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(Chest. 2002;121:906-920.)
© 2002 American College of Chest Physicians

Interaction of Hemostatic Genetics With Hormone Therapy*

New Insights To Explain Arterial Thrombosis in Postmenopausal Women

Joel B. Braunstein, MD; Dawn Warner Kershner, DO; Paul Bray, MD; Gary Gerstenblith, MD; Steven P. Schulman, MD; Wendy S. Post, MD, MS and Roger S. Blumenthal, MD, FCCP

* From the The Johns Hopkins Ciccarone Center for Prevention of Heart Disease (Drs. Braunstein, Kershner, Gerstenblith, Schulman, Post, and Blumenthal), Division of Cardiology, Department of Medicine, Johns Hopkins Hospital, Baltimore, MD; and the Thrombosis Research Section (Dr. Bray), Department of Medicine, Baylor College of Medicine, Houston, TX.

Correspondence to: Roger S. Blumenthal, MD, FCCP, Division of Cardiology, Johns Hopkins Hospital, 600 North Wolfe St, Carnegie 538, Baltimore, MD 21287; e-mail: rblument{at}jhmi.edu


   Abstract
TOP
 Abstract
Introduction
 Estrogen and CVD
 Fibrinogen
 Factor VII
 PAI-1
 FVL
 Prothrombin Gene
 Platelets
 Conclusions and Perspectives
 References
 
Genetic variants of key hemostatic mediators increasingly have been proposed as risk factors for atherothrombosis. The Hormone and Estrogen/Progestin Replacement Study group recently reported that the initiation of estrogen replacement in postmenopausal women with known coronary heart disease is associated with an early increase in cardiovascular events. A putative genetic susceptibility factor has been proposed a potential mediator of this increased event risk. This review outlines the recent literature to support the premise for this important proposal. Genetic profiling has great potential to improve the safety and efficacy of individualized pharmacotherapy in postmenopausal women and other at-risk populations for the prevention of cardiovascular disease.

Key Words: arterial thrombosis • estrogen therapy • genetic polymorphisms • hemostatic factors


   Introduction
TOP
 Abstract
 Introduction
Estrogen and CVD
 Fibrinogen
 Factor VII
 PAI-1
 FVL
 Prothrombin Gene
 Platelets
 Conclusions and Perspectives
 References
 
Arterial thrombosis is the pathophysiologic basis for acute coronary syndrome (ACS). Key mediators in this process are platelets and coagulation factors.1 2 3 4 5 Emerging data have suggested that several polymorphisms of genes encoding for these mediators increase cardiovascular risk.6 In part, these genetic variants may explain some fraction of the familial or ethnic clustering of cardiovascular disease (CVD) and why young individuals with no or few traditional coronary risk factors develop premature CVD events.

Environmental factors likely influence the phenotypic expression of genetic polymorphisms. The environmental factor of exogenous estrogen therapy alters hemostatic protein levels and has a dose-dependent effect on thrombosis. Oral contraceptive therapy (OC) confers a sevenfold venousthrombotic risk that increases to a 30-fold to 50-fold risk in the presence of the procoagulation polymorphism, factor V Leiden (FVL).7 8 In the Heart and Estrogen/Progestin Replacement Study (HERS),9 a 52% increased risk of coronary heart disease (CHD) events after 1 year of hormone replacement therapy (HRT) followed by a trend to CHD reduction in years 3 to 5 was reported in postmenopausal women with known CHD. An explanation for this pattern of early harm and late benefit has heretofore been elusive; however, both HERS investigators10 and others11 have proposed a susceptibility factor such as a prothrombotic genetic variant to be one potential mediator. The public need for discovering a susceptibility factor to arterial thrombotic risk, if indeed there is one, relates to significant paradigm shifts in our approach to the treatment of postmenopausal women with HRT. Genetic profiling, in this regard, could markedly improve the safety and efficacy of individualized pharmacotherapy.

Researchers in the field of HRT agree that this area of drug-gene interaction is a top priority for investigation. The HERS group plans to examine whether certain genetic polymorphisms were associated with an increased risk of CVD events during the first year of the study. This review offers a comprehensive evaluation of the present literature on the topic of hemostatic genetics and estrogen replacement therapy. As this is a relatively novel area of investigation, much of the available data focuses on the separate impact of either estrogen or gene polymorphisms on hemostatic factor levels and/or behaviors contributory to arterial thrombotic risk. Insights and evidence supporting a linkage between the two entities are offered when present.


   Estrogen and CVD
TOP
 Abstract
 Introduction
 Estrogen and CVD
Fibrinogen
 Factor VII
 PAI-1
 FVL
 Prothrombin Gene
 Platelets
 Conclusions and Perspectives
 References
 
Nearly 30 years of observational study data suggest that a variety of HRT regimens are cardioprotective in women. The results of these observational studies, however, may be primarily due to the "healthy cohort effect."10 12 Prospective, randomized trial data do not demonstrate a cardiovascular benefit of HRT use in postmenopausal women with CHD. The HERS trial was the first randomized, double blinded, placebo-controlled secondary prevention trial evaluating the occurrence of nonfatal myocardial infarction (MI) and coronary death in women using HRT (conjugated equine estrogen plus medroxyprogesterone acetate). The results demonstrated no overall cardiovascular benefit for women with heart disease receiving HRT over a mean follow-up of 4.1 years. The cardiac event rate among the women using HRT in HERS was highest during the first 4 months of follow-up (relative risk, 2.3). Long-term therapy was associated with a trend toward a reduced number of cardiac events.9

