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(Circulation. 1995;91:2742-2747.)
© 1995 American Heart Association, Inc.

Articles

Testosterone Increases Human Platelet Thromboxane A₂ Receptor Density and Aggregation Responses

Presented in part at the Clinical Research Meeting, Baltimore, Md, April 29-May 2, 1994, and published in abstract form in Clin Res. 1994;42:252A.

Adesuyi A. L. Ajayi, MD, PhD; Rajesh Mathur, PhD; Perry V. Halushka, PhD, MD

From the Departments of Pharmacology and Medicine (A.A.L.A., P.V.H.), Division of Clinical Pharmacology and Department of Obstetrics and Gynecology (R.M.), Medical University of South Carolina, Charleston.

	Abstract

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Background The incidence of thrombotic cardiovascular disease is greater in men than in premenopausal women. Testosterone has been implicated as a significant risk factor for cardiovascular disease and for acute myocardial infarctions and strokes in young male athletes who abuse anabolic steroids. Thromboxane A₂ (TXA₂) is a vasoconstrictor and platelet proaggregatory agent that has been implicated in the pathogenesis of cardiovascular disease. We therefore tested the hypothesis that testosterone regulates the expression of human platelet TXA₂ receptors.

Methods and Results In a double-blind, placebo-controlled, randomized, parallel-group study, we determined the effects of testosterone cypionate 200 mg IM given twice, 2 weeks apart, or saline placebo in 16 healthy men. Platelet TXA₂ receptor density (B_max) and dissociation constant (K_d) were measured by use of the TXA₂ mimetic ¹²⁵I-BOP. Platelet aggregation responses to I-BOP and to thrombin and plasma testosterone concentrations were measured before treatment (pretreatment phase), at 2 and 4 weeks (active phase), and again at 8 weeks (recovery phase). Treatment with testosterone was associated with an increase in the B_max value from 0.95±0.13 to 2.10±0.4 pmol/mg protein (n=9), with a peak effect at 4 weeks (P=.001), returning to baseline by 8 weeks. There was no significant change in B_max values in the saline-treated group. The K_d values were unchanged. Testosterone treatment was associated with a significant increase in the maximum platelet aggregation response to I-BOP (P<.001) at 4 weeks and returned to baseline at 8 weeks. The EC₅₀ values were not significantly changed. Platelet TXA₂ receptor density was positively correlated (r=.56, P<.001, n=32 measurements) with pretreatment (endogenous) plasma testosterone levels (range, 215 to 883 ng/dL) but not K_d.

Conclusions Testosterone regulates the expression of platelet TXA₂ receptors in humans. This may contribute to the thrombogenicity of androgenic steroids.

Key Words: thromboxane • platelets • testosterone • cardiovascular diseases

	Introduction

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Thromboxane A₂ (TXA₂), a major arachidonic acid metabolite of platelets, acts through membrane surface receptors to aggregate platelets and constrict vascular smooth muscle.^{1 2 3} TXA₂ synthesis is increased in a variety of thrombotic cardiovascular conditions.^{4 5} Human platelet TXA₂ receptor density is increased during acute myocardial infarction and pregnancy-induced hypertension.^{6 7} Thus, TXA₂ and its receptor may play an important pathophysiological role in cardiovascular diseases. This notion is further supported by the observation that inhibition of platelet TXA₂ synthesis by aspirin is an established therapeutic modality that decreases mortality from thrombotic cardiovascular events.⁸

The prevalence of and mortality from thrombotic disorders, especially coronary artery disease, is higher in men than in premenopausal women.^{9 10} At least two possible mechanisms for this sex difference have been proposed. One view proffers that estrogens are cardioprotective and that this protection is lost after menopause owing to postmenopausal estrogen deficit.¹¹ The other holds that testosterone represents a significant risk factor for cardiovascular thrombotic disease in men.¹² These two notions are not mutually exclusive and may both be operative. Recently, Lesko and collaborators¹³ presented epidemiological evidence that linked male pattern baldness, a dihydrotestosterone-mediated condition,¹⁴ with an enhanced risk of ischemic heart disease, which is in agreement with an earlier preliminary observation.¹⁵ The abuse of androgenic steroids in young male athletes has been associated with premature myocardial infarctions and strokes.^{16 17 18 19 20} Additionally, in a nonathlete who received therapeutic testosterone for hypogonadism inadvertently by the intravenous route, an acute myocardial infarction was reported.²¹

