INTRODUCTION

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RELEASE OF GROWTH FACTORS from activated platelets at sites of vascular injury contributes to the repair process (9, 18, 21, 23, 40). Regulation of the content of vasoactive and mitogenic factors in platelets by sex steroids could account for differences in the presentation of coronary artery disease in women after menopause (5) and response to vascular injury (12, 26, 32). Loss of ovarian hormones by ovariectomy increases the expression of estrogen receptors (ER and ER) and ER-associated heat shock proteins (HSP70 and HSP90) as well as the enzyme nitric oxide synthase III, platelet-derived growth factor-BB, and prostacyclin (19, 28). However, the effects of loss of ovarian hormones on platelet aggregation, related dense granule secretion, and content of enzymes involved in vascular remodeling, such as matrix metalloproteinases (MMPs), have not been determined.

MMPs are a family of structurally related zinc- and calcium-dependent enzymes that contribute to the degradation of the extracellular matrix proteins (6) needed for tissue remodeling in wound healing and angiogenesis (8). Excessive production of MMPs leads to accelerated matrix degradation associated with diseases that include atherosclerosis (8, 10). MMP-1, MMP-2, MMP-3, and MMP-9 are expressed in macrophages and cells of atherosclerotic lesions (16, 31). MMPs are thought to participate in weakening the connective tissue matrix in intima, which leads to plaque rupture and acute thrombosis (16, 31). Synthesis and activation of MMPs are regulated by the endogenous proteinase inhibitor ₂-macroglobulin and the family of tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2, TIMP-3, and TIMP-4) (25, 38).

MMPs (MMP-2, MMP-9, and MMP-14) and their inhibitors (TIMP-1 and TIMP-2) also are present in human platelets and megakaryocytes (21, 29). Whereas MMP-2 stimulates platelet aggregation, MMP-9 prevents thrombin- and collagen-induced aggregation (15, 35). Sex steroids are known to regulate MMP expression and activity in reproductive tissues (11, 22). Therefore, changes of MMPs in platelets in response to sex hormones and release at sites of injury could contribute to vascular changes associated with differences in vascular disease in pre- and postmenopausal women. However, the influence of ovarian hormones on MMP expression and activity in platelets is unknown. Therefore, the present study was designed to determine the effects of ovariectomy to represent surgical menopause on platelet aggregation and ATP secretion and to determine the expression and activity of metalloproteinases and their inhibitors in whole platelet lysate.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Antibodies and chemicals. Antibodies were purchased as follows: rabbit anti-human MMP-2, rabbit anti-human MMP-9, rabbit anti-human MMP-14, rabbit anti-human TIMP-1, and rabbit anti-human TIMP-2 were from Chemicon International (Temecula, CA); rabbit anti-human P-selectin antibody was from PharMingen (San Diego, CA); and -actin monoclonal antibody, Tris[hydroxymethyl] aminomethane, glycine, sodium orthovanadate, lauryl sulfate sodium (SDS), and ADP were purchased from Sigma Chemicals (St. Louis, MO). All other reagents and solvents used in this study were of analytic/reagent grade. Equine tendon collagen was purchased from Helena Laboratory (Beaumont, TX). Pig thrombin was prepared as described previously (33, 34).

Animals. Animal studies were approved by the Institutional Animal Care and Use Committee of the Mayo Clinic (Rochester, MN). Sexually mature female Yorkshire pigs (7 mo of age) were used in this study. The external genitalia of pigs this age showed enlargement and discharge associated with estrus. Pigs were divided into two groups: those with ovaries (intact but sham operated; handling of animals was identical to that of operated animals without removal of the ovaries) and those with ovaries removed laparoscopically (ovariectomized). Both groups of animals were fed Lean Grow 93 diet (Land O'Lakes Farmland Feed; Fort Dodge, IA). Four weeks after ovariectomy, age-matched sham-operated intact and ovariectomized pigs were anesthetized by an intramuscular injection of ketamine (2 mg/kg), xylazine (2 mg/kg), and telazol (4 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 and EDTA anticoagulation tubes. The total blood platelet count was obtained in EDTA-anticoagulated blood by a Coulter counter (Mayo Clinic Hematology Lab; Rochester, MN).