Several more recent prospective, randomized trials support the early findings of the HERS. The Estrogen Replacement and Atherosclerosis trial13 reported no angiographic benefit of HRT on the progression of coronary atherosclerosis. The Healthy Women’s Study14 reported no HRT benefit on carotid intimal thickening, and the Women’s Health Initiative15 provided a 1-year interim analysis that healthy women receiving HRT experienced more cardiovascular events including MI, strokes, and venous thromboembolism than did women receiving placebo. Based on the pervasive findings from these well-designed trials, a number of hypotheses may provide a biological rationale to explain the deleterious or null effects of estrogen on cardiovascular outcomes (Table 1 .


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  		Table 1.. Proposed Effects of Estrogen Therapy on Mediators of Atherothrombosis*

 
The inverse, time-dependent risk in HERS might be related to early cardiac events occurring in a subgroup of patients genetically predisposed to thrombosis.10 Estrogen, in this scenario, may act synergistically with this predisposition to increase CHD event risk rather than decrease it. The effect of estrogen replacement on endothelial and vascular function also may differ in women with or without risk factors of age, hypercholesterolemia, hypertension, diabetes, and smoking.

The results of the Postmenopausal Estrogen/Progestin Interventions Study16 suggested that inflammation also may have contributed to the early CHD events observed in HERS. During the 3-year follow-up, C-reactive protein (CRP) levels progressively increased in patients who have received HRT. A cross-sectional study17 and a prospective study18 also reported significantly elevated CRP levels in postmenopausal women receiving HRT. Elevated levels of CRP are an independent risk factor for future cardiovascular events in men and women.19 20 21 22 CRP secreted by the liver in response to inflammatory cytokines increases monocyte tissue factor expression and its exposure to circulating blood. Tissue factor, in turn, is a potent stimulant of platelet activation and local thrombosis. The rise in CRP during the early course of HRT also may reflect and/or result in vulnerable atheromatous plaque destabilization.

Elevated plasma levels of factor VII coagulant activity (FVIIc), fibrinogen, von Willebrand factor (vWF), plasminogen activator inhibitor (PAI)-1, and tissue plasminogen activator (tPA) also seem to predict an increased risk for CHD.1 23 24 25 26 27 Elevations in these factor levels, however, are not likely to be strictly causally related to CHD. For instance, elevated levels of tPA antigen are suggestive of other pathologic conditions imparting CHD risk, including inflammation, vascular endothelial damage, and insulin resistance.27 Several investigators have explained some of the impact of estrogen on cardiovascular health by evaluating its effect on hemostatic factors. However, the action of estrogen on the thrombotic-fibrinolytic system is unlikely to explain its entire impact on cardiovascular pathophysiology. Estrogen independently influences endothelial properties, lipid biochemistry, and mediators of inflammation (Table 1) .

HRT in postmenopausal women may potentiate a thrombogenic risk by increasing coagulation factors, including FVIIc28 29 and factor VII antigen (FVIIag) levels,28 and by decreasing antithrombin III levels.28 30 Favoring a reduction in thrombotic risk, however, is the ability of HRT to reduce fibrinogen levels.29 30 HRT may additionally promote cardioprotection by enhancing fibrinolytic activity through reductions in PAI-1 levels.28 29 The Framingham Offspring Study31 also reported improvements in fibrinolytic activity through favorable changes in PAI-1 and tPA antigen levels in premenopausal and postmenopausal women who were receiving HRT and had high estrogen levels. The balance between the effect of estrogen on increased coagulant and fibrinolytic activity may depend on its prescribed dose, whether or not it is prescribed with progestins or progesterone, and its use in women with certain CHD risk factors and/or genetic polymorphisms of individual hemostatic factors.


   Fibrinogen
TOP
 Abstract
 Introduction
 Estrogen and CVD
 Fibrinogen
Factor VII
 PAI-1
 FVL
 Prothrombin Gene
 Platelets
 Conclusions and Perspectives
 References
 
Fibrinogen is a 340-kd glycoprotein (GP) composed of three distinct polypeptides ({alpha}, ß, and {gamma}) that are linked by disulfide bonds. These polypeptides are encoded by three genes situated on the long arm of chromosome 4.32 The cleavage of fibrinogen to fibrin monomers by thrombin is a crucial step in the sequence of arterial clot formation. Fibrin monomers spontaneously aggregate to form protofibrils that are cross-linked by activated factor XIII to help solidify the thrombus. These monomers also play a role in supporting platelet granule release, adhesion, and aggregation (Figs 1 , 2 ). Fibrinogen also may be prothrombotic by contributing to plasma viscosity. Multiple studies have associated elevated fibrinogen levels with CVD risk,26 33 34 including the results of a recent meta-analysis of six prospective, epidemiologic evaluations.35 However, it remains unclear whether elevated fibrinogen levels are a causal factor for CVD events or just an epiphenomenon of the atherosclerotic process, since high fibrinogen levels also are associated with smoking, advanced age, obesity, hypercholesterolemia, physical inactivity, diabetes mellitus, menopause, and hypertension.



