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J Appl Physiol 91: 2823-2830, 2001;
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Vol. 91, Issue 6, 2823-2830, December 2001

HIGHLIGHTED TOPICS
Genome and Hormones: Gender Differences in Physiology
Selected Contribution: Effects of sex and ovariectomy on responses to platelets in porcine femoral veins

D. A. Lewis, M. P. Bracamonte, K. S. Rud, and V. M. Miller

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Estrogen replacement increases risk of venous thrombosis. In this study, we determined responses in vitro to platelets and platelet products in veins from adult male and intact and ovariectomized female pigs. When contracted with prostaglandin F2alpha , platelets (25,000 platelets/µl) caused relaxation in veins with endothelium. Higher numbers of platelets caused contraction in veins with and without endothelium. In veins without endothelium, contractions were greater in veins from male than in veins from female pigs, and contractions in intact female pig veins were greater than in ovariectomized females pig veins. Platelet products 5-hydroxytryptamine and thromboxane (analog U-46619) caused comparable contractions in all veins; contractions to prostacyclin were less in veins from intact female pigs. ADP caused comparable endothelium-dependent relaxations in all groups. These relaxations were increased by indomethacin in veins from intact males and females; with inhibition of nitric oxide, relaxations were comparable in all groups. These results suggest that venous responses to platelets vary with sex and presence of ovaries in female pigs. These variations reflect differences in type and quantity of substances released from platelets as well as the sensitivity of the smooth muscle to some vasoactive substances. In addition, products of cyclooxygenase may reduce endothelium-dependent relaxations in veins.

estrogen; venous thrombosis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

OBSERVATIONAL AND EPIDEMIOLOGICAL studies indicate that the incidence of arterial cardiovascular disease is less in premenopausal women compared with age-matched men. The incidence of cardiovascular disease increases with menopause, and estrogen-replacement therapy provides primary protection against the development of cardiovascular disease (8, 30, 49, 51). However, although estrogen replacement provides primary protection against development of arterial vascular disease in women, it increases risk for venous thrombosis (16, 21, 24, 29). Estrogen therapy in men also increases the risk of venous thrombosis (13, 50).

Responses of arteries to various vasoactive substances vary by the sex of the animal from which the tissue is derived. Some of these differences are partly due to actions of estrogen on the arterial wall through several mechanisms, including regulation of ion fluxes and receptors on the smooth muscle and production of endothelium-derived factors (6, 7, 12, 15, 18-20, 22, 41-43, 46, 47, 56). No studies have examined sex differences in responses of veins to substances associated with platelet aggregation or thrombosis. This information is important because synthetic estrogen-like compounds (selective estrogen receptor modulators or SERMS) are being developed for tissue-specific therapies for use in both men and women. Two such compounds now in clinical use, tamoxifen and raloxifene, also increase risk for venous thrombosis in women (1, 17). If SERMS are to be used for cardioprotection, it is important to identify potential gender differences not only in arteries but also in veins so that the risk of venous thrombosis can be reduced in both men and women.

Previous work from our laboratory (37) identified differences in the content of platelets from male and female pigs: platelets from adult male pigs contained more thromboxane, whereas platelets from ovariectomized female pigs contained more prostacyclin and 5-hydroxytryptamine (5-HT). However, acute vasomotor responses of the coronary arteries to platelets were found not to vary by sex of the animals (37). The present study was designed to compare responses to platelets and platelet-derived products in veins from gonadally intact male and female animals and to identify whether depletion of ovarian hormone alters the responses in female animals.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Femoral veins from 6-mo-old male (139.5 ± 10.2 kg), female (94.4 ± 2.2 kg), and ovariectomized female (87.4 ± 1.4 kg) Yorkshire pigs were used in these experiments. Ovariectomy was performed when female animals were 5 mo old, and veins were removed 4 wk postoperatively. All animals were fed Lean Grow 93 (Land O'Lakes Farmland Feed, Fort Dodge, IA). Gonadally intact females showed changes in their external genitalia associated with changes in their estrus cycle. Estradiol levels typically range from 5 to 35 pg/ml in intact female pigs, are below the level of assay sensitivity in ovariectomized female pigs, and range from 25 to 55 pg/ml in male pigs (4, 5, 54). Testosterone averages 324 ± 99 pg/ml in male pigs (4, 5, 54). Animals were anesthetized with a combination of ketamine hydrochloride (30 mg/kg), xylazine (6 mg/kg), and butorphanol (0.3 mg/kg) intramuscularly. Blood (500 ml) was obtained from the carotid artery for preparation of platelets (see Platelet Preparation below). Femoral veins were excised bilaterally and placed in cold modified Krebs-Ringer bicarbonate solution (control solution), which contained (in mmol/l) 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, 0.026 edentate calcium disodium, and 11.1 glucose. Coronary arteries from these animals were studied in separate experiments (5, 37).

