The Differential Effect of Estrogen, Estrogen-Progestin and Tibolone on Coagulation Inhibitors in Postmenopausal Women

By Keramaris, N C Christodoulakos, G E; Lambrinoudaki, I V; Dalamanga, A; Alexandrou, A P; Bramis, J; Bastounis, E; Creatsas, G C

Key words: HORMONE THERAPY, ANTITHROMBIN, PROTEIN C, PROTEIN S, TIBOLONE, ESTRADIOL, CONJUGATED EQUINE ESTROGENS ABSTRACT

Objectives Hormone therapy increases the risk of venous thromboembolism, possibly through a negative effect on coagulation inhibitors. The aim of the study was to assess the effect of conjugated equine estrogens alone or in combination with medroxyprogesterone acetate, low-dose 17beta-estradiol combined with norethisterone acetate and tibolone on inhibitors of coagulation.

Methods Two hundred and sixteen postmenopausal women received orally either conjugated equine estrogens 0.625 mg (CEE, n = 24) or tibolone 2.5 mg (n = 24) or CEE + medroxyprogesterone acetate 5 mg (CEE/MPA, n = 34) or 17beta-estradiol 1 mg + norethisterone acetate 0.5 mg (E2/NETA, n = 66) or no therapy (control, n = 68) for 12 months. Plasma antithrombin, protein C and total protein S were measured at baseline and at 12 months.

Results CEE, CEE/MPA and E2/NETA treatment were associated with a significant decrease in antithrombin levels (CEE: baseline 235.6 +- 47.6 mg/l, follow-up 221.3 +- 48.3 mg/l, p = 0.0001; CEE/MPA: baseline 251.1 +- 38.6 mg/l, follow-up 225.0 +- 42.6 mg/l, p = 0.009; E2/NETA: baseline 257.1 +- 59.4 mg/l, follow-up 227.1 +- 50.4 mg/l, p = 0.007; tibolone: baseline 252.6 +- 62.4 mg/l, follow-up 261.9 +- 59.1 mg/l, p = 0.39). Protein C decreased significantly in the CEE and CEE/MPA groups (CEE: baseline 3.64 +- 1.17 mg/l, follow- up 2.48 +- 1.47 mg/l, p = 0.004; CEE/MPA: baseline 3.24 +- 1.23 mg/ l, follow-up 2.61 +- 1.38 mg/l, p = 0.001; E2/NETA: baseline 3.24 +- 1.10 mg/l, follow-up, 3.15 +- 1.11 mg/l, p = 0.08; tibolone: baseline 3.26 +- 1.25 mg/l, follow-up 3.09 +- 1.32 mg/l, p = 0.37). Protein S decreased significantly only in the CEE/MPA group (CEE: baseline 19.4 +- 2.76 mg/l, follow-up 18.0 +- 2.45 mg/l, p = 0.56; CEE/MPA: baseline 18.4 +- 3.42 mg/l, follow-up 14.5 +- 3.43 mg/l, p = 0.005; E2/NETA: baseline 19.0 +- 3.11 mg/l, follow-up 19.5 +- 3.43 mg/l, p = 0.18; tibolone: baseline 18.5 +- 3.09 mg/l, follow-up 18.0 +- 4.09 mg/l, p = 0.32).

Conclusions Estrogen and estrogen-progestin therapy are associated with a reduction in coagulation inhibitors, the extent of which depends on the regimen administered. Tibolone appears to have no effect on inhibitors of coagulation.

INTRODUCTION

Hormone therapy (HT) is extensively administered to postmenopausal women for the relief of climacteric symptoms and the improvement of quality of life1’2. Furthermore, HT decreases bone loss and the risk of osteoporotic fractures3’4. Despite beneficial modulations on the lipidlipoprotein profile, the Women’s Health Initiative (WHI) trial and the Heart and Estrogen/progestin Replacement Study (HERS)4’5 have reported a significant increase in early coronary events. Furthermore, observational6’7 and randomized studies8’9 have reported an increased venous thromboembolism risk among HT users. In a meta-analysis of 12 observational studies10, venous thromboembolic events (VTE) had a relative risk of 2.14, which was augmented to a relative risk of 3.49 during the first year of HT.