Flow cytometry analysis. To determine the percentage of reticulated platelets (youngest platelets in circulation containing mRNA), a 19-gauge needle was inserted into an ear vein, five drops of blood were allowed to drip, and 20 µl of blood were collected with a pipette and diluted (1:100) into 2 ml of the following solution: Hank's balanced salts solution, without NaH₂CO₃, buffered (pH 7.4) with 20 mM HEPES and supplemented with 1 mg/ml bovine serum albumin, 1 µM tick anticoagulant peptide, 25 nM hirudin, and 1 µg/ml prostaglandin E₁. All concentrations are final concentrations in the diluent. Duplicate samples were incubated for 30 min in the dark with 5 µl of porcine glycoprotein Ib (GpIb)-biotin-conjugated mouse monoclonal antibody. Streptavidin-phycoerythrin (PE) (5 µl) was then added for conjugation, and incubation continued for 30 min in the dark. Afterward, control tubes (one from each sample) received 1 ml of 1× PBS. The other tubes (one from each sample) received 1 ml of thiazole orange at a concentration of 20 ng/ml. Tubes were again incubated in the dark for 30 min. All incubations were carried out at room temperature. One milliliter of dilution buffer was added to each tube, and samples were centrifuged at 22°C at 1,600 g for 15 min. The supernatants were discarded, and the pellets were resuspended with 2 ml of 1× PBS. Platelets in the 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) were used to identify platelets. The platelet cloud was gated electronically to exclude red and white blood cells.

Preparation of platelet-rich and -poor plasma. Anticoagulated ACD blood was centrifuged at 200 g at room temperature for 15 min to obtain platelet-rich plasma (PRP). Platelet-poor plasma (PPP) was obtained from PRP by centrifugation at 1,500 g for 10 min. The purity of the PRP was validated by a 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.

Platelet aggregation. Aggregation studies were performed in PRP by a turbidimetric method using the whole blood aggregometer optical mode (model 560-VS, Chrono-log; Havertown, PA). Minimum light transmission was set with 500 µl of PRP (0%) with stirring at 1,000 revolutions/min, and maximum light transmission was set with 500 µl of PPP (100%) without the stirring bar. Platelet aggregation (250,000 platelets/mm³ in PRP) was induced by adding 2.5 µl of 2 mM ADP (10 µM final concentration) for reversible aggregation or 30 µl (100 µg/ml) of collagen (3 µg final concentration) for irreversible aggregation. Aggregation data were acquired and processed through Aggro-Link software (Chrono-log).

Platelet dense body secretion. ATP secretion by platelets was measured by bioluminescence (37). PRP was diluted 1:1,000 in sterile Hanks' balanced salt solution (GIBCO-BRL) buffered (pH 7.4) with 10 mM NaOH-HEPES and containing 1 mg/ml bovine serum albumin and 0.5 g/l glucose. Premixed reagent (10 µl) containing luciferase (0.5 mg/ml Hanks' medium) and luciferin (5 mM in Hanks' medium) was mixed with 40 µl of diluted PRP suspension and equilibrated for 1-2 min. Secretion of ATP was initiated by an injection of 50 µl of pig thrombin (100 nM in Hanks' medium) or 50 µl of collagen (5 µg) to the above mixture. Data were acquired for 2-5 min at 1-s intervals. The rate of release is expressed as ×10⁴ nanomoles of ATP per platelet per minute, whereas total ATP secretion by platelets is expressed as ×10⁴ nanomoles of ATP per platelet.

Platelet isolation. Washed platelets were separated from the ACD anticoagulated whole blood as described previously (19). The purity of washed platelets was validated with a Coulter counter (Mayo Clinic Hematology Lab). For immunoblotting and zymography, platelets were resuspended in lysis buffer (1% SDS, 1 mM sodium orthovanadate, and 10 mM Tris · HCl; pH 7.4) and stored at 70°C for immunoblot and zymography analysis.