Organ Chamber Experiments

After we removed the adventitia, femoral veins were cut into rings; rings that contained valves were excluded from the study. In some rings, the endothelium was removed by gently rubbing the luminal surface with watchmaker's forceps. Because veins vary in length, branching, and the presence of valves, it was not possible to obtain the same number of rings from all animals. However, pairs of rings with and without endothelium or in the absence and presence of either indomethacin (10-5 mol/l) or NG-monomethyl-L-arginine (L-NMMA, 10-4 mol/l) plus indomethacin were studied in parallel from a single animal.

Rings were suspended between a fixed point and a force transducer (Statham UC-2) in organ chambers (25 ml) containing control solution at 37°C and were bubbled with 95% O2-5% CO2.

Passive tension on the rings was progressively increased. Active tension to norepinephrine (3 × 10-7 mol/l) was measured at each passive tension until each ring reached its optimal point on the length-tension curve. Maximal tension to KCl (60 mmol/l) 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 (protocol A, B, or C).

Experimental protocol A. Rings were contracted with prostaglandin F2alpha (2 × 10-6 mol/l), and, once the contraction plateaued, concentration-response curves to ADP (1 × 10-8 to 1 × 10-4 mol/l) were obtained. After a washout and equilibration period, responses to autologous platelets (25,000, 50,000, and 75,000 platelets/µl) were obtained during a second contraction to prostaglandin F2alpha .

Experimental protocol B. Concentration-response curves to 5-HT (1 × 10-10 to 1 × 10-6 mol/l) were obtained from baseline tension. After a washout and equilibration period, rings were contracted with prostaglandin F2alpha (2 × 10-6 mol/l) and cumulative responses to thrombin (0.01, 0.1 & 1.0 U/ml) were obtained, followed by the calcium ionophore A-23187 (1 × 10-9 to 1 × 10-6 mol/l) in rings with endothelium or by nitric oxide (3 × 10-9 to 1 × 10-5 mol/l) in rings without endothelium.

Experimental protocol C. Cumulative concentration-response curves were obtained to either the thromboxane mimetic U-46619 (10-9 to 10-6 mol/l) or prostacyclin (10-9 to 10-5 mol/l) from baseline tension.

In all protocols, at the conclusion of each concentration-response curve measurement, rings were washed with control solution three times. A minimum 20-min equilibration was allowed in all experiments before the next antagonist was tested. Once an inhibitor was added to a chamber, it remained in contact with the tissue for each subsequent stimulation.

Platelet Preparation

Platelets were isolated as previously described (14, 25). Briefly, blood (500 ml) was drawn from the carotid artery into 50-ml centrifuge tubes containing an initial acid citrate dextrose solution [ACD 1, which contained (in mmol/l) 93 sodium citrate, 70 citric acid, and 140 dextrose] so that the concentration was 10 ml of blood to 1 ml of ACD 1. The blood-ACD 1 solution was centrifuged at 55 g for 40 min. The plasma was removed, and an equal volume of a second acid citrate dextrose solution [ACD 2, which contained (in mmol/l) 185.7 sodium citrate, 14 citric acid, 209.8 dextrose, and 9.9 potassium chloride] was added to each tube of plasma. The plasma-ACD 2 solution was centrifuged at 570 g for 40 min at room temperature. The supernatant was discarded, and the platelet pellet was resuspended in ACD 2 (1.25 ml ACD 2 for each 50 ml of original blood). The platelet number in this suspension was obtained by a Coulter counter. Once the platelet count in the suspension was known, volumes could be adjusted so that the final platelet concentration was standard for each experiment (25,000, 50,000, or 75,000 platelets/µl).