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  		Figure 1.. A simplified schematic of the process of arterial thrombosis. Arterial thrombosis is typically initiated with the disruption of the vascular endothelium at sites of vulnerable plaques. The tissue factor, once released into the bloodstream, binds to inflammatory cells and complexes with factor VIIa. This complex converts factor X to factor Xa. Factor Xa, along with factor Va activates prothrombin (II). Thrombin (IIa) is a key procoagulant that activates platelets and other procoagulants (V, XI, and XIII), dissociates factor VIII from vWF, and initiates the generation of fibrin monomers. These monomers aggregate platelets and solidify the thrombus. The activated platelet surface also becomes a site for factors VIIIa, IXa, and XIa to generate additional amounts of factor Xa that interact with Va to further propagate thrombin generation. Subendothelial collagen binding to vWF and platelet GP Ia/IIa, as well as vWF binding to GP Ib-IX-V initiates platelet activation, adhesion, and aggregation. The binding of fibrinogen to GP IIb/IIIa completes the process of platelet aggregation and occlusive thrombosis. The genetic variation and its potential impact on the behavior of the factors highlighted here are discussed in the text.

 


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  		Figure 2.. A simplified schematic of the process of fibrin generation and fibrinolysis. Thrombin leads to the generation of fibrin and the activation of factor XIII. Activated factor XIII, in turn, cross-links fibrin monomers to solidify the platelet-rich thrombus. Endogenous fibrinolysis occurs with the endothelial release of tPA, which converts plasminogen to plasmin. Plasmin lyses the fibrin thrombus. Both {alpha}2-antiplasmin and PAI-1 inhibit endogenous fibrinolysis at various sites of the pathway. Genetic variations and their potential impact on the behavior of the factors highlighted here are discussed in the text.

 
Synthesis of the fibrinogen ß-chain is the rate-limiting step of fibrinogen formation.36 The identification of genetic variants of the fibrinogen polypeptide thus has focused on the ß-chain gene, particularly in its 5'-flanking region since this region contains several transcriptional regulatory elements, including interleukin (IL)-6 responsive elements and hepatic nuclear factor 1. The IL-6 responsive element, in particular, is considered to be an important determinant in mediating elevations of fibrinogen levels as a result of the acute-phase response that occurs during smoking and other proinflammatory states that may predispose a patient to CVD.

The Edinburgh Artery Study37 was the first trial to report an association between a ß-chain fibrinogen polymorphism and CVD risk. The B2 allele of the Bcl-I fibrinogen polymorphism occurred with higher frequency in patients with peripheral artery disease than in age-matched control subjects. The results of this trial were supported by several other evaluations of the B2 allele, including a Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto miocardico-238 case-control report demonstrating its association with MI risk in patients with a familial history of CVD and the Etude Cas-Temoins sur l’Infarctus du Myocarde Study39 correlating it with the severity of coronary atherosclerosis noted on angiographic assessment.

Both large-vessel cerebral atherosclerosis40 and CHD progression41 also have been associated with the A allele of the polymorphism -455G/A (HaeIII). Interestingly, in the study by de Maat et al,41 682 patients with known CHD were genotyped for the -455G/A polymorphism, then were randomized to receive either pravastatin or placebo, and were observed prospectively for 2 years. Patients with the -455A/A genotype demonstrated more progressive disease and higher fibrinogen levels compared to those with the -455G allele. However, pravastatin therapy eliminated both the increase in CHD progression and the fibrinogen rise in individuals with the A allele.

While these data support the existence of a convincing association of fibrinogen polymorphisms with CVD risk, other studies have reported no such associations for the polymorphisms -448 G/A,42 -453 H1/H2,43 and -455 G/A.44 Carter et al,42 while demonstrating no significant relationship between the -448 G/A polymorphism and cerebrovascular risk, noted in a subgroup analysis, that the homozygous form of 2-allele lysine (ie, -448A/A polymorphism) might actually protect women, but not men, from developing vascular disease.

Genotype-dependent elevations in fibrinogen levels, while proposed as a partial mechanism of increased CVD risk in several studies,37 38 41 are unable to explain the observations of others.39 42 45 The Austrian Stroke Prevention Study45 reported a link between carotid atherosclerosis severity and the presence of the ß-chain C148 224 T polymorphism. However, this risk occurred despite the lack of association of the T/T polymorphism with elevated fibrinogen levels. It has been demonstrated that the C148 224 T polymorphism lies in close proximity to an IL-6 responsive element.46 It is speculated that the polymorphism may represent a functional variance sequence that influences a genotype-dependent rise in fibrinogen levels during acute responses to repeated endogenous or exogenous factors that promote atherosclerosis, irrespective of baseline fibrinogen concentrations.39 45 The -455 G/A ß-chain polymorphism is in complete linkage disequilibrium with the -148C/T polymorphism,47 and the Bbeta 44848 and Bcl I49 polymorphisms are in strong linkage disequilibrium with the -455 G/A polymorphism. Thus, while it is conceivable that these polymorphisms may directly modulate the structure or rate and extent of synthesis of the fibrinogen protein, they may also merely be markers for other functional promoter polymorphisms.