VTE may be the result of a local overload of coagulatory activity that exceeds the local capacity of antithrombotic mechanisms, including the inhibitors of coagulation and the fibrinolytic mechanisms. Although the HT-associated risk of VTE is well documented in clinical trials, the results from studies on coagulation are not in unison, as there is still contradiction between them. Different types of HT, namely unopposed estrogen or combined estrogen-progestin may differ in their effect on coagulation. Furthermore, the androgenic potency of the progestin may modulate the effect of a particular HT regimen on the coagulation system11’12.

Tibolone is a synthetic steroid that expresses, via the 3a-and 3beta-hydroxymetabolites and the ?4 isomer, estrogenic, progestogenic and androgenic actions depending on the target tissue. Tibolone has a variable effect on the lipid-lipoprotein profile13, may promote endothelium-dependent vasodilatation and may decrease vascular cell adhesion molecules1 . Tibolone may be administered for a prolonged period of time, expressing an acceptable safety profile regarding the breast and the endometrium15.

The purpose of this study was to investigate the differential effect of various treatment regimens on the coagulation inhibitors antithrombin, total protein C and total protein S among healthy postmenopausal women.

METHODS

Three hundred postmenopausal women were originally enrolled in the study. Subjects were recruited from the Menopause Clinic of the 2nd Department of Obstetrics and Gynecology, University of Athens, Aretaieion Hospital. Patients were at least 1 year menopausal. Women who were past users of HT, tibolone or raloxifene were not included in the study unless they had been off therapy for at least 6 months.

Before commencing therapy, patients had a gynecological and biochemical evaluation which included bimanual examination, PAP smear and transvaginal sonography, breast examination and mammography, thyroid-liver-renal function, as well as blood coagulation tests and bone densitometry. Criteria for inclusion in the study were an endometrial thickness < 5 mm, follicle stimulating hormone > 20 mIU/ml, serum estradiol < 50 pg/ml, the absence of a history of gynecological malignancy, ischemic heart disease, thromboembolism, diabetes mellitus, non-treated thyroid dysfunction and the intake of lipidlowering or antihypertensive medication.

The decision to treat was based on the presence of climacteric symptoms. Women were assigned to one of the following regimens:

(1) Conjugated equine estrogens 0.625 mg (CEE, Premarin(R), Wyeth Ayerst Laboratories, Philadelphia, USA, n = 34);

(2) Tibolone 2.5 mg (Livial(R), Organon, Oss, the Netherlands, n = 36);

(3) Conjugated equine estrogens 0.625 mg plus medroxyprogesterone acetate 5 mg (CEE/MPA, Premelle 5(R), Wyeth Ayerst Laboratories, Philadelphia, USA, n = 50); or

(4) 17beta-Estradiol 1 mg plus norethisterone acetate 0.5 mg (E2/ NETA, Activelle(R), Novo Nordisk, Copenhagen, Denmark, n = 91).

Women without climacteric complaints or women not willing to receive hormone therapy served as controls (n = 89). All subjects signed an informed consent and Institutional Review Board approval was obtained by the Ethics Committee of Aretaieion Hospital.

The study period was 12 months. Blood pressure, weight and height were recorded in the morning in light clothing and the body mass index was computed at each visit. Fasting blood samples were drawn at 9:00, were immediately centrifuged and citrated plasma was stored at – 80[degrees]C until assayed. Antithrombin, protein C and protein S were analyzed using the Immunodiffusion kits RN040.3, GTl 18.3 and GT100.3, respectively of The-Binding-Site Ltd (Birmingham, UK). Intraand interassay coefficients of variation were 0.77% and 0.90% for antithrombin, 3.10% and 3.17% for protein C and 2.13% and 5.96% for protein S.

Statistical analysis was performed by SPSS Version 8.0 (Statistical Package for the Social Sciences, Chicago, Illinois, USA). Skewed variables were logarithmically transformed. Baseline characteristics were compared between therapy groups by analysis of variance (ANOVA) for continuous variables and by chi^sup 2^ test for categorical variables. Baseline and follow-up means were compared across the same therapy group by paired t test. Percentage changes in antithrombin, protein C and protein S between groups were assessed by ANOVA. Statistical significance was set at the 0.05 level.

