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Am J Physiol Heart Circ Physiol 283: H220-H226, 2002. First published March 7, 2002; doi:10.1152/ajpheart.00950.2001
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Vol. 283, Issue 1, H220-H226, July 2002

Ovariectomy upregulates expression of estrogen receptors, NOS, and HSPs in porcine platelets

Muthuvel Jayachandran and Virginia M. Miller

Department of Surgery and Physiology and Biophysics, Mayo Clinic and Foundation, Rochester, Minnesota 55905


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Platelets participate in normal and pathological thrombotic processes. Hormone replacement in postmenopausal women is associated with increase risk for thrombosis. However, little is known regarding how platelets are affected by hormonal status. Nitric oxide (NO) modulates platelet functions and is modulated by hormones. Therefore, the present study was designed to determine how loss of ovarian hormones changes expression of estrogen receptors and regulatory proteins for NO synthase (NOS) in platelets. Estrogen receptors (ERalpha and ERbeta ), NOS, heat shock proteins 70 and 90 (HSP70 and HSP90), caveolin-1, -2, and -3, calmodulin, NOS activity, and cGMP were analyzed in a lysate of platelets from gonadally intact and ovariectomized female pigs. Expression of ERbeta and ERalpha receptors, endothelial NOS (eNOS), HSP70, and HSP90 increased with ovariectomy. NOS activity and cGMP also increased; calmodulin was unchanged. Caveolins were not detected. These results suggest that ovarian hormones influence expression of estrogen receptors and eNOS in platelets. Changes in estrogen receptors and NOS could affect platelet aggregation in response to hormone replacement.

17beta -estradiol; hormones; nitric oxide synthase; endothelial nitric oxide synthase; megakaryocytes; inducible nitric oxide synthase; heat shock protein


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ORAL HORMONAL REPLACEMENT THERAPY reduces the risk of cardiovascular diseases when used for primary prevention in postmenopausal women but increases the risk of venous thrombosis (15, 17, 24, 54) and risk of adverse events when used for secondary prevention of cardiovascular disease (26). Mechanisms by which hormone therapies increase risk for these adverse events are multifactorial and involve changes in coagulation proteins and platelets (9, 33, 36).

Blood platelets are fragments of megakaryocytes and contribute to normal hemostasis and thrombotic disorders (1, 2). Both normal and abnormal (thrombosis) hemostasis depends on various regulation factors within platelets (41). Information regarding interactions among ovarian hormones, platelet functions, and thrombosis is controversial. Ovarian hormones influence platelet functions, e.g., binding of fibrinogen to the platelet surface is greater during the luteal compared with the follicular phase of the menstrual cycle (12). Platelet functions vary by sex, e.g., aggregation is higher in women than in men (20, 29). However, high-dose oral estrogen increases thrombosis in both men and women (10, 16, 53). Alternatively, in vitro platelet aggregation and ATP release from dense granules of platelets are inhibited by 17beta -estradiol and medroxyprogesterone treatment in postmenopausal women (3). Estrogen, when added exogenously to isolated platelets, modulates platelet functions through changes in platelet intracellular calcium and release of nitric oxide (NO) (39, 40, 45). In addition, platelet interactions with endothelial cells are influenced by the hormonal status of the platelet donor (38), suggesting that hormonal status influences platelet functions. Platelets and their precursor megakaryocytes contain estrogen receptor (ER)beta (30). However, nothing is known regarding the regulation of ERs on platelets with depletion of ovarian hormone.

