Changes in Hemostatic Parameters After Oral Hormone Therapy in Postmenopausal Women

Abstract

Objective:

Oral hormone therapy (HT) and menopausal age are both prothrombotic risk factors. The aim of our study was to compare the hemostatic parameters in plasma of postmenopausal women after 6 months of oral HT with parameters of control (without treatment) postmenopausal women.

Methods:

Twenty-seven postmenopausal women were treated with 17β-estradiol (1 mg) and dydrogesterone (5 mg) daily for 6 months. The control group (27 women) did not receive any HT. Hemostatic factors, such as fibrinogen (FG) concentration, activated partial thromboplastin time (APTT), platelet (PLT) count, maximum velocity of clot formation, and fibrin lysis half-time were estimated.

Results:

The hemostatic parameters in both groups differ significantly. After 6 months oral HT, APTT and the level of FG were higher than in the control group (APTT 30.08 seconds vs. 28.18 seconds, p = 0.02; FG 4.14 g/L vs. 3.03 g/L, p < 0.001). However, the higher values of maximal velocity of FG polymerization (153.53 mOD/min vs. 92.87 mOD/min, p < 0.001), maximum absorbance values (0.306 vs. 0.275, p < 0.001), and fibrin lysis half-time (32.33 minutes vs. 18.11 minutes, p < 0.001) compared with values in the control group also were observed. There was no statistically significant difference in PLT counts between control and women treated with oral HT.

Conclusions:

Six months of oral combined HT (17β-estradiol and dydrogesterone) caused increased initial velocity of clot formation and inhibition of fibrinolysis. The increased level of FG and its higher polymerization may help explain the increase in venous thrombosis and cardiovascular events reported after the use of oral HT.

Introduction

Hormone therapy (HT) is aimed at eliminating or alleviating the symptoms of menopause.¹ Postmenopausal HT is an established thrombotic risk factor for venous thromboembolism. It may increase the incidence of myocardial infarction (MI) and stroke in older women. During treatment with oral HT, the risk of venous thromboembolic complications increases by 2–3-fold, particularly within the first 6 months of therapy.^2,3 HT also affects the structure and metabolism of proteins involved in coagulation and fibrinolysis.^4
–6 Because oral HT may cause prothrombotic changes in blood coagulation and fibrinolysis, we studied its effects (17β-estradiol and dydrogesterone) on the kinetics of fibrin formation and fibrinolysis in postmenopausal women using a multiparameter test of clot formation and fibrinolysis.⁷ The purpose of our study was to asses the effects of oral HT on fibrin formation and clot lysis by determinating such parameters as fibrinogen (FG) concentration, maximum velocity of polymerization with maximal extent (maximum absorbance) of clot formation, and fibrin lysis half-time. Our studies were focused on determining selected kinetic parameters that enable characterization of the influence of oral HT on the rate of clot formation and lysis.

Materials and Methods

Study participants

Fifty-four postmenopausal healthy Caucasian women living in an industrial area of central Poland participated in this study. The trial was not randomized. All the women were patients in the Gynaecology and Menopausal Disorders Clinic of Polish Mother's Memorial Hospital–Research Institute in Lodz. Menopause symptoms were the indications for treatment. The exclusion criteria included contraindications to HT: acute or chronic inflammatory diseases, malignant neoplasms, diabetes mellitus, antioxidant or anticoagulant treatment. None of the women had taken any cholesterol-lowering drugs or antioxidant/vitamin supplements in the 2 months preceding the study. All were nonsmokers.

Two groups of healthy postmenopausal women, all of whom had plasma 17β-estradiol levels of <50 pg/mL, participated in this study. Twenty-seven women aged 52.4 ± 4 years (mean ± standard deviation [SD]) were assigned to begin 6 months of treatment with 1 mg 17β-estradiol and 5 mg dydrogesterone daily. In the second (control) group, aged 56.2 ± 4 years, were 27 women without oral HT treatment. The study was approved by the Bioethics Committee at Polish Mother's Memorial Hospital–Research Institute in Lodz (RNN/57/06/KE), and all participants gave consent to our study.

