Effects of different progestin regimens in hormone replacement therapy on blood coagulation factor VII and tissue factor pathway inhibitor

E.M. Bladbjerg1,2,7, S.O. Skouby3,4,6, L.F. Andersen4,5,6 and J. Jespersen1,2

1 Department for Thrombosis Research, University of Southern Denmark and 2 Department of Clinical Biochemistry, Ribe County Hospital in Esbjerg, DK-6700, 3 Department of Obstetrics and Gynecology, Frederiksberg Hospital, DK-2000, 4 Rigshospitalet DK-2100 and 5 Gentofte Hospital DK-2900 and 6 University of Copenhagen, DK-2000, Denmark


    Abstract
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 Abstract
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BACKGROUND: Long-term hormone replacement therapy (HRT) reduces cardiovascular risk, but an early increased risk was reported in women with coronary heart disease. In such women the arterial intima can express tissue factor, and changes in coagulation factor VII (factor VII) and tissue factor pathway inhibitor (TFPI) may be deleterious. METHODS: We measured factor VII clotting activity, activated factor VII, and concentrations of factor VII and TFPI during 12 months in healthy post-menopausal women randomized to: (i) cyclic oral estrogen/progestin (n = 25); (ii) long-cycle oral estrogen/quarterly progestin (n = 32); (iii) continuous oral estrogen/progestin (n = 21); (iv) continuous oral estrogen/intrauterine progestin (n = 22); (v) no HRT (n = 26). Blood was collected at baseline, 3, 6 and 12 months. Additional sampling was done before progestin intake in the long cycle group. RESULTS: No variations were observed in the reference group. There was a substantial decrease in TFPI concentrations in the HRT groups irrespective of the type of progestin. In women receiving long-cycle treatment, all factor VII measures increased during the unopposed estrogen periods, and the increase was reversed after progestin intake. The integrated response, AUC, for TFPI was significantly lower in the HRT groups compared with the reference group. CONCLUSION: The observed changes may increase the early thrombotic risk associated with HRT use.

Key words: factor VII/HRT/progestins/randomized study/TFPI


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
Epidemiological studies suggest that estrogen replacement therapy (ERT) and combined estrogen/progestin replacement therapy (HRT) decrease the risk of coronary heart disease (CHD) in healthy post-menopausal women (Stampfer et al., 1991Go; Grodstein et al., 1996Go), with a relative CHD risk of 0.70 in ERT users compared with non-users, and a relative risk of 0.66 in HRT users compared with non-users (Barrett-Connor and Grady, 1998Go). The cardioprotective effect can be partly explained by the favourable effect on several variables, e.g. blood lipids (Salomaa et al., 1995Go; Writing group for the PEPI trial, 1995Go), hypertension (Writing group for the PEPI trial, 1995Go), glucose tolerance (Barrett-Connor and Laakso, 1990Go), homocysteine (Mijatovic et al., 1998Go), endothelial function (Vehkavaara et al., 2000Go), and plasminogen activator inhibitor type 1 (Koh et al., 1997Go; Scarabin et al., 1997Go).

