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-estradiol, conjugated equine estrogen, and raloxifene on mRNA expression, aggregation, and secretion in platelets
1Department of Physiology and Bioengineering, 2Department of Surgery, 3Section of Hematology Research, and 4Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota
Submitted 2 November 2004 ; accepted in final form 6 January 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-estradiol (2 mg/day), conjugated equine estrogen (0.625 mg/day), or raloxifene (60 mg/day) for 4 wk. Platelet turnover, aggregation, and secretion were assessed before and after treatment. Platelet turnover and mRNA increased significantly only in pigs treated with 17-estradiol. Expression of estrogen receptors increased with ovariectomy and decreased with all treatments. Platelet aggregation and secretion of ATP, platelet-derived growth factor, and matrix metalloproteinase-2 increased with ovariectomy. All treatments reduced both aggregation and secretion. Expression of mRNA for constitutive endothelial nitric oxide synthase (eNOS), but not eNOS protein, increased with ovariectomy. Only eNOS mRNA decreased with all treatments, but only treatment with 17-estradiol increased secretion of nitric oxide from intact platelets. Platelets from 17-estradiol-treated animals caused relaxation of coronary arteries, which was sensitive to inhibition of nitric oxide. Although three different estrogenic treatments reversed increases in platelet aggregation caused by ovariectomy, only 17-estradiol increased platelet RNA and release of platelet-derived nitric oxide. These differences reflect transcriptional and posttranscriptional regulation of protein synthesis in bone marrow megakaryocytes and circulating platelets.
hormones; matrix metalloproteinase; nitric oxide; thrombosis
Platelets contribute to thrombosis in several ways. They provide the membrane surface for the generation of thrombin, express membrane receptors that affect platelet-platelet and platelet-vessel wall interactions, and release vasoactive substances affecting vascular tone, which in turn affect retention of a platelet plug. Platelets and their precursors, megakaryocytes, contain both estrogen receptor (ER)
and ER (21, 22, 26, 40). Therefore, the concentration of circulating hormones, i.e., hormonal status defined by the loss or replacement of ovarian hormones, has the potential to affect platelet functions through genomic mechanisms at the level of the megakaryocyte. Loss of ovarian hormones by surgical ovariectomy increases platelet aggregation, dense body ATP secretion, and content of factors such as PDGF-BB and matrix metalloproteinase-2 (MMP-2) that affect repair and remodeling of the blood vessel wall (4, 23, 24). In addition, the enzyme required for production of NO (endothelial nitric oxide synthase, eNOS) increases with ovariectomy. NO produced by vascular endothelial cells and platelets reduces platelet aggregation and adhesion (20, 30, 36, 38). However, no studies systematically have evaluated and compared how estrogenic treatment of ovariectomized animals affects aggregation, basal and stimulated release of substances from platelet-dense and -granules, and content of other platelet-derived vasoactive and mitogenic substances, like PDGF or NO. Therefore, the present study was designed to determine how treatment with clinically relevant estrogens (17-estradiol, the primary ovarian hormone; CEE, a mixture of metabolic end products of estrogen metabolism; and raloxifene, a SERM) affect platelet functions in hormone-depleted animals.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Anti-human ER monoclonal antibody was purchased from Upstate Biotechnology; anti-human ER monoclonal antibody, Tris, glycine, sodium orthovanadate, SDS, ADP, and ATP were purchased from Sigma (St. Louis, MO). Hanks' balanced salt powder (without NaHCO3) was formulated with 1 g/l D-glucose and 3 mg/ml bovine serum albumin (fatty acid free) obtained from GIBCO Life Technologies (Rockville, MD). Firefly (Photinus pyralis) luciferase and luciferin were purchased from Roche Diagnostic Corporations (Indianapolis, IN). All other reagents and solvents used in this study were of analytical or reagent grade. Pig thrombin was prepared as described previously (34, 35). PDGF-BB and MMP-2 were measured by enzyme immunoassay and total NO by the Griess reaction with commercially available kits (R&D Systems, Minneapolis, MN).