Laboratory assays

Before the study, the following examinations were performed: gynecological examination with cervical cytological analysis, transvaginal ultrasound assessment of the reproductive organs, palpative breast examination, and mammography. Concentrations of glucose; 17β-estradiol; cholesterol; FG estimated by the method of functional FG determination using a Multifibren U kit (Dade-Behring Inc); platelet (PLT) count using a Baker 810 platelet analyzer; activated partial thromboplastin time (APTT) using a Pathromtin SL reagent (Dade-Behring Inc); and triglyceride levels in the blood of all participants were determined. A blood sample (5 mL) obtained from the antecubital vein (between 8 and 9 am after an overnight fast) was added to 0.55 mL of 3.8% sodium citrate (citrate/blood ratio exactly 1:9). To obtain plasma, blood samples were immediately centrifuged (2500g for 15 minutes at 20°C). The analyzed samples contained 300 μL plasma diluted 3-fold in tris-buffered physiological saline solution (TBS), 60 μl bovine thrombin 400 (BIOMED) at a final concentration of 0.5 U/mL, and tissue plasminogen activator (tPA) (commercial Actilyse 20 preparation, Boehringer Ingelheim) at a final concentration of 60 ng/mL.

Kinetics of FG polymerization and clot lysis

Measurement of total clot formation and fibrinolysis in 3-fold diluted plasma was performed with a BIORAD 550 microplate reader in the Department of General Biochemistry at the University of Lodz using a multiparameter test of clot formation and fibrinolysis and a kinetic program (Microplate Manager 4.0.) according to the method described by Kostka et al.⁷ The changes in absorbance at a wavelength of 415 nm within 55 minutes were recorded after the addition of thrombin and tPA using a kinetic program. The microplate reader recorded 132 measurements. For each patient, the estimation was done four times, and assays were performed at ambient temperature. To study FG polymerization, thrombin at a final concentration of 0.5 U/mL was added; to stimulate fibrinolysis, tPA at a final concentration of 60 ng/mL was used. Measurement of changes in the light passing through the well (containing diluted plasma) was performed. The increased absorbance forms a fully established clot when the absorbance maximum is achieved (the plateau in the resulting graph). tPA, added before measurement, activates a process of fibrinolysis of a formed clot concomitant with the decrease in absorbance values (until the baseline is obtained). The graph created by the microplate reader was used for calculation of the maximum velocity of FG polymerization with maximum absorbance and fibrin lysis half-time.

Analysis

All values in this study are expressed as means ± SD using StatSoft Inc. Statistica version 7.0. To check the normality of sample distribution, the data were analyzed using a Shapiro-Wilk test. When the distribution was normal (APTT, PLT count, 50% lysis time), statistical analysis of the difference between the control plasma (without oral HT) and plasma treated with oral HT was performed using a paired Student's t test (StatSoft Inc. Statistica version 7.0). When the distribution was not normal (FG concentration, maximum velocity of polymerization, maximum absorbance), the nonparametric Mann-Whitney U test was used.

Results

The hemostatic parameters determined by a multiparameter test of clot formation and fibrinolysis in the plasma of 54 postmenopausal women without oral HT and women after 6 months treatment with combined (17β-estradiol and dydrogesterone) oral HT differ significantly (Table 1). In postmenopausal women, after 6 months of oral HT consisting of estrogens and gestagens, changes in the kinetics of both polymerization and fibrin lysis were observed (Table 1). Our preliminary results obtained using a multiparameter test of clot formation and fibrinolysis showed marked acceleration of clot formation (V_max increased by about 58%) and strong inhibition of clot lysis in women after oral HT (about 56%) compared with the results obtained for the control group.

Table 1.

Hemostatic Parameters of 27 Postmenopausal Women After 6-Month Treatment With Oral Hormone Therapy and 27 Postmenopausal Women Without Hormone Therapy

	Control group Mean ± SD	Treatment group Mean ± SD	p value
Platelet count	264.000 ± 75.7	228.000 ± 75.9	0.09
Activated partial thromboplastin time (seconds)	28.18 ± 2.15	30.08 ± 3.89	0.02
Fibrinogen concentration (g/L)	3.03 ± 1.22	4.14 ± 1.64	< 0.001
Maximum velocity of polymerization (mOD/min)	92.87 ± 5.17	156.53 ± 8.54	< 0.001
50% lysis time (minute)	18.11 ± 3.38	32.33 ± 10.82	< 0.001
Maximum absorbance Λ = 415 nm	0.275 ± 9.42	0.306 ± 11.21	<0.001

The group treated with oral HT also had increased levels of FG estimated by the method of functional FG determination. Clot formation was presented as the maximum velocity of polymerization value, and inhibition of lysis was determined by means of the time required for half lysis (in the oral HT group, that time was almost twice as long as in the control group). In women after treatment with oral HT, the higher values of maximum absorbance and the longer time of lysis indicate, respectively, that increased fibrin polymerization and inhibition of lysis occurred.

