEG, megakaryocyte; Plt, platelet; BM, bone marrow. Ovary (Ov) and uterus (Ut) were positive controls. (b) Immunoblotting with anti-ER a antibody shows ER a expression in human (1) and mouse (3) platelets. Mouse uterus (2) and COS cells (4) were used as positive and negative control, respectively. Fifty micrograms of platelet lysate were loaded.

Conclusions Estrogen therapy enhances arterial thrombosis risk via complex mechanisms, including stimulating effects on platelets. Not all activation pathways and platelet functions respond to estrogen in a prothrombotic manner, but the sum of these effects in vivo favors arterial thrombus formation. Apparent inconsistencies in the literature regarding the effect of estrogen on platelet function result in part from the preparations (E2 vs. CEE) and agonists used, route of administration, the presence or absence of plasma and different ages of the donors. Additional studies are needed to clarify these issues. Current clinical decision-making regarding HT use must rely on the large population studies until we discover better predictors of harm or benefit for an individual patient. To manage the very common menopausal symptoms of hot flushes and vaginal dryness, many leaders in the field of women’s health are currently recommending short-term HT use, such as three to five years. Such recommendations are based on the increased risk of breast cancer at four years [3] but do not account for the fact that the atherothrombotic risk occurs much earlier (within three months). Since the results of WHI and HERS have been published, fewer women are using HT. However, approximately 57 million HT 90

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