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J Appl Physiol 97: 1445-1452, 2004. First published May 28, 2004; doi:10.1152/japplphysiol.01074.2003
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Sex-specific changes in platelet aggregation and secretion with sexual maturity in pigs

Muthuvel Jayachandran,1,5 Hiroya Okano,1 Ritu Chatrath,2 Whyte G. Owen,3 Joseph P. McConnell,4 and Virginia M. Miller1,5

Departments of 1Surgery, 2Pediatric Cardiology, 3Hematology, 4Laboratory Medicine, and 5Physiology and Biophysics, Mayo Clinic, Rochester, Minnesota 55905

Submitted 2 October 2003 ; accepted in final form 27 May 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Cardiovascular disease may begin early in adolescence. Platelets release factors contributing to vascular disease. Experiments were designed to test the hypothesis that hormonal transitions associated with sexual maturity differentially affect platelet aggregation and secretion in males and females. Platelets were collected from juvenile (2–3 mo) and sexually mature (adult; 5–6 mo) male and female pigs (n = 8/group). Maturation was evidenced by increased weight of reproductive tissue and changes in circulating levels of gonadal hormones. Aggregation to ADP (10 µM) and collagen (6 µg/ml) and ATP secretion to 50 nM thrombin were determined by turbidimetric analysis and bioluminescence, respectively. Total platelet counts, platelet turnover, and mean platelet volume did not change with maturity. Platelet aggregation and ATP secretion decreased in females but increased in males with maturity, whereas total ATP content remained unchanged in platelets from females but increased in platelets from males. Platelet fibrinogen receptor, P-selectin expression, and receptors for sex steroids did not change with sexual maturation. Plasma C-reactive protein and brain-type natriuretic peptide also did not change. Results indicate that changes in platelet aggregation and secretion change with sexual maturity differently in females and males. These observations provide evidence on which clinical studies could be designed to examine platelet characteristics in human children and young adults.

estrogen; platelet activation; sex steroid receptors; testosterone

PUBERTY IS THE DEVELOPMENTAL period of life characterized by increases in production of sex-specific hormones and gonadal maturation (30). Cardiovascular disease begins in early adolescence (34) and begins at earlier ages and with greater prevalence in young men compared with age-matched women (2). Thrombotic diseases, however, are less prevalent in children than adults (3). Therefore, understanding how hormonal changes associated with normal maturation affect components of the vascular system is important for development of strategies for early prevention of cardiovascular disease in young adults.

Platelets contribute to development of thrombotic events, and possibly development of atherosclerosis, by providing an active surface for procoagulant reactions and by secreting vasoactive and mitogenic cytokines (1, 4, 16, 32). Both megakaryocytes, precursors of platelets, and circulating platelets contain estrogen receptors-{alpha} and -{beta} (ER{alpha} and ER{beta}) and androgen receptors (18, 22, 37). Therefore, it might be hypothesized that endogenous sex steroids would affect steroid-associated gene transcription and translation in these cells. In support of this hypothesis, depletion of ovarian hormones by ovariectomy in adult female pigs increases platelet aggregation and dense body ATP secretion and increases content of estrogen receptors, heat shock proteins, endothelial nitric oxide synthase, matrix metalloproteinase-2, and platelet-derived growth factor-BB (9, 19, 20). In addition, platelet aggregation fluctuates during the menstrual cycle, decreasing with increasing estrogen levels (36). However, hormone treatment to postmenopausal women may not affect platelet aggregation even though acute exposure of platelets to 17{beta}-estradiol reduced platelet aggregation in vitro (5, 38). Androgens, on the other hand, increase production and aggregability of platelets (33, 40, 42). Little is known regarding how functions of platelets change with the transition to sexual maturity (puberty) in males and females when secretions of sex-specific hormones are changing.