Animals and Experimental Design
The Institutional Animal Care and Use Committee of Mayo Clinic approved this study. Sexually mature female pigs (4 crossbreeds: Yorkshire, Hampshire, Duroc, and Landrace; 7 mo of age) were used in this study. Ovaries were removed laparoscopically under an anesthesia mixture of xylazine (2 mg/kg), glycopyrrolate (0.44 mg/kg), and telazol (5 mg/kg) given intramuscularly. Blood samples were collected from femoral arteries of anesthetized pigs before ovariectomy (pre-OVX) and 4 wk after ovariectomy (4-wk OVX). At 4 wk after ovariectomy, pigs were randomly assigned to one of four treatment groups: untreated (8-wk OVX), oral 17-estradiol (2 mg/day), oral CEE (0.625 mg/day), or oral raloxifene (60 mg/day) for 4 wk. These drugs and dosages were chosen because they represented dosages that were used clinically at the time the study began. Animals were fed measured portions of Lean Grow 93 diet (Land O'Lakes Farmland Feed, Fort Dodge, IA) twice a day; water was available ad libitum. All medications were mixed into the morning food, which was consumed in the presence of the veterinary or laboratory technician. After 4 wk of treatment or in total 8 wk after ovariectomy (for untreated pigs 8-wk OVX or for treated pigs 4-wk OVX + 4-wk hormone treatment), pigs were anesthetized by intramuscular injection of xylazine (2 mg/kg), glycopyrrolate (0.44 mg/kg), telazol (5 mg/kg), and ketamine (4.5 mg/kg). Blood was collected from the carotid artery into anticoagulated [5 ml of anticoagulant acid citrate dextrose solution USP (ACD) Formula A; Baxter Healthcare] 50-ml polypropylene centrifuge tubes for platelet functional studies and EDTA anticoagulated tubes for plasma studies. Total blood platelet count was obtained in EDTA anticoagulant blood by a three-part Coulter counter at the Mayo Clinic Hematology Lab (Rochester, MN).
Circulating concentrations of 17-estradiol show a cyclic pattern in pigs (13), as would be expected for adult animals with an estrous cycle. In animals with ovaries, plasma estradiol varies from 21.6 ± 3.1 pg/ml to levels below the detection limit of the assay (1). In pigs treated with 17-estradiol, it was not possible to detect 17-estradiol in the plasma obtained 24 h after animals ingested the medication. Therefore, a bioassay of uterine weight and anatomy was used to show treatment efficacy. Uteri were removed and weighed. A section of the uterine horn was prepared for light microscopy with standard techniques for paraffin embedding and staining with hematoxylin and eosin (5-µm sections, prepared on glass slides).
Analysis to Assess mRNA in Platelets
To determine the percentage of platelets containing RNA (reticulated platelets, the youngest platelets in circulation), a 19-gauge needle was inserted into an ear vein, five drops of blood were allowed to drip, and 20 µl of blood was collected with a pipette and diluted (1:100) into 2 ml of the following solution: Hanks' balanced salt solution, without NaHCO3, buffered (pH 7.4) with 20 mM HEPES, supplemented with 1 mg/ml bovine serum albumin, 1 µM tick anticoagulant peptide, 0.05% glucose, 25 nM hirudin, and 1 µg/ml PGE1. The reticulated platelets were determined by flow cytometry (FACSCalibur, Becton Dickinson; Ref. 24).
Platelet Preparation for Western Blotting
Platelets were separated from the ACD-anticoagulated whole blood and washed as described previously (23, 24). The purity of washed platelets was validated with a three-part Coulter counter. The washed platelets were resuspended in lysis buffer (1% SDS, 1 mM sodium orthovanadate, 10 mM Tris·HCl pH 7.4) and stored at –70°C for Western blot analysis. Western blotting of estrogen receptors (ER and ER) and eNOS was determined as described previously (23, 24).
Platelet Aggregation and Dense Body ATP Secretion
Platelet-rich plasma (PRP) was separated from ACD-anticoagulated blood as previously described (24). Purity of PRP was validated by Coulter counter (Mayo Clinic Hematology Lab), yielding <0.1% of leukocyte or red blood cell contamination. Aggregation and dense body ATP secretion studies were carried out in PRP containing 250,000 platelets/µl.
