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Cardiovascular Research 2002 53(3):634-641; doi:10.1016/S0008-6363(01)00410-2
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Copyright © 2002, European Society of Cardiology

Estrogen replacement suppresses function of thrombin stimulated platelets by inhibiting Ca2+ influx and raising cyclic adenosine monophosphate

Yukiko Nakanoa,*, Tetsuya Oshimaa, Ryoji Ozonoa, Atsushi Uedab, Yasuo Oueb, Hideo Matsuurab, Mitsuhiro Sanadac, Koso Ohamac, Kazuaki Chayamab and Masayuki Kambea

aDepartment of Clinical Laboratory Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan
bFirst Department of Internal Medicine, Hiroshima University School of Medicine, Hiroshima, Japan
cDepartment of Obstetrics and Gynecology, Hiroshima University School of Medicine, Hiroshima, Japan

* Corresponding author. Tel.: +81-82-257-5552; fax: +81-82-257-5554 nakano{at}mcai.med.hiroshima-u.ac.jp

Received 9 March 2001; accepted 16 June 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Method
 3. Materials
 4. Results
 5. Discussion
 References
 
Objective: Estrogen replacement therapy (ERT) in postmenopausal women reduces the risk of cardiovascular diseases. Beneficial changes in lipid profiles account for only one part, thereby raising the question of other estrogen induced benefit that may be lost at menopause. The purpose of this study was to determine the effects of estrogen replacement therapy (ERT) on platelet function of postmenopausal women. Methods: The effect of 4 weeks ERT (conjugated estrogens 0.625 mg/day) on platelet function was evaluated ex vivo in 18 postmenopausal women (mean age 53±5 years, after menopause 3.8±1.9 years). Results: After ERT, (1) plasma concentrations of estrone and estradiol significantly increased (estrone: 16±7–211±80 pg/ml, estradiol: 14±3–125±49 pg/ml, P<0.05) and LDL-cholesterol decreased (129±23–94±25 mg/dl, P<0.05). Plasma 6-keto-PG F1 {alpha} significantly increased (7.2±3.4–13.3±6.7 pg/dl, P<0.05). (2) platelet aggregation and positive staining for P-selectin in thrombin- (0.1 and 1.0 U/ml) stimulated platelets were inhibited (Th 0.1 U/ml: 4.0±0.9–2.4±1.0/control, P<0.05), but positive staining for GP IIb-IIIa complex did not alter significantly. (3) Ca2+ influx induced by thrombin decreased (Th 0.3 U/ml: 345±29–298±24 nmol/l, P<0.05). The baseline [Ca2+]i, the release of Ca2+ from internal stores induced by thrombin and the size of internal Ca2+ stores did not alter. (4) platelet c-AMP increased (Th 0.3 U/ml: 66.4±9.4–82.6±13.0 fmol/l, P<0.05), but platelet nitrite/nitrate (NOx) or c-GMP did not alter significantly. Conclusions: These results suggest that modulation of platelet function by decreasing Ca influx and increased production of c-AMP may account in part for the cardiovascular benefit of ERT.

KEYWORDS Calcium (cellular); Ca-channel; Gender; Hormones; Lipid metabolism; Platelets


   1. Introduction
Top
 Abstract
 1. Introduction
2. Method
 3. Materials
 4. Results
 5. Discussion
 References
 
The low prevalence of coronary heart diseases in premenopausal women, and its increase following menopause, is well established [1,2]. Estrogen replacement therapy (ERT) in postmenopausal women reduces the risk of cardiovascular diseases [3–5]. Several mechanisms are postulated for this anti-atherosclerotic effect of hormone replacement therapy (HRT), such as the improvement in the lipid profile [6,7], direct vasodilating effect [8], lowering of blood pressure [5], and decrease in plasma viscosity [9–12].