A variety of basic animal studies have also provided evidence that testosterone can enhance the platelet aggregation response or aortic contractile response to arachidonic acid metabolites or increased mortality to the intravenous injection of arachidonic acid,^{22 23 24 25 26 27} the latter phenomenon being dependent in part on the synthesis of TXA₂.

In vitro and in vivo studies further indicate that androgenic steroids regulate the expression of TXA₂ receptors. Testosterone in vitro increased TXA₂ receptor density in cultured rat aortic smooth muscle cells²⁸ and in human erythroleukemia (HEL) cells, a megakaryocyte-like tumor cell line with platelet marker proteins.²⁹ These effects were attenuated by hydroxyflutamide, a specific androgen receptor antagonist.^{28 29} Treatment of male rats with testosterone resulted in a significant increase in both platelet and vascular TXA₂ receptor density.³⁰ The increase was associated with enhanced aortic contractile responses and platelet aggregation responses to a TXA₂ mimetic. Collectively, these studies suggest that testosterone may contribute to thrombotic cardiovascular disease via an effect on TXA₂ receptors.

The purpose of the present study was to determine whether testosterone can increase the density of platelet TXA₂ receptors and platelet aggregation responses to TXA₂ in humans.

	Methods

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Study Design and Subjects
Sixteen healthy men gave informed consent to participate in a double-blind, placebo-controlled, randomized, parallel-group study. The study protocol was reviewed and approved by the Medical University of South Carolina institutional review board. All the volunteers had a complete physical examination, with biochemical and hematological screening and a 12-lead ECG before the study commenced. The study inclusion criteria were male volunteers between 21 and 35 years old, not on any medications, who neither smoked cigarettes nor imbibed alcohol regularly, with no personal or family history of hypertension or diabetes, and with no contraindication to testosterone injections. The clinical characteristics of the subjects are shown in Table 1.

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics of Subjects

Study Protocol and Drug Administration
The volunteers were studied on six occasions over a 10-week period. They were investigated on two preliminary baseline days before the injections. Thereafter, they received intramuscular injections of testosterone cypionate (200 mg) or saline on day 1 and day 14 of the active treatment phase. These were administered by a research nurse who did not participate in any of the measurements. The two testosterone injections were given to allow an assessment and comparison of the effects after single and cumulative administration. At 2, 4, and 8 weeks after drug administration, the subjects reported at 9 AM for blood sampling. At 1 week, blood sampling was done only to determine plasma testosterone levels to confirm a significant increase. On each of the study days, blood was collected for the determination of plasma testosterone concentration, radioligand binding studies with ¹²⁵I-labeled [1S-1,2ß(5Z),3(1E,3R*),4)]-7-[-3-(3-hydroxy-4-(4''-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5-heptenoic acid (¹²⁵I-BOP), and the ex vivo platelet aggregation response to the TXA₂ mimetic I-BOP³¹ and thrombin. The testosterone cypionate or placebo injections were well tolerated. No untoward reactions or adverse behavioral effects were reported spontaneously or in response to specific inquiry.