Aggregation studies were performed in PRP by a turbidimetric method using a whole-blood aggregometer in optical mode (model no. 560-VS, Chrono-log, Havertown, PA; Ref. 24). Pig platelets do not aggregate to arachidonic acid, epinephrine, norepinephrine, ATP, ristocetin, or super thrombin receptor agonist peptide (Refs. 24 and 43 and unpublished observations) but do aggregate to ADP, collagen, and the calcium ionophore A-23187. ADP (10 µM) and collagen (6 µg/ml) were used as agonists for aggregation. Porcine platelets aggregate reversibly with ADP and irreversibly with collagen.
Secretion of ATP was measured by bioluminescence (24). Thrombin (50 nM pig thrombin) and collagen (3–6 µg final concentration) were used to cause secretion of ATP. Dense body ATP secretion is lower in response to collagen (not shown) than to thrombin.
Analysis for eNOS mRNA by Real-Time PCR
Total RNA was extracted from platelets by TRIzol reagent. DNA contamination in isolated RNA was eliminated with DNase treatment. Expression of eNOS mRNA was determined by quantitative PCR. RT reaction and PCR for eNOS mRNA were performed in accordance with guidelines from Applied Biosystems and as described by our group (6, 7). The following primers were used for eNOS mRNA determination in porcine platelets: forward primer 5'-CAAAGTGACCATTGTGGACCAT-3'; reverse primer 5'-TGCTCGTTCTCCAGGTGCTT-3'; probe sequence 5'-FAM-CCGCCACGGCCTCCTTCATG-TAMPRA-3'.
Stimulated Release of Platelet-Derived Factors
PRP (1 ml containing equal number of platelets) was aliquoted into three different tubes; one served as the control (no activation with agonist), and the other two were activated with either ADP (10 µM) or collagen (6 µg/ml) for 3–5 min. All tubes were then centrifuged at 1,500 g for 10 min to separate platelets and platelet-poor plasma (PPP). The separated platelets reconstituted in 1x PBS and PPP were stored at –70°C for subsequent evaluation. Platelet lysate was prepared from reconstituted platelets by thawing and passing them through a 26-gauge needle several times and sonicated for 6 min. Lysate was separated from insoluble materials by centrifugation at 12,000 g for 5 min. PDGF-BB and MMP-2 were measured in the PPP and/or prepared lysate by enzyme immunoassay according to the manufacturer's instructions. Measurement of factors in platelet lysate of unstimulated platelets is indicative of the effects of hormone treatment on protein synthesis when the platelets were formed and regulation of that protein once the platelet is in the circulation. This value represented baseline for platelets from each pig and was used to calculate how much of a specific protein was released when the platelets were stimulated with an agonist. NO was measured in PPP from unstimulated and stimulated platelets before and after addition of agonists. Release of factors from stimulated platelets was calculated by subtracting the concentration of a substance measured in PPP or platelet lysate of unstimulated platelets from the concentration of that substance measured in PPP or platelet lysate of stimulated platelets in paired samples from each animal.
Interaction of Platelets with Coronary Arteries
The left circumflex coronary artery was removed from each pig. After removal of the adventitia, the artery was cut into four rings. The endothelium was deliberately removed from two rings by rubbing the lumen of the ring with a wetted cotton swab (6). Each ring was suspended between a fixed point and a force transducer (Statham UC-2) in organ chambers (25 ml) containing modified Krebs Ringer bicarbonate solution (control solution; in mM): 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, 0.026 edetate calcium disodium, and 11.1 glucose, bubbled with 95% O2-5% CO2 at 37°C. Pairs of rings with and without endothelium from a single animal were studied in parallel in the absence and presence of NG-monomethyl-L-arginine (L-NMMA, 10–4 M). Rings were incubated with L-NMMA for 40 min before the addition of platelets.
Passive tension on the rings was progressively increased. Active tension to KCl (20 mM) was measured at each level of passive tension until each ring reached its optimal point on the length/tension curve. Maximal tension to KCl (60 mM) was obtained in all rings. From a given animal, rings with and without endothelium or in the absence and presence of inhibitors were then studied in one of three experimental protocols.