Recently, a large randomized, blinded, placebo-controlled secondary prevention trial (HERS) has reported a negative short-term effect of HRT, which should be distinguished from the reported positive long-term effect (Hulley et al., 1998). Overall, there were no significant differences between women receiving HRT or placebo with respect to non-fatal myocardial infarction or CHD death. In subgroup analyses it was demonstrated that there was an increased CHD risk in the HRT group compared with the placebo group during the first year of hormone use, and after 4 years the risk was reversed. This may suggest that in women with established CHD, there is an early increased thrombotic risk associated with the use of HRT. If the risk is restricted to women with CHD, who are more likely to have an atherosclerotic vessel wall with increased tissue factor (TF) expression, it is very tempting to speculate that blood coagulation factor VII (factor VII) and the tissue factor pathway coagulation inhibitor (TFPI) can play a role. Both factor VII and TFPI are key variables of the coagulation system, and when TF is exposed to circulating blood it binds to and activates factor VII (Rapaport and Rao, 1995Go). The central role of factor VII is supported by epidemiological data suggesting that the coagulant activity of factor VII (factor VII:C) is a risk indicator of ischaemic heart death (Meade et al., 1993Go; Junker et al., 1997Go). Factor VII:C is affected by polymorphisms in the factor VII gene (Bernardi et al., 1996Go; De Maat et al., 1997Go) as well as environmental factors, e.g. diet (Bladbjerg et al., 1994Go, 1995Go; Marckmann et al., 1998aGo), weight loss (Marckmann et al., 1998bGo), and exercise (Connelly et al., 1992Go). Furthermore, factor VII:C increases with age (Scarabin et al., 1996Go; Wright et al., 1997Go) and is affected by hormonal changes, i.e. pregnancy (Dalaker, 1968), menopause (Scarabin et al., 1996Go), oral contraceptives (Petersen et al., 1994Go; Mariani et al., 1999Go), and HRT (Nabulsi et al., 1993Go; Kroon et al., 1994Go; Salomaa et al., 1995Go; Writing Group for the Estradiol Clotting Factors Study, 1996Go; Andersen et al., 1999Go; Cushman et al., 1999Go; De Valk-de Roo et al., 2000Go; Folsom et al., 2000Go; Van Baal et al., 2000aGo). Randomized controlled trials looking at the effect of HRT on factor VII have recently been summarized (Van Baal et al., 2000bGo); these workers concluded that the results are contradictory, and that factor VII seems to be affected by the type of hormone therapy, the route of administration, and the factor VII assay used. However, a general trend seems to be a factor VII increase after per-oral unopposed estrogen therapy, an increase which can be modulated by progestin supplementation or transdermal administration of estrogen.

TFPI inhibits activated factor X by forming a complex, which subsequently inhibits factor VIIa bound to TF. The majority of TFPI in plasma is bound to lipoproteins (Lesnik et al., 1993Go), and only ~5% of TFPI in plasma circulates as free, unbound TFPI, which most likely comprises all of the anticoagulant TFPI activity (Hansen et al., 1997Go). Large amounts of free TFPI can be released into blood from the endothelium (Sandset et al., 1988Go). Oral contraceptives markedly reduce TFPI antigen and activity (Harris et al., 1999Go), and also HRT lowers TFPI (Høibraaten et al., 2000Go; Høibraaten et al., 2001Go; Luyer et al., 2001Go; Peverill et al., 2001Go).

In order to get more insight into the effects of estrogen, in combination with different progestin regimens, on factor VII and TFPI for 1 year, we conducted a randomized study in healthy post-menopausal women. We studied the estrogen/progestin effect in: (i) a cyclic oral estrogen/progestin regimen; (ii) a long-cycle oral estrogen/quarterly progestin regimen; (iii) a continuous oral estrogen/progestin regimen; and (iv) a continuous oral estrogen/intrauterine progestin regimen. Furthermore, factor VII and TFPI were measured in women not receiving HRT. In this way we expected to be able to clarify the effect of different progestin regimens on the TF pathway of coagulation.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study design
The current study is part of the Collaborative Danish Climacteric Study (CDCS). The CDCS is a prospective, comparative study performed in one Danish centre consisting of two departments of Obstetrics and Gynecology at Rigshospitalet and Frederiksberg University Hospital in the central Copenhagen urban area. The study consists of 12 month observation periods in a population of 175 healthy post-menopausal women representing an untreated reference group and four HRT groups receiving different types of estrogen/progestin combinations. The study population and study design have been described in detail elsewhere (Skouby et al., 2002Go). Briefly, post-menopausal women, aged 47–55 years, were grouped as follows: reference group (n = 30): no HRT; group 1 (n = 30): cyclic intake of estradiol valerate (E2V), 2 mg/day (1–21/28 days), combined with cyproterone acetate (CPA), 1 mg/day (12–21/28 days), and placebo (22–28/28 days) (Schering AG, Berlin, Germany); group 2 (n = 45): a long-cycle regimen with E2V, 2 mg/day (1–84/91 days), combined with medroxyprogesterone acetate (MPA), 20 mg/day (71–84/91 days), and placebo (85–91/91 days) (Orion Pharma, Espoo, Finland); group 3 (n = 35): continuous combined intake of estradiol (E2), 2 mg/day, and norethindrone acetate (NETA), 1 mg/day (Novo Nordisk, Copenhagen, Denmark); group 4 (n = 35): continuous combined intake of E2, 2 mg/day, plus local intrauterine delivery of levonorgestrel, 20 µg/day (Leiras, Helsinki, Finland).