The trials presented herein examining the associations among fibrinogen polymorphisms, behavior, level of expression, and CVD risk have yielded largely inconsistent results. This may be due partially to the methodological biases that accompanied these small, case-controlled analyses. Fibrinogen polymorphisms may also only impart risk in individuals with certain acquired risk factors, such as smoking, a finding that may be difficult to demonstrate in small population studies. Finally, the effects of these polymorphisms on CVD risk may, in fact, merely be small or negligible.

Effect of Estrogen on Fibrinogen
A decrease in fibrinogen levels is one mechanism by which estrogen therapy has historically been proposed to diminish thrombotic risk in certain patients. Lee et al50 studied the effect of OC and HRT on fibrinogen levels in 4,387 women and concluded that estrogen therapy is associated with lower fibrinogen levels. Both a history of OC use that is unrelated to the duration of therapy and HRT use in postmenopausal women correlated with lower fibrinogen levels (p < 0.001). Levels were lower in premenopausal women with intact higher levels of endogenous estrogen than in postmenopausal women (p < 0.001). Scarabin et al51 also reported that HRT reversed a menopause-related increase in fibrinogen levels. Thus, while these data suggest that HRT would not act through fibrinogen to enhance thrombotic risk, there remain no data examining any gene-estrogen interaction that might influence this risk in select women.


   Factor VII
TOP
 Abstract
 Introduction
 Estrogen and CVD
 Fibrinogen
 Factor VII
PAI-1
 FVL
 Prothrombin Gene
 Platelets
 Conclusions and Perspectives
 References
 
Factor VII (FVII) is an inactive procoagulant synthesized in the liver and secreted as a 48-kd, single-chain GP. On exposure to a tissue factor, FVII undergoes proteolysis to a two-chain active enzyme that complexes with the tissue factor. This complex initiates a cascade that results in thrombin generation and platelet activation (Fig 1) .

Elevated plasma levels of FVII are implicated as a CVD risk factor in some studies,1 52 53 54 but not all.55 56 57 58 59 The Northwick Park Heart Study1 reported a strong, independent association of high FVIIc with fatal CHD events. The Prospective Cardiovascular Münster Study52 observed a similar trend after 8 years of study follow-up, particularly in the presence of additional cardiovascular risk factors, including smoking, family history of MI, angina pectoris, high triglyceride and low-density lipoprotein levels, and low high-density lipoprotein. Both environmental and genetic variables influence FVII plasma levels. The Wembley Dietary Study reported53 54 that FVII levels correlated with total cholesterol levels. Age, increased body mass, and OC use, unopposed HRT, and menopause also may elevate FVII concentrations and/or alter its activity.

Polymorphisms of the FVII gene may affect FVII secretion efficiency,60 the influence of triglycerides on FVII levels,61 and the binding properties of the gene to nuclear proteins and its transcriptional activity.62 Together, these polymorphisms may account for up to one third of the variability of circulating FVIIag and activated factor VII (FVIIa) levels.63 Despite this apparent genetic predetermination for FVII levels and their response to the environment, associations between known polymorphisms and CHD are less substantiated.

Iacoviello et al64 studied the interaction of FVII genetic polymorphisms, FVII levels, and familial CHD in a small case-control study. This study identified three alleles (H5, H6, and H7) on the hypervariable region 4 of intron 7 of the FVII gene. Also identified was a missense mutation that leads to the substitution of arginine by glutamine at position 353 (R353Q). For the R353Q polymorphism, the R/R genotype was associated with the highest cardiovascular event risk, followed by an intermediate risk for the R/Q genotype (p < 0.001). For hypervariable region 4 polymorphisms, the H5 allele predicted the highest risk, the H6 allele predicted an intermediate risk, and the H7 allele predicted the lowest risk (p < 0.001). Patients with Q/Q or H7/H7 variants had lower levels of FVIIag and FVIIc than did patients with higher risk polymorphisms. Also, patients with the lowest FVIIc levels had a lower MI risk than did those with the highest levels (odds ratio [OR], 0.13; 95% confidence interval [CI], 0.05 to 0.34). Unlike studies with null hypothesis findings, the inclusion only of patients with histories of premature familial MIs and the exclusion of control subjects with a history of familial vascular disease likely facilitated the demonstration of a correlation between risk and genotype.

Girelli et al65 recently demonstrated that a polymorphism occurring in the promoter region of the FVII gene (A2) was in strong linkage disequilibrium with the Q allele of the R353Q polymorphism, associated in 80% of the patient cases they studied. Both alleles were associated with lower levels of FVII compared to the levels associated with the wild-type alleles. Also, while the prevalence of alleles A2 and Q among patients with CHD and control subjects did not differ (partially supporting the findings of Wang et al43 that R353Q is not associated with CHD severity), being homozygous A2/A2 and Q/Q or heterozygous A1/A2 and Q/R was associated with a significantly lower MI risk among patients in the CHD population. The genetically predetermined lower levels of FVII in this trial were proposed to reduce the degree or lifespan of thrombus formation and, thus, to reduce the overall risk of developing an MI. The results of two other studies59 66 supported a nonsignificant trend toward a link between R353Q and MI.