Information about platelet function at puberty is required to understand factors that may contribute to early development of cardiovascular disease in young adults. Therefore, the present study was designed to characterize and compare functions (aggregation and secretion) of platelets derived from juvenile (2–3 mo old) and mature or adult (5–6 mo old) male and female pigs. On the basis of data from the literature of effects of sex steroids on platelet function in adults, it was hypothesized that platelet aggregation and secretion would increase with puberty in male animals and decrease in female animals and that the changes would follow changes in sex-specific hormones, hormone receptors, and hormone-receptor-associated proteins (5, 33, 38, 40, 42).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Antibodies and chemicals.   Antibodies were purchased as follows: mouse anti-integrin {alpha}IIb monoclonal and rabbit anti-human integrin {beta}3 polyclonal antibodies from Chemicon International, Temecula, CA; anti-androgen receptor rabbit polyclonal and monoclonal anti-ER-{alpha} mouse antibodies from Upstate Biotechnology (Charlottesville, VA); anti-ER-{beta} mouse monoclonal and {beta}-actin mouse monoclonal antibodies from Sigma Chemical, St. Louis, MO; anti-heat shock protein 27 (anti-hsp27), 32 (hsp32/anti-hemeoxygenase-1), 70 (anti-hsp70), and 90 (anti-hsp90) mouse monoclonal antibodies from StressGen Biotechnologies. Tris (hydroxymethyl) aminomethane, glycine, sodium orthovanadate, lauryl sulfate sodium (SDS), adenosine diphosphate were purchased from Sigma Chemical. Chicken anti-human fibrinogen FITC polyclonal antibody was purchased from Accurate Chemical and Scientific, Westbury, NY. Collagen (equine tendon) was purchased from Helena Laboratories, Beaumont, TX. Hanks' balanced salt powder (without NaHCO3) was formulated with 1 g/l D-glucose, 3 mg/ml BSA (fatty acid free) obtained from GIBCO, Life Technologies, Rockville, MD. Firefly (Photinus pyralis) luciferase and luciferin were purchased from Roche Diagnostic, Indianapolis, IN. Enhanced chemiluminescence (ECL) detection reagents, peroxidase labeled anti-mouse and anti-rabbit antibodies, and Hyperfilm were from Amersham Biosciences, Buckinghamshire, UK. All other reagents and solvents used in this study were of analytical/reagent grade. Pig thrombin was prepared as described previously (25, 26).

Animals and experimental design.   The Institutional Animal Care and Use Committee of Mayo Clinic approved this study. Juvenile (2–3 mo of age; n = 8/sex) and sexually mature (5–6 mo of age; n = 8/sex) male (noncastrated) and female (nonovariectomized) pigs (four crossbreeds of Yorkshire, Hampshire, Duroc, and Landrace) were used in this study. Characteristics of the pigs are given in Table 1. All groups of animals were fed Lean Grow93 diet (Land O'Lakes Farmland Feed, Fort Dodge, IA). The animals were not treated with any exogenous sex steroids at any time.


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  		Table 1. Phenotypic characteristics of pigs used in this study

 
Pigs were anesthetized intramuscularly by injection of ketamine (2 mg/kg), xylazine (2 mg/kg), and telazol (4 mg/kg). Blood was collected from the carotid artery into nonanticoagulated tubes for serum sex steroid determination and anticoagulated [5 ml anticoagulant citrate dextrose solution USP ACD formula A from Baxter Healthcare] 50-ml polypropylene centrifuge tubes for platelet functional studies and EDTA anticoagulated tubes for plasma studies. The blood platelet count was measured in ETDA anticoagulant blood with a Beckman Coulter counter (Coulter Gens, Miami, FL). Serum 17{beta}-estradiol and testosterone were determined by the Clinical Steroid Laboratory of Mayo Clinic Rochester by using chemiluminescent technology (ACS-180 Bayer Diagnosis, East Walpole, MA). The detection limit (with double-volume sample extraction) was 3.6 pg/ml for 17{beta}-estradiol and 10 ng/ml for testosterone. Serum lipids were determined in Mayo Clinic Lipid Lab, Rochester, MN. Plasma C-reactive protein (CRP) was determined by latex particle-enhanced immunoturbidimetric assay on a Hitachi 912 automated analyzer, by use of reagents from Diasorin (Stillwater, MN). The latex particles coated with anti-human CRP antibody aggregates with plasma or serum CRP, forming immune complexes. The formed immune complexes caused increased turbidity measured at 572 nm, which is proportional to the concentration of CRP in the plasma. The plasma CRP concentration was determined from CRP standards of known concentration. Plasma brain-type natriuretic peptide (BNP) was determined by nonequilibrium RIA as described previously (35) with slight modifications. C-8 Bond-Elute cartridges were washed with 4 ml of 100% methanol and 4 ml of distilled water. One milliliter of EDTA plasma was acidified with 1 ml of 0.5% trifluoroacetic acid and applied to cartridges and then washed with 2 ml of saline, 6 ml of water, and 1 ml of 100% methanol. BNP was eluted from the cartridges with 2 ml of 90% methanol in 1% trifluoroacetic acid and dried on a Savant Vacuum concentrator. The dried samples were reconstituted with 300 µl of assay buffer for BNP measurement (Phoenix Pharmaceuticals, Belmont, CA). One hundred microliters of known standards and 100 µl of samples were incubated with 100 µl of diluted porcine antibody for 18–24 h at 4°C. After incubation, 10,000 counts of iodinated BNP were added and again incubated for 18–24 h at 4°C and then added secondary antibody to separate free and bound fractions. The samples were centrifuged at 1,800 g for 15 min, free fractions were aspirated out, and the bound fractions were counted on a gamma counter. A standard curve was generated (range 0.5–128 pg/ml) and used to calculate the unknown samples. This assay is species specific and does not cross-react with atrial natriuretic peptide, C-type natriuretic peptide, or endothelin-1, -2, and -3. Coronary arteries and aortic endothelial cells were collected from these animals and used in separate studies (10, 11).

