(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.
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.
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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 (Bmax) and dissociation constant (Kd) 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 Bmax 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 Bmax values in the saline-treated group. The Kd 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 Kd.
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
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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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.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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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
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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 (Kd) and the
receptor density (Bmax) 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
Bmax and Kd 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 Bmax, and aggregation
responses was 0.8.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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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 (Bmax), 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 Kd (Table 2).
|
) 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.
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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).
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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 (Bmax) (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 Kd
(r=.21, NS).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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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 (Kd) 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 Bmax but not Kd. 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 laboratory28 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 Bmax 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 Bmax (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.
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Acknowledgments |
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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.
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Footnotes |
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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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