Blood Sampling and Platelet Isolation
Blood (60 mL) was drawn by venipuncture via a 19-gauge needle into syringes containing EDTA (5 mmol/L) and indomethacin (10 µmol/L) (final concentrations). Washed platelets were prepared as previously described.^{31 32} Briefly, platelet-rich plasma was prepared by centrifugation at 175g for 20 minutes at room temperature, and after differential centrifugation at 1795g for another 20 minutes, the platelets were suspended in a modified Tyrode's solution buffer containing (in mmol/L): NaCl 137, KCl 3, MgCl₂ 0.6, Na₂HPO₄ 12.5, and dextrose 5.5, and indomethacin 10 µmol/L, adjusted to pH 7.4 for platelet aggregation studies and pH 6.5 for equilibrium radioligand binding assays with ¹²⁵I-BOP.³³

Equilibrium Radioligand Binding Assay
Radioligand binding studies were undertaken as described elsewhere.³² Incubations were in modified Tyrode's buffer at pH 6.5 with a total volume of 200 µL. The optimal assay conditions for platelet TXA₂ receptors occurs at pH 6.5.³² Each reaction mixture contained 100 µL washed platelets (10⁷ platelets per tube), 20 µL of the buffer or unlabeled I-BOP at various concentrations (10^-11 to 10^-6 mol/L), and 80 µL of radioactive ¹²⁵I-BOP (40 000 cpm). These incubations were performed in silanized glass tubes (12x75 cm) at 37°C for 30 minutes. The reaction was terminated by the addition of 4 mL ice-cold 50 mmol/L Tris/100 mmol/L NaCl buffer at pH 6.5, followed by rapid filtration under reduced pressure through Whatman GFC filters with a Brandel cell harvester, and the platelets were washed three times with 4 mL ice-cold buffer within 10 seconds. Nonspecific binding was defined as the amount of radioactivity in the presence of L657925 (10 µmol/L), a stereoselective TXA₂ receptor antagonist. Protein concentrations were determined by the method of Lowry et al.³⁴

Platelet Aggregation Studies
Platelet aggregation studies were carried out with a Chronolog model 300 aggregometer, as described previously.^{6 32} The washed platelet suspension in Tyrode's buffer (pH 7.4) was diluted to 2.5x10⁸ platelets/mL, and 450 µL of this was dispensed into silanized cuvettes to which CaCl₂ (250 µmol/L) was added, stirred, and preincubated at 37°C for 1 minute. This was followed by the addition of various concentrations of I-BOP (0.25 to 100 nmol/L final concentrations), and the aggregation response at 1 minute was recorded. Concentration-response curves were constructed for I-BOP and thrombin (0.00625 to 0.1 U/mL final concentration). The maximum aggregation response at 1 minute for each agonist and each experiment was determined. The aggregometer was standardized by arbitrarily setting 100% aggregation equal to light transmission with the cuvette containing only Tyrode's buffer. The EC₅₀ values for each agonist were calculated directly from a log-logit transformation of the data. The EC₅₀ value was defined as the concentration required to produce 50% of the maximum aggregation response induced by each of the aggregating agents.

Plasma Testosterone Assay
Plasma testosterone concentration was determined with a radioimmunoassay kit (Diagnostic Products). The interassay and intra-assay coefficients of variation were <10%, and the limit of detection was 4 ng/dL.

Data Analyses
Radioligand Binding Data Analysis
The dissociation constant (K_d) and the receptor density (B_max) were calculated by Scatchard analysis and the curve-fitting program LIGAND.³⁵ The receptor density is expressed as pmol/mg protein.

Statistical Evaluation
All data are expressed as mean±SEM. The comparability of baseline characteristics of subjects in the placebo and testosterone groups was evaluated by the unpaired Student's t test. The effects of testosterone or placebo are presented as changes from their respective baseline (average of the two baseline measurements). The effects of testosterone on TXA₂ receptor affinity, density, and platelet aggregation responses were compared with the placebo data by two-way repeated-measures ANOVA. The effects of testosterone or placebo separately on these parameters were also evaluated by one-way ANOVA. The relations between endogenous plasma testosterone concentration and platelet TXA₂ receptor B_max and K_d were evaluated by linear regression analysis. The null hypothesis was rejected at P<.05. The power of the statistical tests for assessing the end points, changes in B_max, and aggregation responses was 0.8.