Analysis of reticulated platelets.   To determine the percentage of reticulated (RNA-containing) 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 then 20 µl of blood were collected with a pipette and diluted (1:100) into 2 ml of HBSS, without NaHCO3, buffered (pH 7.4) with 20 mM HEPES, supplemented with 1 mg/ml BSA, 1 µM/l tick anticoagulant peptide, 25 nM hirudin, and 1 µg/ml prostaglandin E1. The reticulated platelets were determined by flow cytometry (FACScalibur; Becton Dickinson) by using the previously described method (20).

Preparation of platelet-rich and platelet-poor plasma.   Anticoagulated ACD blood was centrifuged at room temperature for 15 min at 200 g to obtain platelet-rich plasma (PRP). Platelet-poor plasma was obtained from PRP by centrifugation at 1,500 g for 10 min. The PRP contained <0.1% of white or red blood cell contamination. Aggregation and dense body ATP secretion studies were carried out in PRP.

Platelet aggregation and dense body ATP secretion.   Aggregation studies were performed in PRP containing a fixed number of platelets (250,000 platelets/mm3) with a turbidimetric method using a whole blood aggregometer in optical mode (model no. 560-VS, Chrono-log, Havertown, PA) as described previously (20). Pig platelets do not aggregate in response to arachidonic acid, epinephrine, norepinephrine, ATP, ristocetin, or thrombin receptor agonist peptide (20, 41, and unpublished observations), so these agonists were not tested. ADP (10 µM) was used as an agonist for aggregation in these studies because porcine platelets aggregate reversibly with ADP and ADP promotes aggregation to other agonists. Collagen (6 µg/ml) induces irreversible aggregation in porcine platelets. ATP secretion by platelets in response to 50 nM porcine thrombin and collagen (6 µg/ml) was measured by bioluminescence as described previously (20).

Preparation of platelet lysate for Western blotting.   Platelets were isolated from whole blood by the method described previously with slight modification (15). In brief, anticoagulated blood was centrifuged at 200 g at room temperature for 15 min to obtain PRP. Platelets were pelleted from PRP by centrifugation at 1,500 g for 10 min. Platelets were then washed twice or thrice 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% BSA. Purity of washed platelets was validated by Coulter counter (Mayo Clinic Hematology Lab, Rochester, MN). The washed-platelet preparation was centrifuged at 1,500 g for 5 min in 22°C to pellet platelets. Platelet pellets were resuspended in 1% SDS, 1 mM sodium vanadate, 10 mM Tris, pH 7.4 (lysis buffer containing protease cocktail inhibitors from Sigma). This preparation was then stored at –70°C.

Western blotting.   The washed platelets stored at –70°C were thawed and passed through a 26-gauge needle five to seven times 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 total protein concentration of the supernatant was determined by BCA-200 protein assay reagents (Pierce, IL). Platelet lysates from juvenile and mature male and female pigs were prepared immediately before a given assay, and lysates from all pigs were performed in parallel to eliminate interassay variability.