Immunoblotting for MMPs (MMP-2/MMP-9/MMP-14), TIMPs (TIMP-1/TIMP-2), and P-selectin. Total platelet lysates were prepared from platelets stored in lysis buffer by brief ultrasonication, by passing the solution through a 26-gauge needle, or both and then centrifugation at 12,000 g for 5 min at 4°C to remove insoluble materials. The supernatants were separated and concentrated using a Centricon (YM10) Centifugal Filter Device from Amicon Bioseparations. The total protein concentration of supernatants was determined by BCA-200 protein assay reagents (Pierce; Rockford, IL). The concentrated samples were mixed with an equal volume of 2× electrophoresis sample buffer [1× = 62.5 mM Tris · HCl (pH 6.8), 2% SDS, 5% glycerol, 0.003% bromophenol blue, and 1% -mercaptoethanol] and heated at 95°C for 5 min. Equal amounts of heated samples (100 µg protein) were loaded in each lane and separated by SDS-PAGE with the use of a 7.5% SDS-polyacrylamide gel (Ready Gels, Bio-Rad) for MMP-2, MMP-9, MMP-14, and P-selectin and a 12% SDS-polyacrylamide gel for TIMP-1 and TIMP-2 protein. After electrophoretic separation, the proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad) with the use of a 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 base, 190 mM glycine, and 20% methanol) for 1 h and were incubated at 4°C with the following specific primary antibodies of the appropriate dilution in transfer buffer overnight: rabbit anti-human MMP-2 (1:1,000 dilution), rabbit anti-human MMP-9 (1:1,000 dilution), rabbit anti-human MMP-14 (1:5,000), rabbit anti-human TIMP-1 (1:1,000 dilution), rabbit anti-human TIMP-2 (1:1,000 dilution), and rabbit anti-human P-selectin (1:500 dilution). Primary antibody-incubated membranes were washed twice in 1× Tris-buffered saline (Bio-Rad) and treated with secondary goat anti-rabbit IgG-horseradish peroxidase conjugates (50 µl in 10 ml of 1× Tris-buffered saline) for 2 h at room temperature. Protein expression on membranes was determined by colorimetric method using an Opti-4CN substrate kit (Bio-Rad). The Opti-4CN substrate was freshly prepared according to the manufacturer's instructions (Bio-Rad). Equal amount of protein transferred from gel to membrane was determined by -actin protein expression (data not shown). Mouse monoclonal antibodies were also used for MMP-2, MMP-9, TIMP-1, and TIMP-2 (R&D Systems; Minneapolis, MN, and Chemicon International) detection in porcine platelet lysate, and similar results were obtained with rabbit polyclonal and mouse monoclonal antibodies. The intensities of protein bands were analyzed by an UN-SCAN-IT gel automated digitizing system through positive segment analysis.

Gelatin zymography. For gelatin zymography, each sample of platelet lysate (preparation same as for immunoblotting) was mixed with an equal volume of 2× Tris-glycine-SDS sample buffer (Novex Invitrogen Life Technologies). Each sample mixture was loaded onto a 10% Ready-Gel zymogram gel (Bio-Rad or Invitrogen) with 0.1% gelatin as the substrate, and electrophoresis was carried out using 1× Tris-glycine-SDS running buffer (Invitrogen or Bio-Rad). After electrophoresis, gels were incubated for 1 h with two changes of 2.5% Triton X-100, for 30 min in 1× zymogram development buffer (Invitrogen or Bio-Rad) with gentle agitation, and finally for 48 h in fresh 1× development buffer at 37°C. Gels were then stained with 0.5% Coomassie brilliant blue R-250 (Sigma) for 1-2 h and destained in 1× destaining solution (Bio-Rad). Gelatinolytic activity of MMPs was revealed as clear bands against a blue-stained background. The cell culture medium U87 was used as a positive control. To measure the activity of MMPs, zymograms were analyzed with use of UN-SCAN-IT negative segment analysis.