Several studies have failed to support elevated FVII levels55 or polymorphisms of FVII59 as CHD risk factors. While Doggen et al59 suggested an association of R353G with FVII levels in the healthy control arm of their trial, the polymorphism had no correlation with CHD risk. In fact, patients with the Q/Q genotype had a low risk of MI despite having high levels of FVIIag and FVIIa. The cumulative results of these studies and several others suggested that although FVII levels are genetically influenced, elevations in this procoagulant do not convincingly contribute to CVD development. It is likely, however, that several of the described polymorphisms conferred an altered risk for cardiovascular events.

Effects of Estrogen on FVII
Both postmenopausal status and age independently predict higher FVIIc and FVIIa levels.67 The effects of HRT on FVII levels are complex and may partially depend on the formulation used. Alterations in FVIIc may occur as a response to variations in FVII mass (ie, FVIIag), the extent of its activation (ie, FVIIa), or both. The results from several analyses67 68 suggest that elevations in FVIIa may account for much of the increase in FVIIc observed with menopause. Unopposed estrogen replacement appears to potentiate postmenopausal elevations in FVII levels,28 68 whereas combination HRT reduces FVII levels to near premenopausal levels.67 68 It remains uncertain whether these actions of HRT significantly impact the thrombotic risk of elderly women with or without known CHD. However, in premenopausal women, OC-induced increases in FVIIag, FVIIa, and FVIIc levels69 are proposed explanations for the increased arterial and venous thrombotic risk reported in women using OC. Furthermore, while no trials have specifically studied the impact of HRT use on CVD risk in the presence of various FVII gene polymorphisms, the ability of estrogen to influence triglyceride levels could impact the regulation of FVII gene transcription.


   PAI-1
TOP
 Abstract
 Introduction
 Estrogen and CVD
 Fibrinogen
 Factor VII
 PAI-1
FVL
 Prothrombin Gene
 Platelets
 Conclusions and Perspectives
 References
 
PAI-1 is present in endothelial and smooth-muscle cells of healthy arteries and, in higher quantities, in atheromatous arteries. PAI-1 inhibits both tPA and urokinase plasminogen activator, thus blocking the activation of plasminogen to plasmin (Fig 2) . Elevated levels, therefore, inhibit fibrinolytic activity, and may be a risk factor for acute coronary events. PAI-1 levels are directly correlated with triglyceride levels, body mass, and acute stressors. They are higher in individuals with insulin resistance, young men with MIs compared to age-matched control subjects, and postmenopausal women compared to premenopausal women. Establishing a causal role for PAI-1 in cardiovascular events is difficult, however, due to its strong clustering with other atherogenic risk factors.

Thus far, the following three polymorphisms have been identified for PAI-1: a 3' HindIII restriction fragment; an intronic CA repeat sequence; and a 4G/5G insertion deletion 675 bp 5' of the start site of transcription in the PAI-1 promoter. Only the 4G/5G variant has been investigated for a possible role in arterial disease. In vitro data from cells containing the 4G/4G genotype have suggested that this variant is associated with enhanced PAI-1 synthesis.70 It also correlates with the highest levels of PAI-1 in hypertriglyceridemic individuals,71 72 suggesting that either the 4G/5G promoter site or an adjacent region represented within the polymorphism may partially mediate the influence of triglyceride on PAI-1 expression.

Mansfield et al72 studied the PAI-1 promoter polymorphism and its association with CHD in type-2 diabetes patients with and without CVD. Patients with CHD had both an increased frequency of the 4G/4G genotype and higher PAI-1 levels than did those without CAD. In a population of postmenopausal women, Grancha et al73 also reported a strong correlation among the 4G allele, PAI-1 levels (antigen and activity), and CHD risk. A significant association between the 4G allele and the risk of MI has been substantiated. Using a case-control study format, among 152 MI survivors and 152 age-matched and sex-matched control subjects, the 4G allele of the PAI-1 gene conferred an OR of 1.4 (p = 0.04) for MI development.74 The presence of the platelet antigen (PlA)-2 GP IIIa polymorphism conferred an OR of 2.11 (p = 0.005) for MI development.74 When these two polymorphisms coexisted, their cumulative risk was markedly greater than the risk for either polymorphism alone (OR, 4.5; p = 0.001).

Numerous additional analyses have associated the 4G allele with CVD risk, including an increased risk of developing an ACS,75 MI risk in persons with a family history of CVD,76 anginal symptoms,77 and premature CVD (ie, occurrence at < 45 years of age) in a Swedish male cohort.78 These data, however, are not supported by the results from several large trials,79 80 81 including a case-controlled cohort analysis from the United States Physicians Health Study,79 that reported no such association of the 4G allele with CHD risk. A consensus opinion as to whether or not PAI-1 polymorphisms are associated with CVD is lacking. A meta-analysis82 evaluating most of the investigative trials on this topic favored a weak, albeit significant, relationship between PAI-1 genotypes and MI. Given the limitations of a meta-analysis in this context, however, PAI-1 polymorphisms at the present time should be considered to have, at best, a minor influence on CVD.