For Western blotting, platelet lysate was mixed with an equal volume of 2x electrophoresis sample buffer (1x = 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. Equal amounts of each sample (100 µg protein) were loaded in each lane and separated by SDS-PAGE using 7.5% SDS-polyacrylamide gel (Bio-Rad) for glycoprotein IIb (GPIIb), GPIIIa, P-selectin, ER{alpha} and {beta}, progesterone receptor-BB, androgen receptor, hsp70, hsp90, and {beta}-actin and 12% SDS-polyacrylamide gel for hsp-27 and hsp-32. After electrophoretic separation, the proteins were transferred onto polyvinylidene difluoride membrane (Bio-Rad) for colorimetric detection and/or Hybond-C extra nitrocellulose membrane for ECL detection 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, 20% methanol) for 1 h and were incubated at 4°C with the following specific primary antibody with appropriate dilution in transfer buffer overnight.

The mouse anti-integrin {alpha}IIb monoclonal IgG1 (1:1,000 dilution), rabbit anti-integrin {beta}3 polyclonal IgG (1:1,000 dilution), rabbit anti-P-selectin polyclonal IgG (1:500 dilution), mouse anti-calf uterus ER{alpha} monoclonal IgG2ak (1:500 dilution), mouse anti-ER{beta} monoclonal IgM (1:500 dilution), mouse anti-progesterone receptor monoclonal IgG2b (1:1,000 dilution), rabbit anti-androgen receptor polyclonal IgG (1:250 dilution), mouse anti-hsp27 monoclonal IgG1 (1:1,000 dilution), mouse anti-hsp32 (hemeoxygenase) monoclonal IgG1 (1:1,000 dilution), mouse anti-hsp70 monoclonal IgG1 (1:250 dilution), mouse anti-hsp90 monoclonal IgG2 (1:250 dilution), and {beta}-actin mouse monoclonal IgG1 (1:1,000) primary antibody-incubated membranes were washed twice (5 min in each) in 1x Tris-buffered saline (Bio-Rad). The washed primary monoclonal antibody- (isotype IgG) treated membranes with secondary goat anti-mouse IgG-horseradish peroxidase- (HRP) conjugated (50 µl in 10 ml 1x Tris-buffered saline) antibody and primary polyclonal antibody-treated membranes with secondary goat anti-rabbit IgG-HRP-conjugated (50 µl in 10 ml 1x Tris-buffered saline) antibody for 2 h at room temperature. The washed primary monoclonal IgM (isotype) antibodies treated membranes were incubated with secondary goat anti-mouse IgM-HRP-conjugated (50 µl in 10 ml 1x Tris-buffered saline) antibody for 2 h at room temperature. The secondary antibody-incubated membranes were washed twice (5 min in each) in 1x Tris-buffered saline (Bio-Rad) and treated Opti-4CN (Bio-Rad) substrate (freshly prepared according to the manufacturer's instructions) for 2–5 min for colorimetric determination of specific protein expression. The colorimetric substrate-treated membranes showed specific protein expression and were washed twice or thrice (5–10 min in each) with reverse-osmosis water to clear off the backgrounds. The specific protein intensity was determined with the use of UN-SCAN-IT positive segment analysis.

The same primary monoclonal and polyclonal antibodies (some antibody dilutions vary from colorimetric) were also used for ECL detection of specific proteins or receptors in platelet lysates. Peroxidase-labeled anti-mouse and anti-rabbit secondary antibodies (1:5,000 or 10,000 dilutions) were used. Similar expression of proteins and receptors were obtained in both colorimetric and ECL methods.

Basal expression of membrane surface receptors.   Blood collected in citrate anticoagulation tubes was allowed to stand for 15–20 min to separate PRP from red blood cells. Twenty microliters of PRP were diluted into 2 ml (diluted 1:100) of HBSS, without NaHCO3, buffered (pH 7.4) with 20 mM HEPES, supplemented with 1 mg/ml BSA, 1 µM tick anticoagulant peptide and 25 nM hirudin (H/H+ medium). One hundred microliters of diluted PRP were aliquoted into flow-cytometric tubes and incubated with rabbit polyclonal anti-human P-selectin antibody (mouse monoclonal anti-human P-selectin antibody did not work in porcine platelets) for 30 min. Platelets were then fixed with 1% formaldehyde for 30 min at room temperature and centrifuged at 2,300 g for 10 min. Unbound antibody was discarded in the supernatant. Fixed platelets were reconstituted with 100 ml of H/H+ medium and incubated with secondary goat anti-rabbit FITC antibody in the dark for 30 min. Two milliliters of dilution buffer (1x PBS) were added to each tube and centrifuged at 2,300 g for 15 min. Supernatants were discarded, and pellets were resuspended with 1 ml of 1x PBS. Platelets in resuspended samples were analyzed by flow cytometry (FACcalibur; Becton Dickinson) within 2 h. All incubations were carried out at room temperature. Log forward scatter (for size characteristic) and log side scatter (for granularity) were used to identify platelets. The platelet cloud was gated electronically to exclude red and white blood cells.