RESULTS

From the 300 women originally enrolled in the study, 216 women completed the study (72%). Of the 84 women who dropped out, 36 were lost to follow-up and 48 withdrew because of unwanted effects (vaginal bleeding, breast tenderness, bloating or weight gain). Baseline characteristics did not differ between women who withdrew and women who completed the study. Baseline demographic characteristics of the 216 women who completed the 12-month study are presented in Table 1. No difference was detected between the therapy groups for any of the parameters assessed.

Table 1 Baseline demographic characteristics of 216 women under various hormone therapies or no therapy. Data are presented as mean (standard deviation) or percentage

Baseline and 12-month means for antithrombin, protein C and protein S according to treatment assignment are presented in Table 2. Antithrombin at 12 months was significantly decreased compared to baseline values in the CEE, CEE/MPA and E2/NETA groups. Protein C decreased significantly in the CEE and CEE/MPA groups, while protein S decreased significantly only in the CEE/MPA group.

Percentage changes from baseline in antithrombin, protein C and protein S between the study groups are presented in Figure 1. The mean antithrombin level was significantly decreased compared to that in the control group in the CEE and E2/NETA groups (CEE: -8.2 +- 4.3%, p = 0.009; E2/NETA: – 11.7 +- 5.6%, p = 0.007). Mean protein C level decreased significantly compared to that in the controls in the CEE and CEE/MPA groups (CEE: -27.4 +- 13.7%, p = 0.002; CEE/ MPA: – 15.0 +- 8.4%, p = 0.006). The mean protein S level decreased significantly compared to that in the control group only in the CEE/ MPA group (-17.8 +- 8.9%, p = 0.04). Tibolone therapy did not modify any of the parameters assessed. DISCUSSION

Following menopause, anticoagulant factors increase and this may represent an serious attempt by the human organism to compensate for the procoagulant state due to the increase of fibrinogen and factor VII. Increased fibrinogen and factor VII levels are associated with an increased risk of cardiovascular disease (CVD) in men, while the increase of anticoagulation factors in postmenopausal women of similar age corresponds to a lower incidence of cardiovascular events. The fact that, during the seventh and the eighth decades, CVD risk is almost identical to that of men may be partly due to the inability to counterbalance the increase in fibrinogen and factor VII16’17. Furthermore, it is important to consider that there is a difference between the arterial and the venous parts of the vascular system. The venous system is more prone to thrombosis compared to the arterial system due to the low pressure, the lack of pulse and the resultant blood stasis. Coagulation is directly implicated as the mechanism of venous thrombosis, whereas, in the arterial system, atherosclerosis functions as the mediator of thrombosis.

Table 2 Baseline and follow-up levels of antithrombin, protein S and protein C in 216 postmenopausal women receiving various hormone therapies or no therapy

In our study, a procoagulant effect was seen among HT users, as suggested by the statistical significant decrease in antithrombin. A procoagulant effect was further seen under CEE and CEE/MPA, both of which contributed to a significant decrease in protein C, while protein S decreased only under CEE/MPA. In contrast, E2/NETA had no effect either on protein C or protein S. The differential effect on protein C between CEE and CEE/MPA, on the one hand, and E2/NETA, on the other, may be attributed to the different regimens administered or to differences in the androgenic potency of the progestin. Our findings are in accord with those reported by Cosman and colleagues18, who observed a decrease in antithrombin and total and free protein S under CEE monotherapy. CEE/MPA was associated with the greatest decrease in the anticoagulation factors investigated, as compared to CEE and E2/NETA. Although the coadministration of progestin is necessary for the prevention of endometrial hyperplasia, its presence has been repeatedly considered to antagonize the beneficial effect of estrogens on various cardiovascular risk markers. In the WHI trial, the relative risk for cardiac events and stroke was lower under CEE compared to CEE/ MPA4’19. Further evidence suggesting MPA as a causative factor lies in the fact that E2 combined with MPA also resulted in a decrease of the anticoagulant factors20. On the other hand, Osmanagaoglu and colleagues21 reported that neither CEE/MPA nor E2/NETA had any effect on markers of anticoagulation in postmenopausal women. This study, however, could be limited by the lack of study power due to the small number of patients.