Conversely, in heart and estrogen/progestin replacement study (HERS), a higher appearance of thrombosis is observed in patients with HRT than without HRT [13]. Besides, epidemiologic investigations have shown that women who take oral contraceptives are at an increased risk of thromboembolic events such as deep vein thrombosis of lower limbs or pulmonary embolism [14,15]. Sparse data exist concerning effects of ERT on platelets function and that has not reached a conclusion. Estrogen, but not progestin, inhibits platelet aggregation in animal models [16,17]. Recently, we reported that 17 β-estradiol inhibited human platelet aggregation in vitro, via promotion of Ca2+ extrusion or re-uptake activity depending upon an increase in nitric oxide synthesis [18]. Accordingly, the main purposes of the present study were to examine the in vivo effects of ERT on human platelet function and to clarify mechanisms of the phenomenon. We investigated the effects of estrogen on the aggregation and expression of P-selectin and GP IIb-IIIa complex of human platelets stimulated by thrombin. We also attempted to determine the effects of ERT on the metabolism of intracellular second messengers such as Ca2+, cyclic nucleotides, and NOx on platelets.


   2. Method
Top
 Abstract
 1. Introduction
 2. Method
3. Materials
 4. Results
 5. Discussion
 References
 
2.1. Study population and design
A total of 25 menopausal women (mean age 54±4 years-old, 3.9±1.7 years after menopause) were studied. Their menopausal status was determined by the absence of menses for at least 12 months and patients who had undergone hysterectomy or oophorectomy were not included. Their mean plasma concentration of 17 β-estradiol was 14±3 pg/ml. A total of 18 patients were recruited from an out-patient clinic of the First Department of Internal Medicine and seven patients were recruited from that of Obstetrics and Gynecology. Five of the subjects had hypertension and seven had hypercholesteremia, whose fasting total cholesterol >220 mg/dl. The subjects had not received any medication for at least 2 weeks prior to the study and they did not take any other medication during the study. Exclusion criteria were current smoking, history of diabetes mellitus, abnormal hepatic or renal function, fasting triglycerides >350 mg/dl on initial screening laboratory test and a contraindication to estrogen replacement therapy.

The study protocol was approved by the ethical committee of the First Department of Internal Medicine at the Hiroshima University School of Medicine. All subjects gave their informed consent for participation in the study.

The subjects were divided into two groups. In the ERT group (n=18) Premarin (Toyojyozo, Japan) at a dose of 0.625 mg daily and in the control group (n=7) placebo was administered for 4 weeks.

Before and after ERT, ambulatory blood pressure monitoring (ABPM) was performed in the left upper arm using a TM2420 (A&D Co.) in accordance with the Korotokoff microphone method. Blood pressure and heart rate measurements were obtained at 30 min intervals. Patients were confined to their ward and maintained on a regular schedule during the study. They woke at 7~8 AM and room light was turned off at 9 PM. Day-time and night-time blood pressure were obtained as the average value during the awake period between 9 AM and 8 PM, and during the sleep period between 9 PM and 8 AM, respectively.

Blood samples were drawn before and 4 weeks after the start of the treatment. After their over-night fast, blood samples were collected at 9:00 AM from the subjects still in their supine position. Serum concentrations of total cholesterol, triglycerides, high density lipoprotein (HDL) cholesterol and glucose were determined by routine chemical methods. β-thromboglobulin, thrombomodulin, platelet factor IV were measured by EIA and von Willebrand factor by using Laurell method. We measured 6-keto-Prostaglandin F1 {alpha} (6-keto-PG F1 {alpha}) by RIA PGE method. Serum concentration of NOx was measured by performance liquid chromatography (HPLC). Venous blood was drawn from each subject into a syringe containing 1/10 volume of sodium citrate (3.8%) using the two-syringe technique for platelet separation. Blood samples were centrifuged at 800 g for 5 min at room temperature to obtain platelet-rich plasma (PRP). PRP was gel-filtered through a Sepharose 2B-CL column (Pharmacia LKB Biotechnology, Uppsala, Sweden) that had been equilibrated with a medium that contained (mmol/l) NaCl 145, KCl 5, MgSO4 1, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) 10, and glucose 5 (pH 7.4).