Discussion

Postmenopausal women have increased levels of risk factors for cardiovascular diseases (CVD). Combined oral postmenopausal HT is related to a weak but clinically important risk of arterial and venous thrombosis.⁸ The increased level of FG in postmenopausal women might partly explain the increase in risk for venous thrombosis and cardiovascular events reported after the use of oral HT. Recently published results by Hellgren et al.⁹ indicate that FG may be decreased after oral HT treatment with estradiol (2 mg) and dydrogesterone (10 mg). Our results demonstrate that after 6 months of oral HT with other doses of hormones than those described by Hellgren et al.⁹ (17β-estradiol, 1 mg, and dydrogesterone, 5 mg), increased initial velocity of clot formation and inhibition of fibrinolysis are observed.

Differences an FG levels between our results and those of Hellgren et al.⁹ may ensue from the different methodology of study group selection. The findings of Hellgren et al. were based on a group of women before and after HT treatment, whereas our research studied two different groups of postmenopausal women, one with and one without oral HT treatment. It is possible that our HT patients had higher levels of FG before treatment, but such data are unavailable. The influence of HT on functioning of the hemostatic system is a very complex and multidirectional process. Thromboembolic complications are among the most dangerous undesired effects in women treated with oral HT.^10,11 During the 6-months HT, no statistically significant changes in coagulation testes (APTT, thrombin and prothrombin time) were found. The observed increase in FG concentration may be associated with increased blood viscosity that hampers its flow in blood vessels.¹² A positive correlation between increased FG levels and the risk of venous thromboembolism were demonstrated in the Leiden Thrombophilia Study (LETS).¹³ Thus, the increased level of that protein may be one of the causes of acceleration of the rate of clot formation.¹⁴ It seems that in postmenopausal women, an abnormal structure of the formed clot can be the cause of hampered clot lysis. The thin and tightly packed fibrin fibers are less accessible to fibrinolytic factors, and clots formed in this way cannot be efficiently removed by the fibrinolytic system. Such impairment of clot removal can evolve into thrombosis.¹⁵ Detection and reduction of cardiovascular risk factors must be among the qualifications for HT and monitoring women during therapy. An important protection is to choose the appropriate route of administration of HT, which is crucial because of possible contraindications. There is no doubt that low-dose HT should be selected for each individual patient.¹⁶

Involvement of the thrombin-activatable fibrinolysis inhibitor (TAFI)—a carboxypeptidase capable of removing C-terminal lysine residues from fibrin and leading to diminution of fibrinolysis—is likely. As it is well known that C-terminal lysine residues of fibrin are essential to bind plasminogen and tPA,^17,18 involvement of TAFI may be important. Thus, this enzyme reduces the cofactor activity of fibrin in plasminogen activation.¹⁹ Fibrin formed in this way has an abnormal structure and is more susceptible to disruption, which can lead to the risk of cardiovascular embolism.²⁰ HT can also reduce antithrombin third (ATIII) plasma levels,^21,22 resulting in uncontrolled thrombin generation.²³

The incidence of clinical thromboembolism during oral HT treatment is relatively low, but it cannot be completely excluded. Based on our results, we can assume that oral HT in postmenopausal women may cause an increase in the rate of clot formation and inhibition of fibrinolysis, although a full explanation of the prothrombotic mechanisms requires further research. It is possible that combined HT (17β-estradiol and dydrogesterone) may be the cause of altered structure of the clot formed, and it can be the cause of hampered fibrinolysis.

Our preliminary results from a small group of postmenopausal women treated with oral HT obtained using a multiparameter test of clot formation and fibrinolysis suggest that the altered lysis of the clot via enhanced fibrinogen polymerization may contribute to a prothrombotic state. Further investigation is required to establish the mechanisms of prothrombotic changes related to HT and to explain how oral HT in postmenopausal women affects clot formation and fibrinolysis.

Footnotes

Acknowledgments

This study was supported by grant 810/373 from the University of Lodz.

Disclosure Statement

The authors have no conflicts of interest to report.

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