Rings were contracted with PGF2 (2 x 10–6 M), and once the contraction plateaued responses to washed, autologous platelets (25,000, 50,000, and 75,000 platelets/µl) were obtained (27). Platelets added to organ baths containing L-NMMA were exposed to the arginine analog.
Statistical Analysis
Data are presented as means ± SE. The number of different animals from which samples were derived is designated by n. Concentrations of released platelet factors are expressed as picograms per milliliter per 106 platelets, and concentration of NO is expressed as picomoles per milliliter per 106 platelets. For relaxations, data are presented as percent change in tension from a submaximal contraction to PGF2. Data were analyzed by one-way ANOVA followed by Dunnett's test to identify differences among groups. Statistical significance was accepted as P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Body weight increased similarly in all treatment groups. Concentrations of plasma 17-estradiol were below the detection limit of the assay in all groups of treated pigs. However, efficacy of treatment is evidenced by qualitatively greater glandular formation and enlargement of endometrium in uteri of treated compared with ovariectomized pigs (Fig. 1). Raloxifene had a statistically greater effect than 17-estradiol or CEE on uterine weight (Table 1).
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-estradiol treatment. Plasma fibrinogen did not change with ovariectomy or hormone treatment (Table 1). Expression of Estrogen Receptors
Expression of both ER and ER increased in platelets after ovariectomy. With 4-wk hormone treatment, expressions of ER and ER decreased and were not different from levels observed before ovariectomy (pre-OVX) (Fig. 2).
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Aggregation. Platelet aggregation in PRP containing the same number of platelets (250,000 platelets/µl) to both ADP (10 µM; Fig. 3A) and collagen (6 µg/ml; data not shown) increased significantly with ovariectomy (4-wk OVX and 8-wk OVX) and decreased to pre-OVX levels with all hormone treatments. Neither lag time nor rate of aggregation in response to either ADP or collagen changed with ovariectomy or hormone treatment (data not shown).
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-estradiol (n = 10) 1.88 ± 0.12, CEE (n = 11) 1.83 ± 0.14, raloxifene (n = 12) 1.86 ± 0.17]. Platelet Content and Release of Growth Factors
Total content of PDGF-BB and MMP-2 increased with ovariectomy (Fig. 4). After hormone treatment, there was a large variability in concentrations of PDGF-BB and MMP-2 in the platelet lysate such that there were no statistically significant differences in platelet content of PDGF-BB and MMP-2 with any treatment compared with concentrations measured at 8-wk OVX (Fig. 4, A and B).
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Content of eNOS and release of NO.
Increases in protein for eNOS paralleled increases in mRNA (Fig. 5, A and B). Hormone treatments did not significantly reduce content of eNOS protein, but mean mRNA for eNOS was reduced by two- to threefold in the platelet lysate. It was not possible to measure NO in platelet lysate. Total NO was measured in the incubation solution (PPP). However, only in platelets from pigs treated with 17-estradiol was there a consistent increase in NO in the incubation medium after stimulation of intact platelets with collagen (5 of 5 samples; mean release of 18.7 ± 9.5 pmol·ml–1·106 platelets–1; Fig. 5C).
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Platelets caused relaxation of coronary arteries with (Fig. 6) and without (data not shown) endothelium. These relaxations ranged between 5% and 50% of the contraction to PGF2 (Fig. 6). In response to the highest concentration of platelets, relaxations of rings from animals treated with 17-estradiol were threefold greater than those relaxations observed in untreated ovariectomized animals. This difference was eliminated in the presence of L-NMMA. Removal of the endothelium did not eliminate platelet-initiated relaxations (data not shown), which were similar to those observed in rings with endothelium in the presence of L-NMMA (Fig. 6). In no case were contractions observed when the platelets were added to arteries with or without endothelium.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-estradiol) metabolized by endogenous enzymes; CEE, a mixture of compounds but primarily estrone and estrone sulfate; and raloxifene, a synthetic SERM. Dosages for treatments were based on those used in humans. Therefore, these pigs may have been underdosed, given that their body weight was greater than that of average women and the metabolism of the drugs may not be the same in pigs as in humans. However, changes in uterine anatomy support that each treatment had some efficacy (28). Differences in how each treatment affected uterine weight and platelet functions probably reflect combined effects of how each drug was metabolized (i.e., natural hormones 17-estradiol, estrone, and estrone sulfate compared with the synthetic compound raloxifene) and the relative affinity of each compound for binding and activating ER and ER. In addition to changes in uterine anatomy, hormone treatments reversed increases in platelet aggregation and ATP secretion that resulted from ovariectomy, thus further supporting treatment effects even though plasma levels of 17-estradiol were below the detection limit of the assay. Body weight increased comparably in all groups, so changes in platelet functions cannot be attributed to changes in weight alone.