A written informed consent was signed by all women before entering the study. The study was conducted in accordance with the guidelines of Good Clinical Practice in the European Community, which incorporates the principles of the Declaration of Helsinki II, and was approved by the local ethics committee.

During the study period, a total of 45 women dropped out from the HRT groups for the following reasons: positive diabetic glucose tolerance test, lack of time, mood changes, arthritis, endometrial hyperplasia with atypia, pruritus, hypermenorrhoea, weight gain, diagnosis of breast cancer, diagnosis of lung cancer, metrorrhagia, nausea, constipation, hirsutism, and increasing episodes of hot flushes. Four women dropped out from the reference group as a result of diagnosis of breast cancer and lack of time. This leaves the following study groups for per-protocol analysis: reference group, n = 26; group 1, n = 25; group 2, n = 32; group 3, n = 21; and group 4, n = 22. In group 2, six women had one missing value for factor VII or TFPI due to a lack of citrate plasma (Table IGo).


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Table I. Plasma concentrations of blood coagulation factor VII (factor VII), tissue factor pathway inhibitor (TFPI), and low density lipoprotein (LDL)-cholesterol during 1 year of different hormone replacement therapy (HRT) regimens or no HRT
 
Blood sampling
Venous blood samples were drawn in the fasting state between 08:00 and 10:00 after 10 min of standardized resting in the supine position. The blood samples were collected with minimal stasis by use of siliconized evacuated tubes, and the first 10 ml of blood was discarded. The following 4.5 ml of blood was collected in 0.5 ml (0.129 mol/l) trisodium citrate and used for analysis of lipids, factor VII and TFPI. The tubes were centrifuged at 20°C for 20 min at 2000 g. Plasma was pipetted into plastic vials in aliquots of 400 µl and stored at –60°C until analysis.

In the reference group and in groups 3 and 4, blood was collected at baseline and after 6 and 12 months. Additional sampling was done after 3 months in groups 1 and 2 and in group 2 also before MPA intake in cycles 2 and 4.

Blood analyses
Plasma samples were rapidly thawed in a water bath at 37°C and analysed in one series for each subject. Samples from all study groups were analysed at the same time. Plasma factor VII:C (%) was measured in a one-stage clotting assay using human brain TF as described previously (Bladbjerg et al., 1994Go). Concentrations of factor VII:Ag (%) were measured with an ELISA using rabbit antihuman factor VII polyclonal IgG as capture and detection antibody (Stago, Asniéres, France). Results for factor VII:C and factor VII:Ag were expressed as a percentage of a reference plasma (citrated plasma pool prepared from 15 fasting women, aged 23–64 years, and 11 fasting men, aged 21–53 years). Determination of factor VIIa (analysed in group 2 only; further details are given in the Discussion section) was performed with Staclot® VIIa-rTF (Diagnostica Stago, Asniéres, France), a clotting assay with recombinant truncated TF specific for factor VIIa co-factor function. Coagulation times were converted to factor VIIa concentrations (mU/ml) by interpolation on a calibration curve prepared from dilutions of human recombinant factor VIIa in buffer. Plasma concentrations of total TFPI (ng/ml) were measured with a commercial ELISA (American Diagnostica, Greenwich, CT, USA). Inter- and intra-assay analytical variations (CV) were: <10 and <4% for factor VII:C, <8 and <7% for factor VII:Ag, <9 and <3% for factor VIIa, <8 and <8% for TFPI. All the coagulation analyses were performed at the Department of Clinical Biochemistry, Ribe County Hospital and Department for Thrombosis Research, University of Southern Denmark, Esbjerg, Denmark. We also included measures of low density lipoprotein (LDL)-cholesterol calculated as the difference between total cholesterols and [high density lipoprotein (HDL)-cholesterol + very low density lipoprotein (VLDL)-cholesterol] (Andersen, 1998Go).