	Results

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Plasma Testosterone Concentrations
Pretreatment (endogenous) plasma testosterone concentrations ranged from 215 to 883 ng/dL. Before treatment, there was no significant difference between the endogenous testosterone concentrations in the placebo group and the group administered testosterone (Table 1). The administration of testosterone cypionate resulted in a substantial rise in plasma testosterone concentration, from 424±34 to 689±64 ng/dL (P<.002) at 1 week. This represents a 63% increase over the pretreatment value. Subsequently, however, plasma testosterone concentration diminished to below the basal levels (278±28 ng/dL) at 2 weeks. Plasma testosterone returned to the pretreatment level by 8 weeks. Placebo administration had no significant effect on plasma testosterone levels.

Platelet Thromboxane A₂ Receptors
¹²⁵I-BOP bound to the TXA₂ receptors on washed human platelets in a saturable manner. Testosterone treatment significantly increased the human platelet TXA₂ receptor density (B_max), with a peak effect at 4 weeks and a return to baseline at 8 weeks (Fig 1). The increase in platelet TXA₂ receptor density was significantly (P<.001) greater in the testosterone-treated group than in the placebo group or its own baseline. The absolute values (in pmol/mg protein) were 1.19±0.23, 1.13±0.23, 1.20±0.20, and 1.21±0.12 for placebo at baseline, 2, 4, and 8 weeks, respectively. Corresponding values for the testosterone-treated group were 0.95±0.13, 1.51±0.22, 2.10±0.43, and 1.10±0.15. Testosterone treatment did not significantly alter the K_d (Table 2).

View larger version (0K): [in this window] [in a new window]   Figure 1. a, Representative Scatchard analysis of equilibrium binding data in a volunteer who received testosterone. The two curves represent the pretreatment () period and the 4-week time point (). The Kd values were 3.4 and 2.3 nmol/L, and the Bmax values were 0.94 and 2.1 pmol/mg protein for pretreatment and 4-week time points, respectively. B indicates bound; F, free. b, Graph showing changes in human platelet thromboxane A2 (TXA2) receptor density. Testosterone treatment was associated with significant increase (P<.001) in Bmax compared with placebo and with the baseline values. Data are mean±SEM. Testosterone, n=9; placebo, n=7.

View this table: [in this window] [in a new window]   Table 2. Influence of Testosterone on Platelet Thromboxane A2 Receptor Affinity and Sensitivity to I-BOP– and Thrombin-Induced Platelet Aggregation

Platelet Aggregation
I-BOP
The pretreatment maximum platelet aggregation induced by I-BOP in the placebo-treated group was 69.0±2.2%, and this was not significantly different from the corresponding value in the testosterone group, 65.0±1.9%. The maximum platelet aggregation response to I-BOP at 4 weeks was significantly (P<.001) increased in the testosterone-treated group compared with the placebo group (Fig 2a). The EC₅₀ values were not significantly changed. The increase in the testosterone-treated group was also significant (P<.001) in comparison with the pretreatment values. The values were +5.2±1.6% at 2 weeks, with a peak effect of +7.3±2.3% at 4 weeks, and a return to pretreatment baseline, -0.44±3.1%, at 8 weeks (P<.001). The EC₅₀ values for the testosterone and placebo groups were not significantly changed by the two treatments (Table 2). Testosterone treatment was associated with significantly (P<.001) augmented aggregation responses to I-BOP at all concentrations at the 4-week time point compared with the pretreatment and posttreatment periods (Fig 2b).

View larger version (0K): [in this window] [in a new window]   Figure 2. a, Graph showing change in the maximum platelet aggregation response induced by I-BOP. The maximum platelet aggregation response induced by I-BOP at 1 minute was determined for each subject. Testosterone significantly (P<.001) increased the maximum aggregation response to I-BOP compared with placebo and with its own baseline value. There were no significant changes in the placebo group. Data are mean±SEM. Testosterone, n=9; placebo, n=7. b, Graph showing I-BOP–induced platelet aggregation in testosterone-treated subjects (n=9). Percent aggregation was determined 1 minute after the addition of I-BOP. Testosterone treatment was associated with a significant (P<.001) increase in the aggregation responses to all I-BOP concentrations at 4 weeks, in comparison with the baseline (week 0) and recovery phase (week 8). Data are mean±SEM. *P<.05 in comparison with weeks 0 and 8.