Although platelet count did not increase significantly with any of the interventions, the percentage of platelets expressing RNA increased in 17-estradiol-treated pigs. The percentage of platelets containing RNA is an estimate of platelet formation. However, as total platelet count did not change with treatment, degradation of platelets may have increased to a comparable degree. Alternatively, 17-estradiol may reduce posttranscriptional degradation of mRNA through cytoplasmic, nongenomic mechanisms or increase degradation of mature platelets with simultaneous production of younger platelets. These observations in pigs are consistent with observations that estradiol treatment increases bone marrow megakaryocytes, proplatelet formation, and platelet release in humans (3, 32). As circulating platelets do not have nuclei, changes in content of RNA reflect changes in gene transcription in megakaryocytes. Therefore, results of the present study suggest, albeit indirectly, that loss of ovarian hormones and institution of estrogenic treatments affect genomic-mediated mechanisms in the megakaryocytes. Evidence to support this conclusion includes changes in expression of total RNA, mRNA for eNOS and expression of estrogen receptors, PDGF-BB and MMP-2 in circulating platelets. As RNA in reticulated platelets has a biological half-life of 
24 h, protein synthesis is limited once the platelet is in the circulation. A greater number of platelets containing mRNA, as was observed in those animals treated with 17-estradiol, would indicate a greater capacity for protein synthesis when the platelet is in the circulation.
ER is the predominant estrogen receptor subtype in human and porcine platelets (21, 23, 26). However, expression of both estrogen receptors increased with ovariectomy and was reduced similarly by all three types of treatment. This is the first demonstration of regulation of expression of estrogen receptors by hormone treatment in platelets.
The present study confirms that platelet aggregation and ATP secretion are elevated 4 wk after ovariectomy (24). However, results of the present study extend those observations to show that platelet aggregation and ATP secretion from dense granules remain elevated up to 8 wk after ovariectomy and that hormone treatment reduces these responses to levels observed in animals with ovaries. These results also are consistent with observations in postmenopausal women, in whom 4 wk of treatment with CEE (0.625 mg/day) reduces platelet aggregation (33). Together, these results demonstrate that three different types of estrogenic treatments affect aggregation and dense granule ATP secretion comparably. Total platelet content of ATP did not change with either ovariectomy or hormone treatment. Therefore, changes in ATP secretion are not due to changes in ATP content but more likely reflect changes in agonist-coupled receptors or signaling pathways needed for activation of dense granules.
Stimulated release of PDGF-BB paralleled changes in total platelet content, as release was greatest 8 wk after ovariectomy, when total content of PDGF-BB was the highest. However, stimulated release of MMP-2 did not parallel changes in total platelet content, as at 4-wk OVX platelet content, but not release, was greater than in platelets from pre-OVX animals. This uncoupling between content and release of MMP-2 compared with PDGF-BB suggests different mechanisms for regulation of synthesis compared with activated release of platelet contents. MMP-2 will activate platelet aggregation and adhesion (29, 37). Therefore, aggregation facilitated by MMP-2 released in conjunction with PDGF might accelerate development of occlusive arterial disease associated with loss of ovarian hormones. Reduction of PDGF and MMP-2 secretion after hormone treatment would reduce these effects and provide yet another possible mechanism by which hormone treatment may reduce development of occlusive arterial disease in animals (8, 39). Decreases in platelet content of PDGF and MMP-2 might have been greater with higher doses of hormones and/or if treatments had continued beyond 4 wk.