   3. Materials
Top
 Abstract
 1. Introduction
 2. Method
 3. Materials
4. Results
 5. Discussion
 References
 
Thrombin (Sigma) was dissolved in deionized water; ionomycin (Sigma) and Fura-2 acetoxymethyl ester (Fura-2/AM) (Molecular Probes, Eugene, OR, USA) were dissolved in dimethyl sulfoxide (DMSO).

3.1. Determination of platelet aggregation
Gel filtered platelets were used in the evaluation of thrombin stimulation. The concentration of platelets was adjusted to 1x108/ml. CaCl2 was added at a final concentration of 1 mmol/l. Thrombin (final concentrations of 0.03, 0.1 or 1.0 U/ml) was then added. Aggregation was recorded for 10 min with NBC HEMA TRACER 601 (MC Medical Inc., Tokyo) using spectrophotometry. Platelet aggregation was expressed as a percent maximal aggregation.

3.2. Determination of P-selectin and GP IIb-IIIa
Fluorescein isothiocyanate (FITC) labeled anti-CD62P antibody (CD62P/FITC) and Phycoerythrin (PE) labeled anti-CD41 (CD41/PE) used in the present study were obtained commercially (Serotec Ltd., Oxford, UK). Anti-CD62P recognizes the {alpha}-granule membrane glycoprotein P-selectin (GMP-140, PADGEM) that surfaces exclusively on activated platelets and is used as a marker for {alpha}-degranulation. Anti-CD41 is raised against the GP IIb-IIIa complex and detects the fibrinogen receptor [19]. After 5 µl platelet suspension was put into polystyrene tubes containing 35 µl HEPES buffer, 5 µl of CD62P/FITC and CD41/PE were added to each tube. Then thrombin (final concentration of 0.03, 0.1 or 1.0 U/ml) was added. After incubation at 37°C for 5 min without stirring, the reaction was stopped with the addition of 500 µl of 1% paraformaldehyde. The stained platelets were analyzed using a flow cytometry (Epics® Elite, Coulter Electronics, HIaleah, FL, USA). The fluorescence intensity of 10 000 platelets per sample was analyzed. A mouse monoclonal antibody, FITC-labeled mouse immunoglobulin G1 (mouse IgG1/FITC; Serotec Ltd., Oxford, UK) was used as an isotype-specific control to set the threshold values.

3.3. Measurement of cytosolic concentration of free Ca2+
For the measurement of [Ca2+]i, the washed platelet solution was adjusted to a concentration of 108 cells/ml and incubated for 30 min at 37°C with fura-2/AM 2 µmol/l, concomitant with 0.02% Pluronic F127 (Molecular Probes). The platelet suspension was refiltered through the Sepharose column to remove the extracellular fura-2/AM. Next, the suspension was adjusted to a concentration of 107 platelets/ml, CaCl2 was added at a final concentration of 1 mmol/l. An aliquot of the cell suspension was stirred in a quartz cuvette at 37°C, and fluorescence was monitored with a spectrofluorophotometer (DM3000CM, SPEX Industries, Edison, NJ, USA). The excitation wavelengths were 340 and 380 nm and the emission wavelength was 510 nm. [Ca2+]i was calculated using the general formula described by Grynkiewics [20], as follows:


Formula

where Kd is 224 nmol/l for fura-2, R is the ratio of fluorescence at excitation wavelengths of 340 and 380 nm in an intact-cell suspension, Rmax is the ratio of absorbance of Ca2+-bound dye at 340 nm to that at 380 nm, Rmin is the absorbance ratio (340/380 nm) of Ca2+-free dye, and Sf380/Sb380 is the fluorescence ratio of Ca2+-bound dye to Ca2+-free dye at 380 nm. The intracellular concentration of fura-2 was determined using an in vitro fura-2 calibration curve, prepared by measuring the fluorescence of known concentrations of fura-2. We measured the basal [Ca2+]i, thrombin- (final concentration of 0.03, 0.1 or 1.0 U/ml) evoked changes in [Ca2+]i in the presence and absence of extracellular Ca2+. We also measured the increase in [Ca2+]i in response to ionomycin, 5 µmol/l, in the absence of extracellular Ca2+, as an index of intracellular Ca2+ discharge capacity [21]. In order to measure the response of [Ca2+]i to agonists in the absence of extracellular Ca2+, after the fluorescence at the resting state had been recorded, ethyleneglycol-bis-(β-aminoethylester)-N,N,N',N'-tetraacetic acid (EGTA), 10 mmol/l, was added to the buffer containing, Ca2+, 1 mmol/l at pH 7.4. Fluorescence was corrected for extracellular dye leakage with EGTA and for autofluorescence by subtracting the fluorescence of the unloaded platelets [22].

3.4. Determination of intraplatelet cAMP, cGMP and NOx (nitrite/nitrate)
Gel-filtered platelets,1x108/ml, were diluted with HEPES buffer that contained 1 mmol/l CaCl2 and then thrombin, 1.0 U/ml was added. After 30 s, the samples were treated with ice-cold 25% PCA to stop the reaction, and were stored at –80°C. After, the samples were thawed at 4°C. The concentrations of cAMP and cGMP were measured using an enzyme immunoassay (EIA) kit with acetylation (Amersham, Arlington Heights, IL, USA) [23]. Platelet levels of NOx were determined with high performance liquid chromatography using anion exchange column (TSK-GEL IC-ANION-PW, Toso, Japan) and detected by UV detector (SPD-10A, Shimazu, Japan).

3.5. Statistical analysis
Values are expressed as mean±S.D. Comparisons between, before and after ERT were made by using paired student T test. A level of P<0.05 was accepted as statistically significant.


   4. Results
Top
 Abstract
 1. Introduction
 2. Method
 3. Materials
 4. Results
5. Discussion
 References
 
In the ERT group, there were three subjects who reported tenderness of the breast and vaginal bleeding but continued the therapy. In the ERT group, plasma concentrations of estrone and estradiol significantly increased (estrone: 16±7–211±80 pg/ml, estradiol: 14±3–125±49 pg/ml, P<0.05) after ERT. Body mass index was not changed by ERT. Total-cholesterol and LDL-cholesterol significantly decreased. Plasma 6-keto-PG F1 {alpha} increased (P<0.05), but the change in NOx was not significant (Table 1). In the control group, after 4 weeks of placebo administration,the lipid profiles and platelet releasing factors did not change significantly.


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  		Table 1 Serum lipid profiles, platelet releasing factors before and after ERTa

 
Results of office BP, mean of 24 h heart rate and ABPM in the ERT group are shown in Table 2. Office BP did not change significantly in the ERT group and in the control group. Mean of 24 h heart rate, 24 h BP, day-time BP or night-time BP did not change significantly in either group.


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  		Table 2 Effect of ERT on office BP and ABPM

 
Fig. 1 shows aggregation in thrombin-stimulated platelets before and after ERT. After ERT, platelet aggregation induced by thrombin was significantly reduced. P-selectin and GP IIb-IIIa expression in thrombin stimulated platelets before and after ERT are shown in Fig. 2. P-selectin expression was significantly reduced after ERT, but GP IIb-IIIa expression did not alter significantly. Fig. 3 shows cytosolic Ca in thrombin stimulated platelets before and after ERT. Thrombin evoked changes in [Ca2+]i in the presence of extracellular Ca2+ was significantly reduced after ERT, but in the absence of extracellular Ca2+, [Ca2+]i did not change significantly. These results suggest that after ERT Ca influx induced by thrombin decreased (Th 0.3 U/ml: 345±29–298±24 nmol/l, P<0.05) but the release of Ca2+ from internal stores induced by thrombin did not change significantly. Additionally, basal [Ca2+]i or Ca2+ discharge capacity did not alter after ERT. Concentration of platelet c-AMP was increased (Th 0.3 U/ml: 66.4±9.4–82.6±13.0 fmol/l, P<0.05), but platelet NOx and c-GMP did not alter significantly (Fig. 4).