For fibrinogen binding, platelets were prepared the same as for P-selectin expression. Chicken anti-human fibrinogen FITC polyclonal antibody (2 µl in 100 µl diluted PRP sample) incubated in the dark for 30 min in room temperature. Platelets were then fixed with 1% formaldehyde for 30 min at room temperature. Two milliliters of dilution buffer (1x PBS) were added and centrifuged at 2,300 g for 15 min. Supernatants were discarded, and pellets were resuspended with 1 ml of 1x PBS. Platelets in resuspended samples were analyzed by flow cytometry (FACcalibur; Becton Dickinson) within 2 h. Log forward scatter (for size characteristic) and log side scatter (for granularity) was used to identify platelets. The platelet cloud was gated electronically to exclude red and white blood cells.

Statistical analysis.   Results are expressed as means ± SE. Densities of protein bands were measured by UN-SCAN-IT automated digitizing software through positive segment analysis. Average pixel values were noted from each protein (receptors and other platelet proteins) band of platelet lysate from young and mature male and female pigs. Mean values of average pixel numbers of each blot and ratio of each protein blot (proteins/{beta}-actin pixels) were analyzed by Student's t-test and one-way ANOVA followed by Newman-Keuls multiple comparison tests to identify differences among groups. Statistical significance was accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Sexual maturity was evidenced by the significant increase in gonad-to-body weight ratio in both males and females (Table 1). There were no statistically significant differences in the levels of either 17{beta}-estradiol or progesterone between juvenile and adult female animals. This may be due in part to the fact that in some animals the measurements were below the detection limit of the assays. Testosterone, however, was significantly greater in adult male compared with juvenile pigs. Total cholesterol was significantly lower in adult compared with juvenile female animals, and there were no significant changes among groups in triglycerides, low-density lipoprotein cholesterol, or high-density lipoprotein cholesterol. BNP did not change significantly with maturation in either males or females. CRP showed high variability in juvenile and adult females and juvenile males. The standard error of measurement for the protein decreased significantly with maturation in the males, and all values were within the distribution of normal range given for humans (Table 1).

Total numbers of circulating platelets and numbers of reticulated (young) platelets in blood did not vary with maturity in either males or females. Mean platelet volume also did not change with maturity (Table 1).

Maximal platelet aggregation in PRP to ADP (10 µM) decreased significantly with maturity in females but increased significantly with maturity in males (Fig. 1). Maximal platelet aggregation was significantly less in platelets from juvenile males compared with juvenile females and significantly greater in adult males compared with adult females (Fig. 1). The same pattern of changes in maximal aggregation was obtained with collagen (6 µg/ml; data not shown) as the stimulus for aggregation.



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  		Fig. 1. A: sample tracings of aggregation of platelets from juvenile and mature male and female pigs in response to 10 µM ADP. B: cumulative results of aggregation in response to 10 µM ADP in platelets from juvenile and mature male and female pigs. Cumulative data (n = number of animals) are shown as means ± SE. Platelet aggregation decreased significantly in females but increased significantly in males with sexual maturity. *Statistical difference between juvenile and adult animals, P < 0.05. {dagger}Difference between platelets from age-matched male and female animals, P < 0.05.

 
The rate of secretion of ATP in response to 50 nM thrombin followed the same pattern as aggregation with secretion decreasing significantly with sexual maturity in females but increasing significantly with sexual maturity in males (Fig. 2A). Dense body ATP secretion was significantly less in juvenile males compared with juvenile females (Fig. 2A). Total dense body ATP content was similar in platelets from mature and juvenile female pigs but increased significantly with maturity in platelets from males pigs (Fig. 2B).