Estrogen receptors cause activation of NO synthase (NOS) in endothelial cells through genomic and nongenomic mechanisms (7, 8, 22, 23, 31, 49). In experimental animals, an association between estrogen replacement and decreased atherosclerosis may occur partially through increases in the production of NO (19, 21). In addition to ERs, platelets are known to express constitutive NOS, and platelet-derived NO alters platelet aggregation and adhesion (27, 37, 43, 48). However, it is not known how the loss of ovarian hormones influences expression of NOS relative to ERs in platelets. It is important to systematically evaluate platelet characteristics (i.e., number and type of ERs, regulation of NOS) relative to the presence or absence of ovarian hormones to further interpret studies of how estrogen affects platelet aggregation and secretion under the influence of hormones. Therefore, experiments were designed to characterize and compare expression of ERs and proteins associated with the regulation of NOS in platelets from sexually mature gonadally intact and ovariectomized pigs. It was hypothesized that depletion of ovarian hormones would increase expression of ERs and decrease expression of NOS.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Female Yorkshire pigs (6 mo of age) were used in this study. External genitalia of pigs this age show enlargement and discharge associated with estrus. Pigs were divided into two groups: those with ovaries (intact but sham operated, n = 4) and those with ovaries removed laparoscopically (ovariectomized, n = 4). This experimental design would represent a surgical menopause. Both groups of animals were fed Lean Grow 93 diet (Land O'Lakes Farmland Feed LLC; Fort Dodge, IA) each morning and had free access to water throughout the day. Four weeks after sham surgery/ovariectomy, age-matched intact and ovariectomized pigs were anesthetized by intramuscular injection of ketamine (12 mg/kg) and xylazine (8 mg/kg). Blood was collected from the carotid artery into anticoagulated [anticoagulant citrate dextrose solution USP (ACD) Formula A from Baxter Healthcare] 50-ml polypropylene centrifuge tubes. Previous studies indicate that plasma concentrations of estrogen in intact females of similar age range from 10 to ~30 pg/ml and that ovariectomy reduces both circulating estrogen and progesterone to below the detection limit of the assay (4, 5, 55). Therefore, on the basis of this historical data, uterine weight was used as a bioassay for hormonal status and removal of ovaries was validated by direct observation. Animal studies were approved by the Institutional Animal Care and Use Committee of Mayo Clinic.

Preparation of washed platelets and lysate. The total platelet count in the blood was obtained for each pig by a Coulter counter (Mayo Clinic Hematology Lab; Rochester, MN). Platelets were isolated from whole blood by a method described previously with slight modifications (25). In brief, anticoagulated blood was centrifuged at 200 g at room temperature for 15 min to obtain platelet-rich plasma. Platelets were pelleted from platelet-rich plasma by centrifugation at 1,500 g for 10 min. The platelets were then washed two or three times with ACD buffer (pH 6.5) containing 185.7 mM sodium citrate, 14 mM citric acid, 209.8 mM dextrose, 9.9 mM KCl, and 0.3% bovine serum albumin. The purity of washed platelets was validated by a Coulter counter. The washed platelet preparation was centrifuged (1,500 g for 5 min at 22°C). Platelet pellets were resuspended in 1% SDS, 1 mM sodium vanadate, and 10 mM Tris (pH 7.4) (lysis buffer). This preparation was then stored at -70°C. For assays requiring platelet lysates, the frozen platelet preparation was thawed and passed through a 26-gauge needle and sonicated for 6 min. The resulting platelet lysate was then centrifuged at 4°C at 12,000 g for 5 min to remove insoluble materials. The supernatant was separated and concentrated by using a Centricon (YM10) Centrifugal Filter Device from Amicon Bioseparations. Total protein concentration of the supernatant was determined by BCA-200 protein assay reagents (Pierce). Platelet lysates from intact and ovariectomized animals were prepared immediately before a given assay, and lysates from all pigs were performed in parallel to eliminate interassay variability.