Calculations and Statistical Analysis

Data are expressed as means ± SE; n equals the number of pigs from which rings were taken. Data for contractions are expressed as the increase in grams of tension or as a percentage of the contraction to KCl. For relaxations, data are expressed as percent change in tension to prostaglandin F2alpha from a submaximal contraction. In some veins, spontaneous activity was observed. Peak tension was used as the value for which further changes in tension were measured. One-way ANOVA was performed on the calculated areas under individual concentration-response curves, maximal tensions, or, when appropriate, the concentration at 50% maximal relaxation or contraction (EC50), expressed as a negative log molar concentration. If a significant F value was found, a Bonferroni test was used to determine differences among means. Alpha level was set at 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The number of platelets in the prepared suspension did not differ significantly among groups: 93 ± 6 × 103 platelets/µl in male pigs (n = 14), 106 ± 10 × 103 platelets/µl in female pigs (n = 16), and 100 ± 7 × 103 platelets/µl in ovariectomized female pigs (n = 10). All rings contracted to KCl (Table 1). Contractions to KCl were similar between rings with and without endothelium within each group. However, contractions of veins with endothelium from intact female pigs were significantly less than those from veins with endothelium from male pigs (Table 1).

                              
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  		Table 1.   Contractions to KCl (60 mM) in porcine femoral veins

Responses to Platelets

Platelets at a concentration of 25,000 platelets/µl caused endothelium-dependent relaxations in veins from all groups of pigs, whereas higher concentrations of platelets caused contractions in rings with and without endothelium. In rings without endothelium, contractions were as follows: male > female > ovariectomized female pigs (Figs. 1 and 2).


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  		Fig. 1.   Sample tracings of responses to platelets in femoral veins with (left) and without (right) endothelium from male (A), intact female (B), and ovariectomized (OVX) female (C) pigs. Veins were contracted with prostaglandin F2alpha (PGF2alpha , 2 × 10-6 mol/l).



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  		Fig. 2.   Responses of porcine femoral veins to autologous platelets. Veins were contracted with prostaglandin F2alpha (2 × 10-6 mol/l). Data are represented as a percent change in tension from this contraction and are shown as means ± SE; n = number of animals from which rings were taken. Contractions to prostaglandin F2alpha did not differ significantly among groups: 4.3 ± 0.8 g (n = 14) in male pigs, 2.7 ± 0.5 g (n = 16) in female pigs, and 4.4 ± 0.6 g (n = 10) in OVX female pigs. Contractions also did not differ between rings with (A) and without (B) endothelium. *Differences in contractions to 75,000 platelets/µl by one-way ANOVA (P < 0.05). For some responses, the standard errors were small, and the bars are obscured by the symbols.

Contractions to Platelet-Derived Factors

5-HT caused comparable concentration-dependent contractions of rings with and without endothelium from all groups of pigs. Absolute tension developed to 10-6 M 5-HT was less in veins with endothelium from females compared with male and ovariectomized pigs: 6.3 ± 0.9 g (n = 11) in males, 2.4 ± 0.4 g (n = 15) in intact females, and 6.5 ± 1.7 g (n = 9) in ovariectomized females. Tensions were not altered significantly by removal of the endothelium. When expressed as a percentage of the maximal tension to KCl, the sensitivity of veins without endothelium from ovariectomized pigs was less than that of veins from either intact male or female pigs (Fig. 3).


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  		Fig. 3.   Concentration-response curves to 5-hydroxytryptamine (5-HT) in veins with (A) and without (B) endothelium from male, female, and OVX female pigs. Data are expressed as a percent of contraction to KCl (60 mM, see Fig. 1) and are shown as means ± SE; n = number of animals from which rings were taken. max, Maximal. *Significant difference in EC50 of contractions of veins from OVX females compared with intact males and females (ANOVA, P < 0.05).