Addition of platelets to arterial rings from these healthy animals caused variable relaxations in all groups. Because platelets were added directly to the rings, it is not possible to distinguish between responses initiated directly by NO released from platelets and responses initiated by platelet-stimulated release of NO from the arterial endothelium. Aggregating platelets release other vasoactive substances, like ADP, that cause endothelium-dependent and -independent relaxation of vascular smooth muscle and 5-hydroxtryptamine, which causes endothelium-dependent relaxations, and endothelium-independent contractions (9, 10). It is possible that relaxations in response to platelets represent a combination of platelet- and endothelium-derived NO because relaxations observed in tissue derived from pigs treated with 17-estradiol were sensitive to inhibition by L-NMMA. Sensitivity of responses to inhibition by L-NMMA is consistent with greater release of NO from platelets derived from pigs treated with 17-estradiol (27, 31). Relaxations observed in the presence of L-NMMA or in rings without endothelium may be due to direct effects of prostacyclin or ADP on vascular smooth muscle tone (9, 18, 19, 27, 31).
Greater release of NO in platelets from pigs treated with 17-estradiol but not other treatments was unexpected, and the mechanisms by which estrogen treatment facilitates release of NO from platelets remain to be determined. Because mRNA for eNOS decreases with all treatment groups, estrogenic treatments may affect transcriptional regulation of eNOS in megakaryocytes. However, release of NO from the circulating platelet would reflect posttranscriptional or posttranslational regulation of eNOS in platelets. Whether or not this regulation is associated with nongenomic activation of eNOS through estrogen receptors as is proposed to occur in endothelial cells remains to be determined (5).
In summary, the present study demonstrates that platelet functions are modulated by hormonal status. Hormonal status affects expression of estrogen receptors, aggregation, and content and secretion of some platelet-derived proteins such that short-term treatment with estrogenic compounds reverses effects of ovariectomy. It is unlikely that these changes in platelet functions alone would account for increased risk of thrombotic events with oral hormone therapy in humans. Changes in production and release of platelet-derived mitogenic and vasoactive factors vary with the type of estrogenic treatment, as only treatment with 17-estradiol increased total RNA and collagen-stimulated release of NO. Differences in efficacy of 17-estradiol compared with CEE and the SERM raloxifene may reflect differences in metabolism of these compounds, differential receptor affinity required for genomic, transcriptional actions at the level of megakaryocytes, and nongenomic, posttranslational regulation of eNOS in the platelets. Differences in affinity of estrogen receptors for the natural hormones 17-estradiol and its metabolites estrone and estrone sulfate compared with the synthetic compound raloxifene may have clinical implications for cardiovascular effects of these products when used in postmenopausal women for treatment of menopausal symptoms.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, caveolin-1 and estrogen receptor associated proteins. Platelets 14: 75–81, 2003.[CrossRef][Web of Science][Medline]
and androgen receptor (AR): testosterone regulates AR expression. Blood 95: 2289–2296, 2000.This article has been cited by other articles:
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 M. Jayachandran, G. J. Brunn, K. Karnicki, R. S. Miller, W. G. Owen, and V. M. Miller In vivo effects of lipopolysaccharide and TLR4 on platelet production and activity: implications for thrombotic risk J Appl Physiol, January 1, 2007; 102(1): 429 - 433. [Abstract] [Full Text] [PDF]
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M. Jayachandran, A. Sanzo, W. G. Owen, and V. M. Miller Estrogenic regulation of tissue factor and tissue factor pathway inhibitor in platelets Am J Physiol Heart Circ Physiol, November 1, 2005; 289(5): H1908 - H1916. [Abstract] [Full Text] [PDF]
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 M. Jayachandran, K. Karnicki, R. S. Miller, W. G. Owen, K. S. Korach, and V. M. Miller Platelet Characteristics Change With Aging: Role of Estrogen Receptor {beta} J. Gerontol. A Biol. Sci. Med. Sci., June 1, 2005; 60(7): 815 - 819. [Abstract] [Full Text] [PDF]
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