Thrombin
The baseline maximum thrombin-induced platelet aggregation was 56±3% in the testosterone group and 61±2% (P=NS) in the placebo group. There was no significant change in thrombin-induced platelet aggregation in the testosterone-treated group (P=.07). EC₅₀ values for thrombin-induced aggregation for the placebo and testosterone groups were not significantly changed by treatment (Table 2).

Correlations Between Pretreatment (Endogenous) Plasma Testosterone Concentrations and Platelet Thromboxane A₂ Receptor Density and Dissociation Constant
There was a significant and positive correlation between the endogenous testosterone concentrations and platelet TXA₂ receptor density (B_max) (r=.56, P=.001, n=32 measurements) (Fig 3). The correlation was still significant when the average of the two baseline readings was used (r=.63, P<.01, n=16). There was no statistically significant correlation between the endogenous plasma testosterone and the K_d (r=.21, NS).

View larger version (0K): [in this window] [in a new window]   Figure 3. Scatterplot showing correlation between pretreatment (endogenous) plasma testosterone concentrations and platelet thromboxane A2 (TXA2) receptor density. r=.56, P<.001 by ANOVA, n=32 measurements.

	Discussion

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This study demonstrated that administration of testosterone to young men at a clinical replacement dose³⁶ was associated with a significant increase in human platelet TXA₂ receptor density and the maximum platelet aggregation response to the TXA₂ mimetic I-BOP. Testosterone did not affect the receptor affinity (K_d) or the EC₅₀ value for I-BOP–induced platelet aggregation. There was also a positive and significant correlation between the endogenous plasma testosterone concentrations before treatment and the platelet TXA₂ receptor B_max but not K_d. These results are consistent with the notion that testosterone regulates the expression of platelet TXA₂ receptors in humans and are also in accord with earlier in vitro and in vivo findings from our laboratory^{28 29 37} and those of other groups demonstrating increased vascular responses to TXA₂ mimetics in experimental animals treated with testosterone.^{23 27}

The peak plasma levels after intramuscular administration of testosterone cypionate are attained 4 to 5 days after injection.³⁸ Thereafter, they decline to basal levels by 2 weeks and sometimes to hypogonadal values.^{38 39} This profile for plasma testosterone was seen in this study and indicates that the administered testosterone exerted a biologically relevant effect on the endocrine system. Blood samples for the platelet studies were always collected between 8:30 and 9 AM to minimize the diurnal variability in platelet aggregation⁴⁰ as well as the nyctohemeral variation in plasma testosterone levels owing to the pulsatile nature of its secretion.³⁹ Plasma samples were taken at 1 week in the subjects to verify that the administered testosterone was absorbed, and they demonstrated the expected pharmacokinetics.³⁸

Platelet turnover from megakaryocytes after testosterone injection will still be incomplete at 1 week, so that assay of circulating platelet TXA₂ receptors would reflect a mixed platelet population at 1 week. The 2- and 4-week samples permitted an evaluation of testosterone treatment on platelet TXA₂ receptors and aggregation after complete turnover of the circulating platelet population and no residual elevations in testosterone. Thus, any effect seen could not be attributable to a direct effect of testosterone on the assays. Given a platelet circulation half-life of 5 days, complete turnover should occur in approximately five half-lives, or 3 weeks. The effect of cumulative doses of testosterone injection could therefore be seen at 4 weeks. At 8 weeks, 6 weeks after the second testosterone injection, the effect of the exogenous testosterone treatment should be gone, since there should be a new population of circulating platelets. If there were a residual effect of testosterone at the 8-week time point, then it would be unlikely that it was a direct effect on the platelets; rather, it would be an effect on some other plasma component that in turn produced the observed effect. The fact that the B_max values and aggregation responses returned to values not significantly different from baseline supports the contention that the effect of testosterone was directly on megakaryocytes.