Both U-46619 and prostacyclin caused concentration-dependent contractions in veins with and without endothelium from all groups of pigs (Fig. 4). There was large variability in responses, especially in rings without endothelium. However, maximal contractions to prostacyclin in female pig veins without endothelium were significantly less than the contractions in ovariectomized female pig veins. Prostacyclin did not cause a significant change in tension if the veins were first contracted with prostaglandin F2alpha (data not shown, n = 5/group).


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  		Fig. 4.   Concentration-response curves to U-46619 (top left and top right) and prostacyclin (PGI2; bottom left and bottom right) in veins with (A) and without (B) endothelium from male, female, and OVX female pigs. Data are expressed as grams increase in tension and are shown as means ± SE; n = number of animals from which rings were taken. * Significant difference in maximal contraction of veins from intact and OVX female pigs (ANOVA, P < 0.05).

Relaxations to Platelet-Derived Factors

In veins with endothelium contracted with prostaglandin F2alpha , ADP caused comparable concentration-dependent relaxations in veins from all groups of pigs (Fig. 5). However, incubation of the rings with indomethacin caused a significant increase in sensitivity and maximal relaxation in veins with endothelium from male pigs. Maximal relaxations were also increased significantly in veins from intact females. Inhibition of production of nitric oxide with L-NMMA significantly reduced maximal relaxation and decreased sensitivity in veins from male pigs but not from either intact or ovariectomized females (Fig. 6). Relaxations of rings without endothelium were not different among groups, with a maximal relaxation averaging a decrease of ~25% of the contraction to prostaglandin F2alpha . Relaxations of rings without endothelium were not altered significantly by indomethacin alone or by the combination of indomethacin plus L-NMMA (data not shown).


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  		Fig. 5.   Concentration-response curves to ADP in veins with endothelium from male (A), female (B), and OVX female (C) pigs in the absence (control) and presence of indomethacin (Indo; 10-5 mol/l) or indomethacin plus L-NMMA (Indo & L-NMMA; 10-4 mol/l). Data are expressed as percent decrease in tension from the contraction to prostaglandin F2alpha (2 × 10-6 mol/l) and are shown as means ± SE; n = number of animals from which rings were taken. Contractions to prostaglandin F2alpha did not differ among groups (4.5 ± 0.8 g in males, 2.8 ± 0.4 g in females, and 4.4 ± 0.5 g in OVX females). *Significant difference in area under the curve and maximal relaxations from control condition (ANOVA, P < 0.05).



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  		Fig. 6.   Concentration-response curves to thrombin in veins with endothelium from male (A), female (B), and OVX female (C) pigs in the absence (control) and presence of indomethacin (10-5 mol/l) or indomethacin plus L-NMMA (10-4 mol/l). Data are expressed as percent decrease in tension from the contraction to prostaglandin F2alpha (2 × 10-6 mol/l) and are shown as means ± SE; n = number of animals from which rings were taken. Contractions to prostaglandin F2alpha did not differ among groups (5.1 ± 1.0 g in males, 2.3 ± 1.4 g in females, and 4.5 ± 0.8 g in OVX females).

Thrombin also caused concentration-dependent relaxations in veins from all groups of pigs. However, unlike responses to ADP, relaxations to thrombin were not significantly inhibited by incubation of veins with either indomethacin alone or with a combination of indomethacin plus L-NMMA (Fig. 6).

Both A-23187 and nitric oxide caused comparable relaxations of rings with and without endothelium, respectively, in veins from all groups (Fig. 7). These relaxations were unaffected by incubation of the veins with either indomethacin or a combination of indomethacin plus L-NMMA (data not shown).