Statistical analysis. Total blood platelet count, percentage of reticulated platelets, densitometric analysis of immunoblots, and zymography are presented as means ± SD. Statistical significance was evaluated by Student's unpaired t-test, and differences at a level of P <0.05 were considered to be significant. All analyses were carried out independently using a minimum of four preparations of platelets from different animals; n represents the number of animals.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Ovariectomy had no effect on the number of circulating platelets (intact: 291 ± 37 × 10³ platelets/µl, ovariectomized: 296 ± 41 × 10³ platelets/µl, n = 7 animals/group), but the percentage of reticulated platelets increased significantly (intact: 1.76 ± 0.915%, ovariectomized: 5.14 ± 1.62%, n = 7 animals/group). Platelet aggregation in PRP in response to ADP (10 µM) and collagen (3 µg) increased significantly after ovariectomy, as did the rate of secretion of ATP in response to 50 nM thrombin (Fig. 1). Maximal ATP release in response to collagen (5 µg) reached one-half of the maximal release initiated by thrombin (data not shown). Total ATP content was unaffected (intact: 1.99 ± 0.49 nM · 10⁴ ATP · platelet¹, ovariectomized: 1.92 ± 0.49 nM · 10⁴ ATP · platelet¹, n = 7 animals/group).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Effect of ovariectomy on platelet aggregation in adult female pigs. A-D: sample tracing of platelet aggregation in response to 10 µM ADP (A), cumulative results of aggregation in response to 10 µM ADP (B), response to 3 µg collagen (C), and rate of ATP secretion in response to thrombin (100 mM; D) in gonadally intact and ovariectomized (Ovx) female pigs. Cumulative data are shown as means ± SD. Platelet aggregation and rate of ATP secretion were increased significantly (P < 0.05) after 4-wk ovariectomy. * Statistically significant difference from intact females, P < 0.05.

Expression of proenzyme MMP-2 (72-kDa protein) did not change, but 64-kDa (active enzyme) protein increased significantly in platelets after ovariectomy (Fig. 2). Expression of active MMP-9 (82-kDa protein) and proenzyme MMP-9 (92-kDa protein) did not change significantly after ovariectomy (Fig. 3).

View larger version (31K): [in this window] [in a new window]   Fig. 2.   A: representative Western blot of latent [pro-matrix metalloproteinase (MMP)-2] and active forms of MMP-2 protein in platelet lysate from intact and ovariectomized female pigs. B: densitometric data representing immunoblots of latent and active MMP-2 in platelet lysate from seven (n = 7) different animals, shown as means ± SD. Expression of active MMP-2 was significantly greater in platelets from ovariectomized females. * Statistically significant difference from intact females, P < 0.05.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   A: representative Western blot of latent (pro-MMP-9) and active forms of MMP-9 protein in platelets from gonadally intact and ovariectomized female pigs. B: densitometric measurements representing immunoblots of latent and active MMP-9 in platelet lysate from seven (n = 7) different animals, shown as means ± SD. Expression of active MMP-9 was not statistically different between intact and ovariectomized pigs.

Gelatin zymography showed greater activity of MMP-2 but not MMP-9 in platelets from ovariectomized pigs (Fig. 4). Platelet membrane-bound MMP-14 decreased significantly after ovariectomy (Fig. 5). MMP-2 and MMP-9 activities are regulated by specific tissue inhibitors, called TIMPs (TIMP-1/TIMP-2). Expression of TIMP-1, TIMP-2, and P-selectin did not change with ovariectomy (Fig. 6).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   A: representative gelatin zymography of latent and active MMP-2 and MMP-9 activity in platelets from gonadally intact and ovariectomized female pigs. B: densitometric analysis representing zymography of MMP-2 and MMP-9 activity in platelet lysate from four (n = 4) different animals, shown as means ± SD. U-87 cell culture medium was used as a positive control to determine MMP-2 and MMP-9 activity in porcine platelets. Expression of active MMP-2 significantly increased after ovariectomy. * Statistically significant difference from intact females, P < 0.05.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   A: representative Western blot of latent (pro-MMP-14) and active forms of MMP-14 protein expression in platelets from gonadally intact and ovariectomized female pigs. B: densitometric data representing immunoblots of latent and active MMP-14 in platelet lysate from five (n = 5) different animals, shown as means ± SD. Expression of latent and active MMP-14 was significantly lower in platelets from ovariectomized females. * Statistically significant difference from intact females, P < 0.05.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Representative Western blot of tissue inhibitors of matrix metalloproteinase [TIMP-1 (A) and TIMP-2 (B)] and P-selectin (C) expression in platelets from gonadally intact and ovariectomized female pigs. Expression of TIMP-1 and TIMP-2 proteins and P-selectin was not changed after ovariectomy. Similar results were obtained using platelets from five (n = 5) additional animals.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Platelets are released from the bone marrow into the bloodstream by fragmentation of megakaryocytes. Newly formed platelets contain a small endoplasmic reticulum and mRNA, and thus retain the ability to synthesize small amounts of protein (24). However, lacking nuclei, platelets are unable to synthesize new mRNA, and therefore the so-called reticulated (young) platelet mRNA decays quickly (~24 h) (2, 14). Whereas the total platelet count did not change after ovariectomy, reticulated platelets increased, suggesting that platelet turnover was accelerated by the loss of ovarian hormones. These results differ from another study from our group where ovariectomy did not affect the percentage of reticulated platelets (9). Differences in husbandry of pigs from different suppliers may account for this disparity (20).