The molecular mechanisms by which exogenous testosterone increased platelet TXA₂ receptor density has not been elucidated by this study. It is unlikely to result from a nonspecific anabolic action of testosterone, causing increased platelet protein synthesis, since the B_max (pmol/mg protein) normalized to protein concentration was significantly increased. The increase in receptor density is also unlikely to be a consequence of the lipophilicity of testosterone. Lipophilic substances or maneuvers that cause acute platelet swelling increase platelet surface area in vitro, permitting greater expression of TXA₂ receptor number.³³ It is unlikely, however, that the increase was simply due to an increase in platelet size.

The gene encoding the human TXA₂ receptor has recently been cloned and found to possess a glucocorticoid-responsive element.⁴¹ This suggests that anabolic steroid regulation of human TXA₂ receptors may be exerted at the genomic level. Earlier studies revealed that actinomycin D and cycloheximide, which inhibit transcription and translation, respectively, attenuated the effects of testosterone in increasing TXA₂ receptor density in HEL cells, a tumor cell line with megakaryocyte marker proteins,²⁹ and in rat aortic smooth muscle cells. Collectively, these observations suggest that testosterone may increase platelet TXA₂ receptor density through the synthesis of new receptors, probably in the megakaryocytes in the bone marrow.

The increase in platelet TXA₂ receptor density was accompanied by a small but significant increase in the maximum aggregation response to I-BOP. There was a discrepancy in the magnitude of platelet TXA₂ receptor density increase (twofold) and the increase in I-BOP–induced maximum platelet aggregation (+7±2.3%) after testosterone treatment. In HEL cells, the testosterone-induced increase in TXA₂ receptor density was associated with comparable maximum TXA₂ agonist–induced increases in cytosolic calcium.²⁹ In this study, since there is a ceiling (100%) for platelet aggregation responses, a direct quantitative association between change in receptor density and aggregation might not be assessable. The possibility also exists that some of the receptors are not coupled and, therefore, there was not a proportionate increase in the aggregation response. The maximum thrombin-induced platelet aggregation responses were not statistically significantly altered by testosterone, although there was a trend toward enhancement. This is similar to observations in rats, in which testosterone did not alter thrombin-induced platelet aggregation.³⁰ Thus, testosterone augments platelet aggregation induced by TXA₂. Whether this effect of testosterone extends to other aggregatory receptors remains to be determined.

The pretreatment (endogenous) plasma testosterone concentration was positively and significantly correlated with platelet TXA₂ receptor density but not affinity. This raises the possibility that endogenous testosterone is one of the factors that regulates the expression of platelet TXA₂ receptors.

The direct clinical implications of this study with reference to the sex difference in thrombotic cardiovascular disease is unclear. The results, however, raise the possibility that testosterone-induced increases in TXA₂ receptor density may contribute to the premature cerebrovascular accidents and coronary thrombosis in young male athletes who abuse anabolic steroids.^{16 17 18 19 20} It may also provide further insight into potential pathogenic mechanisms by which endogenous androgens may contribute as a cardiovascular risk factor in humans. The results raise the need for further studies on the role of endogenous testosterone in modulating TXA₂ receptor expression in humans.

	Acknowledgments

 
This study was supported in part by NIH grants HL-36838 and RR-1070. Dr Ajayi is a Merck Sharp & Dohme International Fellow in clinical pharmacology. We gratefully acknowledge the helpful discussions of Dr Alan J. Gross and the technical assistance of Julie Griffin, the nursing and administrative help provided by Martha Donohue and Jonetta Lee, and typing by Susan Higerd.

	Footnotes

 
Reprint requests to P.V. Halushka, MD, PhD, Division of Clinical Pharmacology, Medical University of South Carolina, 171 Ashley Ave, Charleston, SC 29425-2251.

Received October 10, 1994; revision received November 21, 1994; accepted December 3, 1994.

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