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  		Fig. 7.   Concentration-response curves to A-23187 with endothelium (A) and nitric oxide without endothelium (B) in femoral veins from male, female, and OVX female pigs. Data are expressed as percent decrease in tension from the contraction to prostaglandin F2alpha (2 × 10-6 mol/l) and are shown as means ± SE; n = number of animals from which rings were taken. In veins with endothelin (A), contractions to prostaglandin F2alpha averaged 4.5 ± 0.7 g in males, 2.4 ± 0.5 g in females, and 4.8 ± 0.7 g in OVX females. In rings without endothelium (B), contractions to prostaglandin F2alpha averaged 4.8 ± 1.0 g in males, 3.2 ± 0.6 g in females, and 4.5 ± 0.8 g in OVX females.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Results of this study suggest that the response to platelets in veins vary depending on the sex of the animal from which they are derived and, for female animals, on the presence of ovaries. These differences were most obvious in expression of contractions to the platelets in the absence of endothelium: contractions of veins from ovariectomized female pigs were less than those from intact male and female pigs. These observations in part confirm previous studies that indicated that factors released from aggregating platelets cause endothelium-dependent relaxations in the presence of endothelium and contractions in the absence of endothelium (14, 25, 26, 32, 35). Thus an aggregation of platelets may not be retained after dilation of a blood vessel with intact endothelium. However, with either mechanical or chemical damage to the endothelium, platelet aggregation, once initiated, would cause constriction of the venous wall. The constriction-decreased cross-sectional area results in increased flow velocities through the site, which would tend to wash off excess platelets and prevent an occlusive thrombus (14). Irritability of platelets, i.e., the tendency to form an aggregate, is also increased with ovariectomy (unpublished observations from our laboratory). Ovarian hormones modulate the content of vasoactive substances in platelets (shown here and in Ref. 37) as well as other factors of the coagulation cascade involved in thrombus formation (34). Therefore, under conditions of hormone depletion and/or replacement, additional factors may affect expansion and retention of the platelet aggregate, although a normal part of hemostasis may contribute to thrombosis in pathological situations.

Contractions to platelets in veins without endothelium from ovariectomized females were significantly less than contractions of veins from intact males. This result was unexpected. Contractions to KCl were similar between veins from males and ovariectomized females, thus demonstrating similar contractile characteristics of the smooth muscle. Therefore, reduced contractions to platelet products in veins from ovariectomized compared with male pigs most likely represent differences in the various factors released from the aggregating platelets. For example, release of 5-HT and prostacyclin is greater from platelets of ovariectomized pigs compared with either intact male or female pigs (37). Platelet-derived 5-HT causes platelet-associated contractions of other blood vessels (14, 25, 26, 32, 35); indeed, 5-HT directly caused contraction of the femoral veins from these animals, albeit with a decreased sensitivity compared with veins from intact male or female animals. In addition, prostacyclin caused contraction of the veins from ovariectomized females. Therefore, prostacyclin, if released from platelets, would contribute to contraction rather than relaxation. ADP is also released from platelets (25, 26, 31) and, although not quantified in this study, causes relaxation of veins with and without endothelium (25, 26, 31). In addition, platelets contain other vasoactive and mitogenic factors, including nitric oxide, cyclic guanosine monophosphate, and platelet-derived growth factor, some of which are modulated by ovarian hormones (M. Jayachandran and V. M. Miller, unpublished observations, and Ref. 45). Nitric oxide released from platelets of ovariectomized pigs would functionally antagonize contractions to 5-HT and prostacyclin. Other studies need to quantify nitric oxide from platelets of animals of differing gonadal and hormonal status. Unpublished observations from our laboratory indicate that megakaryocytes, the precursors of platelets, contain estrogen receptors. Therefore, gonadal hormones may alter content of platelets through genomic mechanisms in these cells. Despite the lack of such quantification of all factors in platelets in the present study, it is likely that the response to aggregating platelets in veins, as in arteries, represents the cumulative effect of all platelet-derived products released during aggregation.

The diet of the pigs contained soy as one protein source. Phytoestrogens can bind to estrogen receptors and act as antioxidants. It is not known at this time how these compounds regulate estrogen receptor expression or compete with binding to receptors when endogenous estrogen is present. At least in the ovariectomized pigs that lacked endogenous ovarian hormones including estrogen, the dietary source of phytoestrogens may have modulated platelet responses such that even greater differences may have been observed between responses to platelets in veins from intact and ovariectomized pigs if the diet did not contain soy.