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  		Fig. 2. Dense body ATP secretion in response to 50 nM pig thrombin from platelets of juvenile and mature female and male pigs (A) and total ATP content from the same platelets (B). Data (n = number of animals) are shown as means ± SE. Platelet dense body ATP secretion decreased significantly in females with sexual maturity, but it increased significantly in males with sexual maturity. *Statistical difference between juvenile and adult animals (i.e., juvenile male vs. female; adult male vs. female), P < 0.05. {dagger}Difference between platelets from age-matched male and female animals, P < 0.05.

 
Concentration of platelet surface proteins [platelet fibrinogen receptor (GPIIb/IIIa) and cellular adhesion molecule P-selectin, Fig. 3], steroid hormone receptors (estrogen receptors ER{alpha} and ER{beta}), progesterone receptor-BB and androgen receptor (Fig. 4), or steroid hormone-associated proteins (hsp27, hsp32, hsp70, and hsp90, Fig. 5) in the platelet lysate did not change with maturity in platelets from male or female pigs (Table 2). Nor were there significant differences in content of these proteins in lysate between age-matched male and female animals. However, expression of P-selectin on the platelet surface as measured by flow cytometry increased with maturity only in female pigs (Table 3).



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  		Fig. 3. Representative Western blots of glycoproteins (GPIIb/IIIa) and P-selectin expression in platelet lysate from juvenile and mature male and female pigs. A: glycoprotein IIb (GPIIb) or integrin {alpha}2. B: glycoprotein IIIa (GPIIIa) or integrin {beta}3. C: P-selectin. D: {beta}-actin as a control for protein. Similar experiments were carried out in platelet lysate from 5 different individual animals of each group. Expression of fibrinogen receptors (GPIIb/IIIa) and P-selectin did not differ between groups of pigs.

 


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  		Fig. 4. Representative Western blots of sex steroid receptors in total platelet lysate. A: estrogen receptor-{alpha} (ER{alpha}). B: estrogen receptor-{beta} (ER{beta}). C: progesterone receptor-BB (PRBB). D: androgen receptor (AR). E: {beta}-actin as a control for protein loading. Similar experiments were carried out in platelet lysate from 5 different individual animals of each group. Expression of sex steroid receptors did not differ among groups of pigs.

 


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  		Fig. 5. Representative Western blots of heat shock proteins in total platelet lysate. A: heat shock protein 27 (hsp27). B: heat shock protein 32 (hsp32). C: heat shock protein 70 (hsp70). D: heat shock protein 90 (hsp90). E: {beta}-actin as a control for protein loading. Similar experiments were carried out in platelet lysate from 5 different individual animals of each group. Expression of heat shock proteins did not differ among groups of pigs.

 

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  		Table 2. Cumulative results for expression of proteins, sex steroid receptors, and heat shock proteins in platelets from juvenile and mature male and female pigs

 

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  		Table 3. Basal P-selectin expression and fibrinogen binding in platelets from juvenile and mature female and male pigs

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Results of this study support the hypothesis that platelet aggregation and dense body ATP secretion change differentially during sexual development in males and females. Although circulating numbers of blood platelets and percentage of reticulated platelets (newly formed platelets containing a residual amount of RNA) were not altered by sexual maturity in pigs, platelet aggregation and dense body ATP secretion were lower in adult females compared with juveniles, whereas platelet aggregation and secretion were higher in adult males compared with juveniles. Animals in the present study demonstrated sexual maturity by changes in uterine or testes weight and sex-specific hormones. Hormone secretion is more sensitive to environmental conditions in females compared with males, which may account for the variability in hormone measurements and patterns of estrus in adult animals (13). Increases in uterine weight and decreases in total cholesterol are consistent with changes expected with sexual maturity in adult females.

BNP and CRP are used clinically to monitor progression of cardiovascular disease, and there is some controversy regarding sex or gender specificity of reference values in "normal" populations (12, 17, 24, 29). In the present study, plasma levels of BNP and CRP did not change significantly with sexual maturity, but variability in distribution of values decreased with maturity in males. In adult animals, mean values for both CRP and BNP tended to be greater in females than males, which is similar to reference values for these proteins in humans. Additional studies are needed to determine specific sex-related hormonal regulation of these factors.