Western blotting. For Western blotting, platelet lysate was mixed with an equal volume of 2× electrophoresis sample buffer [1× = 125 mM Tris · HCl (pH 6.8), 2% SDS, 5% glycerol, 0.003% bromophenol blue, and 1% beta -mercaptoethanol] and heated at 95°C for 5 min. Protein prepared from MCF-7 (ER +ve cells) cells and COS-7 or BT-20 cell lysates were used for positive or negative controls, respectively, for ERs. Equal amounts of heated samples (100 µg protein) were loaded in each lane and separated by SDS-PAGE using 7.5% SDS-polyacrylamide gels (ready gels from Bio-Rad) for ERalpha and -beta , endothelial NOS (eNOS), inducible NOS (iNOS), neuronal NOS (nNOS), heat shock protein (HSP)70, and HSP90 and 12% SDS-polyacrylamide gels for caveolin-1, -2, and -3 and calmodulin protein. After electrophoretic separation, the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad) using Trans-Blot SD, semidry transfer cell (Bio-Rad). The protein-transferred membranes were blocked with 5% nonfat dry milk (Bio-Rad) dissolved in transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol) for 1 h and were incubated at 4°C with specific primary antibodies with appropriate dilution in transfer buffer overnight.

The anti-calf uterus ERalpha mouse monoclonal IgG2ak (1:500 dilution), anti-bovine calmodulin mouse monoclonal IgG1 (1:500 dilution), mouse anti-HSP70 monoclonal IgG1 (1:250 dilution), anti-mouse HSP90 monoclonal IgG2 (1:250 dilution), and beta -actin mouse monoclonal IgG1 (1:1,000) primary antibody-incubated membranes were washed twice in 1× Tris-buffered saline (Bio-Rad) and treated with secondary goat anti-mouse IgG-horseradish peroxidase conjugates (50 µl in 10 ml of 1× Tris-buffered saline) for 2 h at room temperature. Protein expressions on membranes were determined by colorimetric method using Opti-4CN Substrate kit (Bio-Rad). Opti-4CN substrate was freshly prepared according to manufacturer's instructions (Bio-Rad).

The anti-mouse ERbeta monoclonal IgM (1:500 dilution), anti-human eNOS mouse monoclonal IgG1 (1:1,000 dilution), anti-mouse iNOS monoclonal IgG1 (1:250 dilution), anti-human nNOS polyclonal IgG (1:250 dilution), anti-rous sarcoma virus-transformed chick embryo fibroblasts caveolin-1 mouse monoclonal IgG1 (1:1,000 dilution), anti-human caveolin-2 mouse monoclonal IgG1 (1:250 dilution), and anti-rat caveolin-3 mouse monoclonal IgG1 (1:1,000 dilution) antibody-incubated membranes were washed in transfer buffer three times (5 min each). The washed membranes were incubated at room temperature for 45 min with biotinylated secondary anti-mouse IgG antibody for primary monoclonal IgG antibodies, biotinylated secondary anti-mouse IgM for primary monoclonal IgM antibodies, and biotinylated secondary anti-rabbit IgG antibody for primary polyclonal antibodies at a dilution of 50 µl in 10 ml transfer buffer. The secondary antibody-treated membranes were washed in transfer buffer three times (5 min each) and then conjugated for 45 min with avidin-biotin complex (ABC) reagent (ABC kit, Vectastain) prepared 30 min before treatment in a combination of 100 µl reagent A and 100 µl reagent B in 5 ml transfer buffer. The conjugated membrane was washed in transfer buffer three times (5 min each) and then incubated with diaminobenzidine reagent prepared according to the manufacturer's instructions (Vectastain, Vector Laboratories) for 3-4 min (28). The specific protein was visualized on the membrane.

NOS activity. Platelet lysate was passed through a 213-µm nylon sieve onto an equilibrated 10-DG desalting column (Bio-Rad) and eluded according to the manufacturer's instructions. A small aliquot was used to determine protein concentrations using BCA-200 protein assay reagents (Pierce). eNOS activity in platelet lysates was determined by the stoichiometric conversion of L-[3H]arginine to L-[3H]citrulline using a method described previously by our group (52, 55).