Effects of Estrogen on PAI-1
Several studies have examined the effect of HRT on PAI-1 levels. Koh et al83 reported that oral estrogen both alone and in combination with medroxyprogesterone acetate decreased PAI-1 levels by approximately 50% (p = 0.003) in 30 women, while transdermal estrogen did not significantly affect PAI-1 levels in 30 other postmenopausal women. Grancha et al73 also reported that oral HRT reduced PAI-1 levels, even though this occurred only in women with a 4G PAI-1 allele. One additional analysis51 reported that postmenopausal women not using HRT had elevated PAI-1 levels when compared to premenopausal women (p < 0.05). These results suggest that estrogen is generally associated with decreases in PAI-1 levels and a fibrinolytic benefit.

It is biologically plausible, however, that estrogen has a different effect on fibrinolysis in several subgroup female populations, such as those with both the insulin resistance syndrome and genotypically determined (as in the presence of the 4G allele) high PAI-1 levels. The PAI-1 4G allele is transcriptionally regulated by triglycerides,84 and estrogen replacement causes elevations in triglyceride levels.85 Increased PAI-1 levels as a result of such an interaction could lead to impaired endogenous fibrinolysis and could further augment atherothrombotic risk in an already high CVD risk population.


   FVL
TOP
 Abstract
 Introduction
 Estrogen and CVD
 Fibrinogen
 Factor VII
 PAI-1
 FVL
Prothrombin Gene
 Platelets
 Conclusions and Perspectives
 References
 
FVL is a point mutation in the factor V gene resulting in a substitution of arginine for glutamine at position 506.86 87 88 89 This abolishes a cleavage site in factor V for activated protein C, causing a functional activated protein C resistance. This defect leads to a 7-fold increase in the risk for venous thromboembolism in heterozygous carriers and up to a 100-fold increased risk in homozygous carriers. FVL is the most common genetic mutation causing venous thromboembolism in individuals of Northern European ancestry, in whom the prevalence of heterozygous carriers is 3 to 6%. While the role of FVL in venous thrombosis is well-established, its role in arterial thrombosis is much more controversial and likely is related to environmental interactions.90

Rosendaal et al91 reported in a population of young women that the combination of smoking and FVL led to an OR for a first MI that was an increase of 32-fold compared to that for nonsmoking noncarriers. The impact of the polymorphism on MI risk was nonsignificant among nonsmoking women. Male patients with MIs92 and angiographic CHD93 have been reported to have a 2-fold to 2.5-fold higher carrier rate of FVL than their healthy counterparts. FVL also is associated with earlier saphenous vein graft occlusion after coronary artery bypass grafting94 and premature or recurrent cerebrovascular accidents (CVAs).95 96 All of these positive association trials have limitations that warrant attention, including small sample sizes, case-controlled study designs, and selective investigations of survivors of CVD events. These concerns seem justified in light of the numerous reports97 98 99 100 101 102 103 that fail to support FVL as an independent risk factor for arterial thrombosis.

Interaction of Estrogen Therapy With FVL
FVL may interact with exogenous estrogen to predispose women to an increased risk of vascular thrombosis. This is supported by the results of one trial104 that demonstrated a strong association among FVL, exogenous estrogen use, and the risk of arterial occlusion causing bone death. Another trial105 reported that estrogen replacement therapy was a significant risk factor for atherothrombosis in hyperlipidemic women with FVL. Interestingly, however, in this analysis estrogen replacement therapy was protective against thrombotic events in women without FVL. The authors proposed that this FVL interaction with HRT was one possible mechanism for the failure of HERS to show HRT benefit in secondary prevention. This proposal lends justification for the future study of FVL as an independent risk factor in postmenopausal women.90


   Prothrombin Gene
TOP
 Abstract
 Introduction
 Estrogen and CVD
 Fibrinogen
 Factor VII
 PAI-1
 FVL
 Prothrombin Gene
Platelets
 Conclusions and Perspectives
 References
 
Prothrombin (factor II) is a precursor to thrombin that is activated in one of the final steps of the clotting cascade leading to fibrin formation (Fig 1 , 2) . A recently discovered prothrombin variant allele is associated with an increased risk for venous thrombosis and is found in 1 to 2% of the population. The mutation of the prothrombin gene, located on chromosome 11, is at position 20210 where there is a G->A transition. Similar to FVL, its role is less established in arterial thrombosis than in venous thrombosis.

Rosendaal et al91 demonstrated in a population-based case-control study of 79 women aged 18 to 44 years who had experienced MIs and 381 randomly chosen control subjects that the women with a history of MI had a 3.2-fold higher carriage rate (5.1% vs 1.6%) for the factor II 20210A allele than did control subjects (OR, 4.0).106 All carriers of the mutation were white women, and the results were unaltered by menopausal status or OC use. While the prothrombin variant failed to confer risk in nonsmokers and women without traditional CVD risk factors, smokers with the variant experienced a 43-fold increased risk of MI (95% CI, 6.7 to 281) compared to nonsmoking women without the mutation.

Several additional studies107 108 109 110 have reported no significant association between the prothrombin variant and arterial thrombosis. The failure to record traditional CVD risk factors in one of these trials,108 however, limited the detection of any potential interaction between such risk factors and the polymorphism. Another of these trials110 failed to correlate the G20210A prothrombin mutation either with angiographically documented CHD or MI, despite a significant enhancement of prothrombin activity that did independently associate the mutation with the presence of CHD.