Figure 1 Mean percentage changes (SEM) in antithrombin (a), protein C (b) and protein S (c) according to treatment assignment. *p < 0.05; **p < 0.01 compared to control group by Student's t test for unpaired observations, adjusted for multiple comparisons. CEE, conjugated equine estrogens; CEE/MPA, CEE combined with medroxyprogesterone acetate 5 mg; E2/NETA, 17beta-estradiol 1 mg/ norethisterone acetate 0.5 mg

Although the combined estrogen-progestin therapy may have a more pronounced impact 22, our findings indicate that estrogen monotherapy also decreases the coagulation inhibitors, albeit to a lesser degree. Previous studies23’24 have reported similar findings. Estrogens are capable of directly increasing the hepatic synthesis of factors involved in the hemostatic cascade. Furthermore, estrogens, by exercising a relaxing effect on the vasomotor tone and through an effect on the vascular endothelium, result in changes in blood flow, activation of coagulation and fibrinolysis and blood rheology, all of which indirectly affect the hemostatic system25.

In this study, tibolone had no effect on any of the parameters assessed. Our results on the effect of tibolone on coagulation are consistent with those reported in the literature15’26-29. In a recent report comparing CEE/MPA to tibolone with respect to coagulation inhibitors, tibolone was found to induce fewer pharmacological alterations on blood coaguability than CEE/MPA30. Tibolone converts to the 3alpha- and 3beta-hydroxymetabolites and the Delta4 isomer, which express variable affinity for the estrogen, progesterone and androgen receptors, depending on the tissue targeted15. It may be that the negative effects resulting from the binding to the estrogen and progesterone receptors are counterbalanced to some degree by an anticoagulant effect expressed by the ?4 isomer binding to the androgen receptor. Low-dose androgens, in contrast to high-dose, can be beneficial for the normal function of the coagulation system31’32. Although these pathophysiological pathways have not been elucidated, recent evidence confirms our findings on tibolone’s anticoagulation profile27. Cardiovascular risk is influenced by the net effect of heritable and environmental factors on lipids and on the vascular wall and endothelium. Despite the fact that tibolone does not appear to trigger the coagulation cascade, recent evidence suggests that it does not retard cardiovascular disease progression33. Furthermore, although the LIFT study reported that tibolone increased ischemic stroke risk, it should be noted that the women investigated had a mean age of 68 years; this factor may relate to the presence of advanced atherosclerotic lesions 34.

The variability of the studies addressing the matter cannot be explained only by the heterogeneity of medications and study design. It is possible that genetic susceptibility factors are involved in the pathophysiology of CVD and VTE. Furthermore, environmental factors, such as age, menopausal age, obesity, smoking, dyslipidemia, insulin resistance and hypertension, may interact with inheritable factors35. In our study, no correlation was seen between anticoagulant factors and most of these environmental parameters. This finding, however, may be due to the small number of women in each group, which may have prevented the surfacing of differences between therapy groups.

In conclusion, CEE, CEE/MPA and E2/NETA regimens decreased coagulation inhibitors. The extent of the decrease varied among these groups. CEE/MPA decreased all three anticoagulants investigated. CEE-only decreased antithrombin and protein C, while E2/NETA decreased only antithrombin. Tibolone had no impact on coagulation inhibitors. However, the risk of thromboembolic disease among postmenopausal women under HT is defined not only by the regimen administered but also by the genetic and phenotypic factors predisposing to the disease. Therefore, large clinical trials are needed to clarify the interplay between all these factors involved in the pathogenesis of thromboembolic disease.

Conflict of interest Nil.

Source of funding University of Athens Special Research Account.

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N. C. Keramaris, G. E. Christodoulakos* , I. V. Lambrinoudaki* , A. Dalamanga[dagger], A. P. Alexandrou[double dagger], J. Bramis[dagger], E. Bastounis and G. C. Creatsas *

Vascular Clinic, 1st Department of Surgery, University of Athens Medical School, Laikon Hospital,

Athens; *Menopause Clinic, 2nd Department of Obstetrics and Gynecology, University of Athens

Medical School, Aretaieion Hospital, Athens; [dagger]Hematology Department, University of Athens Medical

School, Aretaieion Hospital, Athens; [double dagger]St Department of Propaedeutic Surgery, University of Athens

Medical School, Hippocrateion Hospital, Athens, Greece

Correspondence: Dr I. V. Lambrinoudaki, 27 Themistokleous Street, Dionysos, GR- 1 4578, Athens, Greece

Received 03-10-06

Revised 20-02-07

Accepted 22-02-07

Copyright Taylor & Francis Ltd. Oct 2007

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