To determine eNOS activity in the lysates, reactions were started by adding 150 µl protein soluble fraction to 150 µl buffer containing 14.7 nM L-[3H]arginine, 5 µM L-arginine, 54 nM valine, 1.2 mM MgCl2, 1 mM NADPH, 50 U/ml calmodulin, 2 mM FAD, and 10 µM terahydrobiopterin with or without 0.83 mM CaCl2, 1 mM EGTA, and 2 mM NG-monomethyl-L-arginine (L-NMMA) to assess total, calcium-independent, and nonspecific activity. The reactions were carried out by incubating at 27°C for 1 h, and reactions were stopped by the addition of 1.5 ml ice-cold HEPES buffer containing 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and 8 mM EDTA (pH 5.5). Poly-Prep chromatography columns (Bio-Rad) were prepared for the separation of L-[3H]citrulline from L-[3H]arginine by the addition of Dowex suspension, which retains L-[3H]arginine but allows L-[3H]citrulline to pass through. The elute was collected in scintillation vials containing Opti-Fluor solution (Packard; Meriden, CT). L-[3H]citrulline activity was measured in a Beckman 6800 liquid scintillation counter. Incubations containing 150 µl protein-free lysis buffer (blank) previously passed over a desalting column were used as controls. NOS activity was expressed as a percentage of the control from the values of picomoles of L-[3H]citrulline produced per milligram of protein per hour. Calcium-dependent activity was calculated as the total minus calcium-independent activity after correcting for nonspecific activity.

Platelet cGMP determination. An aliquot of the washed platelet preparation was diluted in phosphate-buffered saline to obtain a standard platelet count (1 × 109 platelets/ml). This preparation was stored at -70°C. These frozen platelets were thawed, and an equal volume of 6% trichloroacetic acid at 4°C was added. The mixture was homogenized and centrifuged at 2,500 g at 4°C for 15 min. The supernatant was collected and extracted four times with a 5-ml portion of water-saturated ether and a discard of the ether phase. Extract from each sample (5 ml) was dried at 70-80°C and evaporated to dryness under a steam of air or was lyophilized overnight. The dried samples were reconstituted with 0.5 ml sodium acetate buffer. Reconstituted samples (100 µl) were used for cGMP determination by a cGMP 125I-labeled RIA kit (Cat. No. NEX-133, New Life Sciences Products; Boston, MA).

Antibodies and chemicals. Antibodies were purchased as follows: monoclonal ERalpha and calmodulin antibodies were from Upstate Biotechnology; ERbeta monoclonal antibody was from Sigma (St. Louis, MO); monoclonal anti-eNOS (anti-NOS3), iNOS (anti-iNOS), and anti-caveolin-1, -2, and -3 antibodies were from Transduction Laboratories (Lexington, KY); polyclonal nNOS was from Cayman Chemicals (Ann Arbor, MI); anti-HSP70 and anti-HSP90 monoclonal antibodies were from Stress Gen Biotechnologies; beta -actin monoclonal antibody was from Sigma; and Tris-[hydroxymethyl] aminomethane, glycine, sodium orthovanadate, and lauryl sulfate (SDS) were purchased from Sigma. All other reagents and solvents used in this study were of analytic/reagent grade.

Statistical analysis. Results of eNOS activity and densitometric analysis of Western blots are presented as means ± SD. Statistical significance was evaluated by Student's t-test unpaired observations, and differences at a level of P < 0.05 were considered to be significant. All Western blot experiments were carried out independently using a minimum of four preparations of platelets from different animals.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Uterine weight decreased significantly with ovariectomy (intact, 86.6 ± 12 g; ovariectomized, 51.2 ± 7.8 g). Total platelet count did not differ between intact (325 ± 71 × 103 platelets/µl) and ovariectomized (303 ± 65 × 103 platelets/µl) pigs. Both ERalpha and -beta were detected in platelet lysates from intact and ovariectomized pigs by Western blotting using ER-positive MCF-7 cells and ER-negative COS-7 cells as positive and negative controls, respectively (Fig. 1). Expression of both ERs increased significantly after ovariectomy (Fig. 1).


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  		Fig. 1.   Representative Western blots of estrogen receptor (ER)alpha (A) and -beta (B) expression in blood platelet lysates from gonadally intact and ovariectomized female pigs. Both receptors were identified using monoclonal antibodies. ER-positive MCF-7 cells and ER-negative COS-7 cells were used as the positive and negative controls, respectively. Expression of both receptors increased significantly after ovariectomy. C: densitometric measurement representing lysates from at least 4 separate animals (n = 4). * P < 0.05 compared with intact pigs.