Interaction of Estrogen Therapy With Prothrombin
Another trial11 recently reported the first interaction between HRT use and prothrombin variants concerning the risk of MI. This population-based, case-control study evaluated the presence of prothrombin 20210G->A variant and FVL mutation in a population of 232 postmenopausal women aged 30 to 79 years who had recently experienced a first MI. There were 723 age-matched and hypertension status-matched postmenopausal women who had not experienced MIs who served as control subjects. The prothrombin variant conferred an OR of 4.32 (95% CI, 1.52 to 12.1) for a first MI compared to genotypically wild-type women. Current HRT use compounded this risk and conferred an OR of 10.9 (95% CI, 2.15 to 55.2) for a nonfatal MI. While no interaction was observed in nonhypertensive women or in women with the FVL mutation, the risk of a first MI related to the presence of the prothrombin variant in hypertensive women was highly specific to HRT use. While this study was limited by a low prevalence of the prothrombin variant and an inability to assess confounding factors in a multivariate model, its results highly support further evaluation in another high-risk CVD risk population like the HERS cohort.11


   Platelets
TOP
 Abstract
 Introduction
 Estrogen and CVD
 Fibrinogen
 Factor VII
 PAI-1
 FVL
 Prothrombin Gene
 Platelets
Conclusions and Perspectives
 References
 
Platelets are a key component of the pathologic thrombotic process at sites of atheromatous plaque disruption (Figs 1 , 2) . Several major platelet surface GPs and adhesion receptors bind ligands at different flow shear rates to promote clot formation. The most important of these are the GPs IIb/IIIa (also known as integrin {alpha}IIb'2fß3), Ib-IX-V, and Ia/IIa (also known as VLA-2 and integrin {alpha}2'2fß1). While each of these integrins has genetic variants that may influence the risk for ACS, the GP IIIa integrin polymorphism has stimulated the greatest clinical interest.

The platelet membrane GP IIb/IIIa functions as a receptor for fibrinogen and vWF, and plays an important role in platelet adhesion, aggregation, and response to injury. PlA2 is a common polymorphism of the GP IIIa integrin, present in an estimated 25% of all white individuals. Weiss et al111 published the first study to suggest an independent relationship between the GP IIIa PlA2 allele and ACS. The frequency of the PlA2 variant in 71 consecutive patients admitted to the hospital with an MI or unstable angina was 39% vs 19%, respectively, in 68 age-matched, sex-matched, and race-matched hospitalized control patients without known heart disease (p = 0.01).111 The presence of at least one PlA2 allele was even more frequent in patients < 60 years of age (50% vs 14% of control subjects; p = 0.002). This association of the PlA2 genotype with a risk both for ACS and premature CHD has been reported in subsequent analyses.112 113 114 115 Two studies113 116 reported a higher frequency of percutaneous coronary intervention in patients with the PlA2 allele, and one prospective evaluation117 observed that the allele predicts higher risks of saphenous vein graft occlusion, MI, and death after coronary artery bypass grafting.

Coexistent CVD risk factors may potentiate the risk of patients with the PlA2 polymorphism, as is supported by a study of young survivors of MI.118 Patients who carried the PlA2 allele had a 13-fold increased risk of premature MI if they were smokers, and the interaction between smoking and the polymorphism accounted for 46% of premature MIs.118 One epigenetic analysis reported that the combination of the PlA2 allele and the 4G allele of the PAI-1 gene confers an additive risk for MI development (OR, 4.5; p = 0.001).74

Not all studies have reported an association between PlA2 polymorphism and vascular risk. The United States Physicians Health Study119 observed similar prevalences (ie, 13 to 15%) of the PlA2 allele in patients who had experienced MIs, CVAs, and venous thromboses, but did not have CVD. While a limitation of this analysis is that nearly all of its subjects were middle-aged white men in generally good health with a low incidence of smoking, several other studies120 121 122 123 corroborated such results in a number of other populations. Two angiographic analyses124 125 reported no association of PlA2 with the severity of coronary stenoses, one of which125 also found no association of PlA2 with restenosis after percutaneous coronary intervention.

In search of biological mechanisms to elucidate a still controversial link between the PlA2 polymorphism and CVD, the PlA2 polymorphism appears to be associated with enhanced platelet reactivity in patients with ACS. Thresholds for platelet activation and aggregation to adenosine diphosphate stimulation,126 127 {alpha}-granule release,126 activation of GP IIb/IIIa,126 and fibrinogen binding126 128 are reduced in the setting of the PlA2 polymorphism. How these modifications ultimately influence cardiovascular events is currently under investigation.

Estrogen and Platelets
Platelets from healthy women129 and from the female siblings of patients who have premature CHD (unpublished data from our group) bind significantly more fibrinogen than do platelets from men. This finding is likely estrogen-dependent, since binding varies with the phase of the menstrual cycle and is enhanced in women receiving OC.129 Human megakaryocytes and platelets express the estrogen receptor-ß (not estrogen receptor-{alpha}), raising the possibility of a direct hormonal effect on this cell lineage.130 This evidence supports estrogenic compounds having an activating effect on platelets.