Statistical methods
Non-parametric statistical methods were used due to non-Gaussian distribution of the results. Differences within groups were analysed with Friedman’s analysis of variance. When significant time effects were found, the results were compared with the results at baseline with the Wilcoxon signed rank sum test. Differences between groups (baseline values and the integrated response) were compared with the Kruskal–Wallis test. Correlations were analysed using Spearman’s rank correlation test. The integrated response was calculated as area under the curve (AUC), a valid summary measure for repeated measurements (Matthews et al., 1990Go). When significant differences were found in AUC, each hormone group was compared with the reference group with the Mann–Whitney test. P < 0.05 was considered statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Changes in concentrations over time
The measured variables were stable over time in the reference group (Table IGo). There was a marked decrease in total TFPI concentrations and LDL-cholesterol in each of the hormone groups (P < 0.001) irrespective of the type of progestin (Table IGo). A minor increase (P < 0.01) was observed in factor VII:C after 12 months with cyclic oral estrogen/progestin therapy at the end of the combined estrogen/progestin phase (group 1). In women receiving long-cycle estrogen/quarterly progestin (group 2) there was a marked increase in factor VII:C (P < 0.01), factor VII:Ag (P < 0.01), factor VIIa (P < 0.01) and factor VIIa/factor VII:Ag (P < 0.01) during the unopposed estrogen periods, and the increase was reversed after progestin intake (Table IGo, Figure 1Go).



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Figure 1. Effects of hormone replacement therapy on coagulation factor VII (factor VII):C (%) (•), factor VII:Ag (%) ({circ}) and factor VIIa (mU/ml) ({blacksquare}) during 1 year of long-cycle treatment with estradiol valerate, 2 mg/day (1–84/91 days), combined with medroxyprogesterone acetate, 20 mg/day (71–84/91 days), and placebo (85–91/91 days) (group 2). Values are medians (25th–75th percentiles) (n = 32). *P < 0.05, **P < 0.01, ***P < 0.001 compared with baseline values.

 
Integrated responses
There was no significant difference in baseline values between groups for any of the variables measured. The integrated response, AUC, for TFPI was significantly lower in each of the hormone groups compared with the reference group (P < 0.001) (Table IGo, Figure 2Go). There was no difference in TFPI response between the hormone groups. The integrated response for LDL-cholesterol was significantly lower after continuous combined therapy (groups 3 and 4) compared with the reference group (P < 0.001) (Table IGo). There was a difference in LDL-cholesterol response between the hormone groups (P < 0.001) with lower values after continuous combined therapy (groups 3 and 4) (Table IGo). No differences in AUC were observed for factor VII.



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Figure 2. Box plots for integrated tissue factor pathway inhibitor (TFPI) responses, area under the curve (AUC), during 1 year of different hormone replacement therapy (HRT) regimens or no HRT. Values are medians with boxes representing 25th–75th percentiles and whiskers representing minimum and maximum values (without outliers). Group 1: cyclic intake of estradiol valerate (E2V), 2 mg/day (1–21/28 days), combined with cyproterone acetate, 1 mg/day (12–21/28 days), and placebo (22–28/28 days); group 2: long-cycle treatment with E2V,2 mg/day (1–84/91 days), combined with medroxyprogesterone acetate, 20 mg/day (71–84/91 days), and placebo (85–91/91 days); group 3: continuous combined intake of E2, 2 mg/day, and norethindrone acetate, 1 mg/day; group 4: continuous combined intake of E2, 2 mg/day, plus local intrauterine delivery of levonorgestrel, 20 µg/day. References: no treatment. ***P < 0.001 compared with the reference group.

 
Correlations
There was a highly significant association between baseline concentrations of LDL-cholesterol and TFPI (r = 0.41; P < 0.01), between the integrated response of LDL-cholesterol and TFPI (r = 0.58; P < 0.01), and between changes in LDL-cholesterol and changes in TFPI during 12 months (r = 0.35; P < 0.01). Significant associations were also observed between baseline concentrations of LDL-cholesterol and factor VII:C (r = 0.28; P < 0.01) and factor VII:Ag (r = 0.21; P < 0.01). No other correlations were observed between LDL-cholesterol and factor VII, and no associations were observed between TFPI and factor VII.