HSP70 and HSP90 compared with intact also increased significantly in platelet lysates after ovariectomy (Fig. 2). Only eNOS, but not iNOS and nNOS, was present in platelet lysates. eNOS protein expression increased significantly in platelets after ovariectomy (Fig. 3). All proteins detected by Western blotting in this study were normalized to beta -actin. eNOS activity (0.81 ± 0.13 in intact and 2.74 ± 0.37 pmol [3H]citrulline · mg protein-1 · h-1 in ovariectomized) and cGMP also were significantly greater in platelet lysates after ovariectomy (Fig. 4).


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  		Fig. 2.   A and B: representative Western blot of heat shock protein (HSP) expression in platelet lysates from intact and ovariectomized female pigs. C: densitometric measurements representing Western blots of HSP70 and HSP90 in platelet lysates from 4 separate animals (n = 4). Expression of HSP70 and HSP90 was significantly greater in lysates from ovariectomized compared with gonadally intact female pigs. * P < 0.05 compared with intact pigs.



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  		Fig. 3.   A: representative Western blot of endothelial nitric oxide synthase (eNOS) protein expression in platelet lysates from intact and ovariectomized female pigs. B: densitometric measurements of Western blots of eNOS protein expression in platelet lysates from 4 separate intact and ovariectomized female pigs (n = 4). eNOS protein was significantly increased with ovariectomy. * P < 0.05 compared with intact female pigs.



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  		Fig. 4.   eNOS activity and cGMP levels in platelets from gonadally intact and ovariectomized female pigs. A: eNOS activity was increased significantly in platelet lysates from ovariectomized female pigs compared with gonadally intact female pigs. eNOS activity in platelet lysates was determined by stoichiometric conversion of L-[3H]arginine to L-[3H]citrulline and expressed as a percentage of intact female pigs (activity for intact female pigs was 0.815 ± 0.127 pmol [3H]citrulline · mg protein-1 · h-1 and that for ovariectomized female pigs was 2.74 ± 0.375 pmol [3H]citrulline · mg protein-1 · h-1). Data represent assays of lysates from four separate animals (n = 4). B: cGMP concentrations in platelets from intact and ovariectomized pigs. cGMP concentrations were increased significantly in platelets from ovariectomized pigs compared with intact pigs. * P < 0.05 compared with intact female pigs.

The eNOS regulatory proteins caveolin-1, -2 and -3 were not detected (data not shown), but the eNOS activation protein calmodulin was detected by Western blotting in lysates of porcine platelets. Expression of calmodulin was similar in platelet lysates from intact and ovariectomized animals (Fig. 5).


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  		Fig. 5.   A: representative Western blot of calmodulin expression in platelet lysates from gonadally intact and ovariectomized female pigs. There was no change in expression of calmodulin protein in platelet lysates from female pigs with and without ovaries. B: densitometric analysis of calmodulin expression in platelet lysates from 4 separate pigs (n = 4).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results of the present study show, for the first time, the expression of ERalpha on platelets and demonstrate that both ERalpha and ERbeta increase significantly with acute loss of ovarian hormones. The significant decrease in uterine weight with ovariectomy supports the removal of the primary source of estrogen and progesterone as would occur with surgical or natural menopause in women. These results at first appear to be in contrast with those of Khetawat et al. (30), who identified only ERbeta in human platelets. These differences in results may be due to the specific antibody used for ERalpha detection. In previously published papers, negative data for various antibodies against ERalpha were not reported. Preparations of the platelets may also affect ability to identify proteins by Western blot because concentrated protein from platelet-rich plasma was used in the present study, whereas gel-filtered platelets were used by others (30). Because of potential differences in the affinity of antibody-receptor interactions, it was not possible to quantify the relative ratio of the alpha - to beta -subtype.