While polymorphisms of the major platelet adhesive membrane proteins all have been associated with MIs and CVAs,131 not all epidemiologic studies have found these associations. This may be attributable, however, to the design of many of these inconclusive studies, including the two largest,119 122 which studied men only. There is reason to believe that these inherited platelet risk factors have the greatest effect in women, which may account for some of the differing results. In fact, when women are excluded from the analysis of the original study by Weiss et al,111 significance is lost.132 Several other studies74 133 have observed that PlA2 conferred a substantially greater risk in women than in men. Also, in vitro estrogen modulates platelet aggregation in a PlA2-dependent manner.134 These gender discrepancies are intriguing and justify future studies to better define the role of estrogen and platelet polymorphisms in CVD risk.


   Conclusions and Perspectives
TOP
 Abstract
 Introduction
 Estrogen and CVD
 Fibrinogen
 Factor VII
 PAI-1
 FVL
 Prothrombin Gene
 Platelets
 Conclusions and Perspectives
References
 
Extensive biological data support the idea that estrogen favorably impacts various determinants of CVD risk. These data include improvements in endothelial function, overall improvements in lipoprotein composition, and, possibly, enhanced fibrinolysis. In addition, nearly 30 years of observational data suggest that estrogen is beneficial for the cardiovascular system. Data from HERS and other large, well-designed, prospective trials now refute earlier reported clinical benefits from estrogen therapy and suggest that exogenous estrogen may even be harmful when initiated in various subgroups of women. The early increase in cardiovascular events among postmenopausal women with prior CHD has led to speculation about the presence of a susceptibility factor that contributed to the observed events.

The results of simulation studies11 suggest that this susceptibility factor must display both a prevalence of 3 to 5% and a risk ratio of 13 to 25 in HRT users to reproduce the clinical outcomes seen in the HERS. It is unlikely, however, that just one susceptibility factor can entirely explain the early increased event risk. Acquired atherogenic risk factors such as obesity, smoking, dyslipidemia, insulin resistance, and hypertension have complex interplays with hemostatic, thrombotic, and proinflammatory mediators that lead to the promulgation of atherothrombosis. Inheritable factors of atherogenic risk not only interact with one another, but also with the environment to which they are exposed.

Arriving at a clear consensus as to which genetic variants emerge as "attributable risk factors" for atherothrombosis in the HERS study group promises to be challenging. Beyond their complex interplay with one another and their environment, the polymorphisms implicated to play a role in arterial thrombosis previously have demonstrated highly variable and inconsistent associations with CVD risk (Table 2 ). A general lack of understanding of the function of these polymorphic proteins also impairs our ability to rationalize the findings of conflicting trials. In addition, while the large sample size of the HERS cohort will offer better power than most previously published case-control reports, the likely low prevalence rates of the genetic variants in the planned study population will still lead to small absolute numbers from which to derive risk estimates. Nonetheless, the HERS will be superior to most smaller trials in its ability to adequately adjust for important confounders of risk. Most prior published studies have maintained lenient patient and control inclusion criteria that subjected them to numerous biases in their search for clinically significant end points.


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  		Table 2.. Genetic Polymorphisms and Impact on Arterial Thrombosis*

 
The impetus driving the evaluation of HRT use in individuals with various genetic profiles relates to the boon of pharmacogenomics. Provided that there is pervasive use of HRT in women of all populations, identifying subgroups who have genetic predispositions to atherothrombotic risk and/or adverse events while receiving HRT has substantial implications for the design of safer and more effective therapies that target individualized risk reduction. Several reports have already demonstrated the practicality of this approach. Statins prescribed for patients with several documented genetic polymorphisms have led to greater reductions in the number of recurrent MIs.41 135 Platelets in ACS individuals with heterozygous PlA1/PlA2 are suspected to be more sensitive to the antiplatelet effects of aspirin and therapeutically dosed abciximab125 than those with homozygous PlA1. At present, several advocates already recommend using DNA genotyping to detect the presence of FVL and/or the prothrombin variant. This approach is relegated to assessing the thrombotic risk and safety of OC or HRT use in women of childbearing age with a personal history of venous thromboembolism or with a family history of thrombophilia and additional transient environmental risk factors (eg, smoking).136 Time will tell whether an elusive susceptibility factor can provide us equally important insights into cardiovascular risk for all of our postmenopausal female patients who are considering the initiation of estrogenic therapy.


   Footnotes
 
Abbreviations: ACS = acute coronary syndrome; CHD = coronary heart disease; CI = confidence interval; CRP = C-reactive protein; CVA = cerebrovascular accident; CVD = cardiovascular disease; FVII = factor VII; FVIIa = activated factor VII; FVIIag = factor VII antigen; FVIIc = factor VII coagulant activity; FVL = factor V Leiden; GP = glycoprotein; HERS = Heart and Estrogen/Progestin Replacement Study; HRT = hormone replacement therapy; IL = interleukin; MI = myocardial infarction; OC = oral contraceptive therapy; OR = odds ratio; PAI = plasminogen activator inhibitor; PlA = platelet antigen; tPA = tissue plasminogen activator; vWF = von Willebrand factor

Received for publication July 18, 2001. Accepted for publication September 18, 2001.


   References
TOP
 Abstract
 Introduction
 Estrogen and CVD
 Fibrinogen
 Factor VII
 PAI-1
 FVL
 Prothrombin Gene
 Platelets
 Conclusions and Perspectives
 References
 

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