    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Our results demonstrate that the estrogen component of HRT increases plasma factor VII levels, and the progestin component opposes this effect. The most pronounced effect on factor VII was observed during the long-cycle regimen with 70 days of unopposed estrogen (group 2) (Table IGo, Figure 1Go), whereas normal cyclic treatment only had a minor effect on factor VII (Table IGo). With continuous combined treatment (systemic or intrauterine) the effect on factor VII was abolished (Table IGo). In contrast, total TFPI decreased by >25% in all the hormone groups, irrespective of the progestin regimen (Table IGo, Figure 2Go). Also, LDL-cholesterol concentrations decreased in all the hormone groups (Table IGo).

In keeping with our observations, cross-sectional studies have demonstrated that factor VII is higher in women using ERT than in women using HRT (Nabulsi et al., 1993Go; Wright et al., 1997Go; Cushman et al., 1999Go), and a dose–response effect on factor VII of the estrogen component of HRT has been observed (Schlegel et al., 1999Go). The factor VII increase is most pronounced with oral administration, but also transdermal estrogen therapy increases factor VII (Kroon et al., 1994Go). In contrast, transdermal combined therapy may even lower factor VII (Writing Group for the Estradiol Clotting Factors Study, 1996Go). The mechanism has not been clarified yet, but most likely estrogen affects the hepatic synthesis and/or clearance of factor VII. Whether progestin has anti-estrogenic effects or specific effects by itself is not known, but we observed that a marked increase in factor VII after prolonged estrogen therapy can be reversed after only 2 weeks of progestin supplementation. We only measured factor VII before progestin intake in the long-cycle group, and it is therefore not known whether factor VII is increased after only 11 days of unopposed estrogen therapy in group 1. Our results do, however, underline that very different results for factor VII are obtained during the different phases of combined therapy. Only continuous combined therapy was without effect on factor VII in our study, and we found no difference between supplementation with oral progestin (NETA) and intrauterine progestin (levonorgestrel), indicating that even local progestin application has a systemic effect that prevents the factor VII increase. In the same study, intrauterine progestin application was unable to interfere with the estrogen-induced increase in C-reactive protein (Skouby et al., 2002Go). These observations indicate that local progestin application affects one system (factor VII) but not another (CRP). Possibly, the amount of progestin absorbed locally is sufficient to affect the factor VII system but not CRP. Alternatively, the intrauterine device may cause an ongoing low-grade inflammation, thereby opposing a possible progestin effect on CRP.

Some of the discrepancy in studies on HRT and factor VII is due to the different factor VII assays used. We have included three factor VII measures: the factor VII coagulant activity, factor VII:C; activated factor VII, factor VIIa; and the factor VII protein concentration, factor VII:Ag. Also, the ratio factor VIIa/factor VII:Ag was calculated as a measure of the specific activity of the factor VII protein. During long-cycle therapy, estrogen induced a highly significant increase in factor VII:C, factor VII:Ag, and factor VIIa, but also an increase, though smaller, in factor VIIa/factor VII:Ag. This demonstrates that the estrogen in HRT increases the protein concentration of factor VII (either through increased synthesis or decreased catabolism), thereby increasing factor VII:Ag, factor VIIa and factor VII:C, and the specific activity of the factor VII protein is also slightly increased. The increase in protein concentration is also found in other studies (Kroon et al., 1994Go; Salomaa et al., 1995Go; De Valk-de Roo et al., 2000Go; Van Baal et al., 2000aGo), but elevated factor VIIa during estrogen therapy has until now only been observed in a cross-sectional study (Wright et al., 1997Go). In contrast, De Valk-de Roo et al. found no significant change in factor VIIa (0.31 ng/ml before treatment, 0.38 ng/ml after treatment; n = 12) after 8 weeks of unopposed estrogen therapy (De Valk-de Roo et al., 2000Go). Recent findings suggest that continuous combined HRT lowers factor VIIa (Høibraaten et al., 2001Go; Peverill et al., 2001Go). In both studies, women received 2 mg estradiol and 1 mg NETA, either for 6 weeks (Peverill et al., 2001Go) or 2 years (Høibraaten et al., 2001Go). This was accompanied by no change in factor VII:Ag (Høibraaten et al., 2001Go) or a reduction in factor VII:C (Peverill et al., 2001Go). Women participating in our study group 3 also received 2 mg estradiol and 1 mg NETA, but we observed no changes in factor VII:C or factor VII:Ag. We have now analysed the samples also for factor VIIa, and we find that factor VIIa is lowered by 7–8% compared with baseline (P < 0.05) (results not presented). In our study this minor reduction did not influence factor VII:C. Unfortunately, we cannot analyse factor VIIa in hormone groups 1 and 4, because we have no citrated plasma left. However, the fact that women who receive the same HRT regimen react differently with respect to factor VII underlines the importance of environmental factors, e.g. time of blood sampling, age, diseased versus healthy women, and standardization of the factor VII assays, but also polymorphisms in the factor VII gene may modulate the individual response to HRT (Meilahn et al., 1995Go).