Activation of ERs modulates cellular functions through genomic and rapid or nongenomic mechanisms (6, 8, 35, 46, 57). For platelets, genomic effects of estrogen such as would be observed with sustained loss of ovarian function would occur only in megakaryocytes because these precursors of platelets contain nuclei, whereas circulating platelets do not (57). Therefore, changes in ERs and other regulatory proteins measured in this study would most likely reflect transcriptional changes in megakaryocytes before platelets reach the circulation (30, 50).

HSPs are molecular chaperones expressed constitutively at physiological temperature (56, 58). In the absence of ligand, or in an inactive form, ERs are associated with a number of HSPs (e.g., HSP70 and HSP90). It has been proposed that receptor-associated proteins keep receptors in a conformation that makes the receptor have a high affinity for hormone and a low affinity for DNA binding and are important for the transport of ligand-receptor complex (13, 32). HSP90 and HSP70 expression were increased with ovariectomy. Therefore, increases in HSP70 and HSP90 are consistent with increases in ERalpha and ERbeta with ovariectomy. Binding of a ligand to cytosolic estrogen receptor results in a conformational change of the receptor, which may include dissociation of HSPs from the receptor-protein complex (13, 42). Whether or not platelet receptors are membrane bound or only cytosolic remains to be determined.

In addition to being associated with ER binding, HSP90 also regulates the activity of eNOS (14). Recruitment of HSP90 to eNOS stimulates NO production in endothelial cells (14, 46). Relationships among HSP90 binding to ER and activation of NOS relative to production of NO in circulating platelets remains to be defined. However, changes in the expression of ER and eNOS protein with ovariectomy would be expected to alter production of NO and subsequently platelet aggregation when hormone replacement therapy is instituted in ovarian-depleted animals. Increased production of NO would be expected to inhibit platelet aggregation, adhesion, and thrombus formation (43, 44, 47). Only eNOS was observed in circulating platelets. However, these were healthy pigs. It remains to be determined whether or not the inducible isoform of NOS would be present in platelets from immunologically challenged animals (i.e., lipopolysaccharide stimulated, high cholesterol feeding, or active infection or rejection).

In addition to HSPs, eNOS activity is regulated by other proteins. For example, unlike HSP90, which enhances eNOS activity (18), caveolin-1 maintains eNOS in its inactive state. Caveolin-1 was not detected in porcine platelets, a finding also consistent with what has been reported in human platelets (11). Therefore, regulation of eNOS in platelets may differ from that of endothelial cells.

Calmodulin expression in the platelet lysates was not changed with ovariectomy. This result suggests that eNOS activity in ovariectomized pig platelets may not be related 1:1 with calmodulin expression. Furthermore, the observation that calmodulin expression did not change with ovariectomy provides support for specific rather than nonspecific regulation of all proteins with ovariectomy.

An important question for future studies is how these changes in ERs and protein expression with loss of ovarian function affect platelet functions like irritability, aggregation, and secretion. Differences in expression of ERs would alter responses of the platelets to exogenous or replaced hormone. Indeed, deep vein thrombosis and venous thromboembolism represent a risk of oral hormone replacement in postmenopausal women (10, 17). Both venous thrombosis and arterial vascular events increase in women with preexisting atherosclerotic disease during the first year of hormone replacement (26). Procoagulant effects of hormone replacement in conjunction with changes in platelet functions could increase risk of thrombotic events (34). Results of studies of effects of hormone replacement on platelet functions are controversial (3, 51).

In conclusion, differential expression of ERs, ER-associated HSPs, and eNOS protein and activity in response to ovarian hormone depletion probably reflect genomic regulation of these proteins in megakaryocytes.


    ACKNOWLEDGEMENTS

The authors thank Margarita Bracamonte, Kevin Rud, and Sandy Severson for kind help and cooperation during the study.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-51736.

Address for reprint requests and other correspondence: V. M. Miller, Dept. of Surgery and Physiology and Biophysics, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905 (E-mail: miller.virginia{at}mayo.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published March 7, 2002;10.1152/ajpheart.00950.2001

Received 1 November 2001; accepted in final form 2 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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