Responses to platelets do not only reflect differences in the content of vasoactive factors in platelets but also differences in functions of the smooth muscle and endothelium. Contractions to KCl were decreased in veins from intact female pigs compared with other groups. Ovarian hormones modulate expression of potassium channels in uterine smooth muscle (9), but similar regulation needs to be demonstrated in venous smooth muscle. Inhibition of potassium channels decreases endothelium-dependent relaxations in veins from intact female animals to a greater extent than in veins from ovariectomized female animals (M. P. Bracamonte, M. Jayachandran, K. S. Rud, and V. M. Miller, unpublished observations, and Bracamonte, Rud, and Miller, unpublished observations). Therefore, ovarian status may influence mechanisms associated with regulation of potassium and membrane polarization in veins. Sex differences in activation of potassium channels were also observed in coronary arterial smooth muscle (3, 22). It is unclear why contractions to prostacyclin are less in veins from intact female pigs compared with the other groups. Differences in activation of cAMP cannot be excluded at this time. Because relaxations to nitric oxide were similar among groups, it is unlikely that activation of soluble guanylate cyclase is altered by sex or gonadal status.

Endothelium-dependent relaxations to ADP, thrombin, or A-23187 were similar among groups. However, indomethacin had different effects on these responses in veins from male compared with female pigs, especially as related to increased relaxation to ADP in males and the tendency for decreased relaxations to thrombin in females. As in coronary arteries, endothelium-dependent relaxations in veins from male animals increased with inhibition of cyclooxygenase (5), suggesting a greater contribution of contractile products of arachidonic acid metabolism in male than in female animals. Exogenous arachidonic acid causes endothelium-dependent contractions of femoral veins from gonadally intact male dogs (39). Thromboxane synthase may be modulated by testosterone, since amounts of thromboxane measured from activated platelets were greater in male than in female animals (37). Increased amounts of thromboxane in platelets and evidence of a cyclooxygenase contractile factor from the endothelium of males suggest that products of arachidonic acid metabolism may be modulated similarly in several cell types by sex steroids. If similar conditions could be demonstrated in humans, results could account in part for the effectiveness of aspirin to limit cardiovascular disease in men (23).

Observations that inhibition of neither cyclooxygenase nor L-NMMA blocks endothelium-dependent relaxations to thrombin or A-23187 in veins are not new (36, 38, 52) and suggest release of other endothelium-derived factors; these remain to be characterized. Possible candidates for other endothelium-derived factors include products of lipoxygenase and/or C-type natriuretic factor (2, 33, 55). These factors would not only affect venous tone but also would act to release factors from the endothelium that would in turn affect platelet aggregation (40, 44, 48). Therefore, changes in risk for venous thrombosis with initiation of estrogen-replacement therapies would represent the combined effects of the hormones on all components of Virchow's triad (53), that is, venous endothelium, smooth muscle, coagulability of the blood including platelets (irritability as well as content), and components of the coagulation cascade (M. P. Bracamonte, M. Jayachandran, K. S. Rud, and V. M. Miller, unpublished observation, and Refs. 27, 34).

In summary, sex and hormonal status influence responses of veins to platelets and platelet-derived products. These differences represent effects of ovarian hormones on the smooth muscle, as contractions to KCl, 5-HT, and prostacyclin differ between veins from intact and ovariectomized female animals. Differences in responses are also affected by sex and/or testosterone, as contractions to platelets and endothelium-derived contractile factors differ between veins from male and female ovariectomized animals.


    FOOTNOTES

Address for reprint requests and other correspondence: V. M. Miller, Depts. of Surgery, Physiology and Biophysics, Mayo Clinic and Foundation, Medical Sciences Bldg. 4-62A, 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.

Received 21 June 2001; accepted in final form 17 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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J APPL PHYSIOL 91(6):2823-2830
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