Figure 1
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  		Fig. 1 This figure shows platelet aggregation in thrombin-stimulated platelets before and after ERT. Open bar represents status before ERT and closed bar represents that after ERT. After ERT, platelet aggregation induced by thrombin was reduced significantly. *P<0.05 vs. before ERT.

 

Figure 2
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  		Fig. 2 This figure shows P-selectin and GP IIb-IIIa expression in thrombin stimulated platelets before and after ERT. Open bar represents status before ERT and closed bar represents that after ERT. As shown in the left figure, P-serectin expression was significantly reduced, but in the right figure, GP IIb-IIIa expression did not change significantly. *P<0.05 vs. before ERT.

 

Figure 3
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  		Fig. 3 This figure shows intracellular Ca in thrombin stimulated platelets before and after ERT. Open bar represents status before ERT and closed bar represents that after ERT. In the left figure, thrombin evoked changes in [Ca2+]i in the presence of extracellular Ca2+ was significantly reduced. Conversely, in the right figure, thrombin evoked changes in [Ca2+]i in the absence of extracellular Ca2+ did not change significantly. *P<0.05 vs. before ERT.

 

Figure 4
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  		Fig. 4 This figure shows concentration of cyclic nucleotides and NOx in thrombin-stimulated platelets before and after ERT. Open bar represents status before ERT and closed bar represents that after ERT. Platelet c-AMP increased, but platelet NOx and c-GMP did not alter. *P<0.05 vs. before ERT.

 
In the control group, after 4 weeks of placebo administration, platelet aggregation did not change significantly and also there were no significant differences in the expression of P-selectin and GP IIb-IIIa before and after placebo administration. Therefore, we did not examine the metabolism of intracellular second messengers such as Ca2+, cyclic nucleotides, and NOx on platelets in the control group.


   5. Discussion
Top
 Abstract
 1. Introduction
 2. Method
 3. Materials
 4. Results
 5. Discussion
References
 
Cardiovascular diseases are the leading cause of death in postmenopausal women. Data from observational studies suggest that the risk of coronary heart diseases in postmenopausal women can be reduced by ERT. The mechanisms by which ERT contributes to cardiovascular benefit have not completely been clarified. The consensus has been obtained that ERT conferred a benefit on the lipid profile. Also, in the present study, total-cholesterol and LDL-cholesterol were significantly decreased after ERT. In HERS study, treatment with oral conjugated estrogen plus medroxyprogesterone acetate did not reduce the overall rate of coronary heart diseases in spite of decreasing the level of LDL cholesterol and increasing that of HDL cholesterol in the hormone group [13]. Beneficial changes in lipid profiles may account for only a part of estrogen induced vasoprotection. It has been suggested that non-lipid effects are either direct effects on the blood vessel wall or effects on the clotting mechanism.

Thromboembolic complications reported earlier in association with oral contraceptive use have not been discussed with HRT regimens, probably due to the smaller dose of estrogen used. Additionally, some studies have reported that HRT decreases the concentration of coagulation factors such as fiblinogen [5] and factor VII [9], and an inhibitor of thrombolysis such as PAI-1 [10] but increases that of anticoagulants like AT III [11] and lowers plasma viscosity [12]. Unexpectedly, HERS study recently reported that a higher appearance of thromboembolism is observed in patients with HRT than placebo [13]. The present study demonstrated platelet aggregation was reduced after 4 weeks ERT. The suppressive effect of ERT on platelet activity may be among the mechanisms responsible for its beneficial effects in reducing the incidence of cardiovascular events in postmenopausal women.

We also investigated the mechanism of the antiplatelet effect of ERT.