Most studies that have examined effects of sex steroids on platelet function have been conducted using platelets from adult animals with manipulation of sex steroids through gonadectomy and subsequent hormone replacement. In the present study, platelet aggregation increased in males and decreased in females with maturation of testes and ovaries, respectively. These observations are consistent with studies of platelet from gonadectomized adult animals that were treated with hormones (21). For example, platelet aggregation initiated by ADP was greater in platelets from male compared with female rats, and castration reduced aggregation in males but increased aggregation in females. Aggregation of platelets from humans, rats, guinea pigs, and mice was enhanced by treatment with androgens (21, 27, 31). However, in the present study in which changes in platelet functions were observed with natural puberty, unlike specific hormone treatments to adults, it is not possible to rule out effects of hormones of the pituitary gonadal axis (luteinizing hormone, follicle-stimulating hormone), which were not measured, in addition to hormones produced by the gonads.

Although platelet aggregation initiated by specific agonists changed with maturity in both male and female pigs, neither lysate content or surface expression of fibrinogen receptor glycoproteins GPIIb/IIIa changed with maturity in either male or female pigs. However, surface binding of P-selectin increased with maturity in platelets derived from female pigs even though content of P-selectin protein in the platelet lysate did not change. A similar result was observed after ovariectomy in adult female pigs (19, 20). These observations point out that regulation of proteins within the lysate and membrane surfaces differs and that hormones may regulate compartmentalization of the various proteins and receptors as well as their transcriptional and translational regulation and synthesis.

Androgen and estrogen receptors act as transcription factors and mediate genomic (receptor-mediated changes in gene expression) and/or rapid (seconds to minutes) nongenomic (not dependent on changes in gene expression) effects in targeted cells. Genomic effects of sex steroids in platelets would occur only in megakaryocytes because these precursors of platelets contain nuclei, whereas circulating platelets do not (19, 20, 37). Expression of sex steroid receptors in total platelet lysate is not changed with puberty. This result is unexpected because estrogen receptors increase after ovariectomy in adult females (19). These latter observations suggest that ovarian hormones modulate estrogen receptor concentration in females. It may be that in adults methlyation of DNA affects transcription of estrogen receptors more than in younger animals (28). However, much remains to be learned regarding regulation of steroid receptor expression in megakaryocytes of both males and females. Whether platelet sex steroid receptors are membrane bound or only cytosolic remains to be determined.

In the absence of a ligand, or in an inactive form, androgen and estrogen receptors are associated with a number of heat shock proteins (e.g., hsp27, hsp32, hsp70, and hsp90). It has been proposed that receptor-associated proteins keep receptors in a conformation that increases the affinity of the receptor for hormone and decreases the affinity of the receptor for DNA binding as well as transport of ligand-receptor complex from the cytosol of the nucleus (14, 23, 40). Expression of hsp27, hsp32, hsp70, and hsp90 does not change with maturity in male and female pigs, suggesting that hormones or factors other than sex steroids may regulate expression of these proteins.

A limitation of this study is that mature female pigs were studied without regard to stage of estrus. Larger differences between juvenile and adult females may have been observed if the adult pigs had been established on a fixed light cycle in the absence of male animals for a longer period of time, i.e., 12 mo, and/or examined at a fixed stage of their estrus cycle. However, previous studies suggest that fluctuations in estrogen and progesterone associated with estrus do not affect endothelium-dependent relaxations of coronary arteries or endothelial content of endothelial nitric oxide synthase in adult female pigs of the same age used in the present study (7, 39) but do affect affinity of endothelin-3 receptors (8). Additional studies are needed to differentiate effects of estrogen, progesterone, and hormones of the pituitary axis on platelet and other cardiovascular functions in female animals.

In conclusion, results from this study suggest that increases in gonadal hormones or other hormones of the pituitary-gonadal axis during sexual development alter platelet functions. Platelet aggregation and secretion decreased with maturity in females whereas platelet aggregation and secretion increased with maturity in males. In humans, thrombotic diseases are less prevalent in children than adults (3). There are no studies of thrombotic disease in pigs as they age. However, it may be hypothesized that increased aggregatibility of platelets in adult males would accentuate platelet-vascular wall interactions under conditions of endothelial dysfunction, potentially contributing to earlier development of cardiovascular disease in age-matched males compared with females. Results of the present study provide the basis for development of a clinical study of platelet functions in humans with transitions to sexual maturity.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-51736 and the Mayo Foundation, Rochester, MN.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors thank Denise Heublein for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. M. Miller, Dept. of Surgery, Physiology and Biophysics, Mayo Clinic Rochester, 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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
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