In addition to turnover, platelet aggregation in PRP and ATP release by platelets increased significantly with ovariectomy. The increase in aggregation was observed with both a reversible agonist (ADP) and an irreversible agonist (collagen). Reticulated platelets may be hyperreactive. Because the percentage of reticulated platelets increased with ovariectomy, this could account for increased aggregation. This increase probably reflects the loss of ovarian hormones rather than surgical intervention, as the controls were sham operated. That is, animals had surgery and manipulation of the ovaries without their removal. At this time, it is unclear whether increases in aggregation reflect changes in the expression of surface receptors or intraplatelet processes. However, the expressions of P-selectin (this study), calmodulin (19), and transforming growth factor- (9) do not change with ovariectomy. Pig platelets do not aggregate in response to arachidonic acid and epinephrine (42), so these agonists were not tested.

ATP release was also increased in response to thrombin with ovariectomy even though the total ATP content was not altered. Pig platelets do not secrete contents of dense granules in response to ADP and epinephrine (1). Collagen acts as a stimulating substance to induce ADP and thromboxane A₂ release (27). Pig platelet dense body ATP secretion in response to collagen (5 µg) was lower (one-half) than to thrombin (50 nM)-induced secretion (data not shown). This may be due to insufficient ADP induction by collagen to secrete ATP in pig platelets, or collagen-induced ADP may not work on ATP release in porcine platelets.

In human platelets, MMP-2 is proaggregatory, whereas MMP-9 is antiaggregatory (15, 35). MMP-2 increased in pig platelets after ovariectomy, which may have also contributed to the increased aggregation observed in this study. Because MMP-9 was unchanged, an imbalance in the antagonistic action of MMP-2 and MMP-9 may contribute to physiological and pathological mechanisms of platelet functions after loss of ovarian hormones and is consistent with an increase in aggregation after ovariectomy.

Expression and activities of MMPs are tightly regulated, and most MMPs are expressed only when their activity is required. These proteases are synthesized zymogens that require the proteolytic removal of a 10-kDa amino-terminal domain to become active. MMP-14 is present on cell membranes and acts as a receptor to activate pro-MMP-2 (36, 39, 44). Expression of MMP-14 was decreased after ovariectomy, suggesting that binding of pro-MMP-2 with MMP-14 for activation may be decreased. Further study is needed to clarify MMP-2/MMP-14 association and dissociation.

Activated MMPs are inhibited by the endogenous proteinase inhibitors ₂-macroglobulin and TIMP-1, -2, -3, and -4 (25, 38). TIMP-1 and TIMP-2 did not change in platelets after ovariectomy. These inhibitors have been shown to promote cell growth, inhibit angiogenesis, and induce apoptosis (10). Disruption in the balance between MMPs and TIMPs may result in diseases associated with an increased turnover of matrix, resulting in tissue ulceration (17, 30, 43). The cell adhesion molecule P-selectin did not change with ovariectomy. This observation provides support for specific rather than nonspecific regulation of MMPs and other proteins (19) with ovariectomy in female pigs.

In conclusion, 4 wk of ovariectomy increases platelet turnover, aggregation, ATP secretion, and content of MMP-2 and decreases MMP-14. Normal physiological processes of angiogenesis and tissue repair require extracellular proteolysis. At sites of vascular injury, platelets become activated and release several factors including platelet-derived growth factor and MMPs that modify tissue integrity (7, 9, 13). The results of the present study support the hypothesis that increases in platelet aggregation and ATP secretion, content, and activity of growth factors and MMPs after the loss of ovarian hormones may enhance platelet-vessel wall interactions contributing to increased response to injury, as would be associated with arterial occlusive diseases after menopause.