Firstly, we analyzed expression of P-selectin and GP IIb-IIIa using flow cytometry. After ERT, secretion of P-selectin was decreased but surface expression of GP IIb-IIIa did not alter significantly. P-selectin, associated with the platelet membrane, was used as a marker for platelet degranulation in the present study. Together with P-selectin, other granule-stored compounds are released into the plasma compartment. These mediators have been shown to trigger proliferation and migration of vascular smooth muscle cells, resulting in fibrocellular intimal hyperplasia [24,25].

Secondly, we studied Ca2+ handling in platelets before and after ERT, because [Ca2+]i is one of the most important cellular second messengers that determine platelet function. After ERT, thrombin-induced Ca2+ influx decreased, but baseline [Ca2+]i, thrombin-release of Ca2+ from internal stores or the size of internal Ca2+ stores did not change. It is consistent with past studies using vascular smooth muscle cell of rat tail arteries [26] and coronary arteries of pigs [27], 17 β-estradiol inhibits Ca2+ entry from extracellular space but not Ca2+ release from intracellular stores.

It has been suggested that increased levels of cAMP and cGMP activate the resequestration of Ca2+ into internal stores and the extrusion of Ca2+ across the plasma membrane and decreased cytosolic Ca2+ [28–30]. To clarify the relevance of the altered Ca2+ handling caused by ERT and by cyclic nucleotides, we measured the platelet levels of cAMP and cGMP. Four weeks of ERT increased cAMP significantly but the increase in cGMP by ERT did not. Thus, the suppressive effect of ERT on platelet function appeared to be mediated mainly by cAMP.

A comparison study in postmenopausal women reduced thromboxane B2 (TxB2), the metabolite of the platelet derived TxA2, which causes vasoconstriction and platelet aggregation [31]. Platelet aggregation and ATP release were significantly lower in postmenopausal women with HRT than those without HRT [32]. Estrogen's effect on prostacyclin (PG I2) remains unclear. In estrogen treated atherosclerotic rabbits, greater PG I2 synthesis and lower TxB2 levels have been reported [33]. In the present study, plasma 6-keto-PG F1 {alpha}, a PG I2 degradation product increased, suggesting elevated levels of circulating endogeneous PG I2. Prolonged exposure to elevated endogeneous PG I2 may induce a decrease in platelet responsiveness.

Recent studies have confirmed a relationship between estrogen and NO [34–36]. Platelets have been reported to possess an L-arginine/NO pathway [37,38]. Previously, we reported that 17 β-estradiol plays an important role in inhibiting human platelet aggregation in vitro, via promotion of Ca2+ extrusion or re-uptake activity depending upon an increase in nitric oxide synthesis [18]. In the present study, we analyzed the NOx concentration in platelets before and after ERT but found that 4 weeks of ERT did not change the platelet concentration of NOx significantly. The present study did not support the hypothesis that the effect of ERT to suppress platelet aggregation may result from direct stimulation of NO synthesis in platelets. This discrepancy between the results of in vitro and ex vivo may be explained due to a number of reasons. The first may be the difference of the type of estrogen, namely we used 17 β-estradiol in vitro but conjugated equine estrogen ex vivo in the present study. Secondly, the inhibitory effect of ERT on platelet function may be through the change of other metabolic factors such as lipid profile. Thirdly, 4 weeks of ERT may have an influence upon transcription of megacaryocytes.

In conclusion, these results suggest that modulation of platelet function via decreasing Ca influx and increased production of c-AMP may account in part for the cardiovascular benefits of short term ERT.

Time for primary review 34 days.


   Acknowledgements
 
This research was supported in part by a grant-in-aid for Scientific Research (11470518) from Ministry of Education, Science and Culture, Japan.


   References
Top
 Abstract
 1. Introduction
 2. Method
 3. Materials
 4. Results
 5. Discussion
 References
 

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I. C Villar, A. J Hobbs, and A. Ahluwalia
Sex differences in vascular function: implication of endothelium-derived hyperpolarizing factor
J. Endocrinol., June 1, 2008; 197(3): 447 - 462.
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