We observed a highly significant decrease in TFPI in all hormone groups, irrespective of the type of progestin regimen. This is the first study on the effect of cyclic HRT on TFPI, and total TFPI was reduced by >25% during 1 year, leading to significantly lower integrated responses (AUC) in HRT users (Figure 2Go). Our observations confirm recent findings in women treated with continuous combined therapy for 6 weeks (Peverill et al., 2001Go) or 2 years (Høibraaten et al., 2001Go) and with estrogen only for 3 months (Luyer et al., 2001Go). In contrast, transdermal combined therapy has only a minor effect on TFPI (Høibraaten et al., 2000Go).

The effect on total TFPI concentration may in part be secondary to an effect on LDL-cholesterol, which was lowered in all hormone groups (Table IGo). We observed a significant correlation between changes in LDL-cholesterol and changes in concentrations of TFPI, as have others (Luyer et al., 2001Go). It has previously been demonstrated that a statin-mediated reduction in LDL-cholesterol lowers TFPI activity (Sandset et al., 1991Go). However, a reduction in TFPI concentrations does not seem to be caused by a reduction in lipids only (Harris et al., 1999Go; Høibraaten et al., 2001Go), and other mechanisms may contribute. Thus, it has been demonstrated in vitro that physiological concentrations of 17ß-estradiol decrease the release of TFPI from human umbilical vein endothelial cells (Bilsel et al., 2000Go). Whether progestins have any effects on TFPI is unknown, but the very similar TFPI responses with estrogen alone and with different progestin regimens suggest that TFPI is only influenced by the estrogen component.

The observed changes in factor VII and TFPI may increase the risk of thrombosis by shifting the haemostatic balance towards a procoagulant state. Theoretically, the risk is highest in progestin-free periods because of the increase in concentrations of factor VII protein and activated factor VII. Since factor VIIa and TFPI are only enzymatically active in the presence of TF, the risk is augmented in women with atherosclerosis. It is known that TF is present in high amounts in atherosclerotic plaques (Ardissino et al., 1997Go), and intimal TF expression is therefore more likely to occur in atherosclerotic women. In the HERS study, women with CHD were treated with a continuous combined therapy, i.e. conjugated equine estrogen and MPA (Hulley et al., 1998), while in our study we used continuous combined E2 and NETA, which had no effects on factor VII. However, if sufficient amounts of intimal TF are exposed to circulating blood, a substantial reduction in TFPI may in itself explain the increased thrombotic risk.

In conclusion, we have demonstrated that HRT during 1 year lowers TFPI substantially, irrespective of the progestin supplementation, and increases concentrations of factor VII protein and activated factor VII during unopposed estrogen. The effect on factor VII is reversed by progestin supplementation, and HRT should therefore be preferred to ERT with respect to factor VII. The observed changes may increase the thrombotic risk associated with HRT use. Our findings might also provide a possible explanation of the different effects of HRT in primary and secondary cardiac prevention.


    Notes
 
7 To whom correspondence should be addressed at: Department of Clinical Biochemistry, Ribe County Hospital, Østergade 80,DK-6700 Esbjerg, Denmark. E-mail: emb{at}ribeamt.dk Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on February 2, 2002; accepted on August 19, 2002.