© 1998 American Heart Association, Inc.
Original Contributions |
Oral Contraceptives and Hormone Replacement Therapy Do Not Increase the Incidence of Arterial Thrombosis in a Nonhuman Primate Model
From the Center for Thrombosis and Hemostasis, Department of Pathology and Laboratory Medicine, and the Division of Laboratory Animal Medicine, School of Medicine, University of North Carolina at Chapel Hill (D.A.B., D.E.B.); and Department of Comparative Medicine, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC (J.K.W., M.R.A., E.K.H.).
Correspondence to Dr Dwight A. Bellinger, The School of Medicine, Division of Laboratory Animal Medicine, The University of North Carolina at Chapel Hill, CB#7115, B12 Berryhill Hall, Chapel Hill, NC 27599-7115. E-mail dbsbsb{at}med.unc.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—Older oral contraceptive (OC) formulations containing high doses of potent synthetic estrogens and progestins are associated with increased risk of thrombosis. To examine the effects of current low-dose OC and hormone replacement therapy (HRT) regimens on arterial thrombosis, premenopausal and surgically postmenopausal cynomolgus monkeys were divided into four treatment groups. Premenopausal monkeys were given either no OCs or ethinyl estradiol and levonorgestrel as an OC at a dose equivalent to that currently given to women. Postmenopausal monkeys were given either no HRT or conjugated equine estrogens and medroxyprogesterone as an HRT at a dose equivalent to that currently given to women. The monkeys were fed an atherogenic diet containing these treatments for 27 to 30 months. At the end of this time, arterial thrombosis was evaluated with a standardized stenosis/injury procedure in the left carotid artery. Blood flow velocity was monitored for cyclic or permanent occlusive thrombosis. The current OC and HRT regimens did not increase the susceptibility of the artery wall to develop an occlusive thrombus following injury and stenosis. In fact, there was a reduction in the incidence of thrombosis in the OC animals compared with untreated controls. Increased amounts of atherosclerosis were associated with an increased incidence of occlusive arterial thrombosis. Several selected coagulation parameters [von Willebrand factor, protein C, lipoprotein(a), and platelet aggregation] did not appear to be associated with either the amount of atherosclerosis or incidence of arterial thrombosis.
Key Words: oral contraceptives • hormone replacement therapy • arterial thrombosis • atherosclerosis • cynomolgus monkeys
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The effects of OCs and HRT on arterial thrombosis are unclear. Older OC formulations containing high doses of potent synthetic estrogens and progestins are associated with increased risk of thrombosis. This association is based on a number of reports of increased venous thrombosis, coronary heart disease, and thrombotic strokes.1 2 3 However, the current lower-dose OC formulations are considered to have less thrombotic risk compared with previous formulations.4 5 Furthermore, reports of increased coagulation parameters (plasma fibrinogen, prothrombin, and factors VII and VIII)6 7 associated with use of OCs have not correlated well with risk of thrombotic events. Present thought would support the notion that OC use is associated only with increased thrombotic risk in women who smoke8 9 and in selected populations prone to thrombosis.10
In contrast, HRT in postmenopausal women is associated with reduced risk of coronary heart disease.11 How HRT lowers coronary heart disease risk is unclear, but evidence exists that HRT has beneficial effects on plasma lipoprotein concentrations,12 13 14 inhibits progression of atherosclerosis,15 inhibits arterial wall accumulation of LDL,16 and reduces plasma Lp(a) concentrations.17 18 However, there is still concern about the effect of HRT on thrombotic events. This concern is sufficient to direct many physicians to stop HRT in women with a history of thrombotic disease. Much of this concern seems to be based on preconceived notions about exogenous estrogen/progestin administration in premenopausal women taking OCs. There is a paucity of information available about the effects of current OC and HRT formulations on risk of arterial thrombosis. Therefore, the purpose of this study was to examine the effects of current commonly used OC and HRT formulations on arterial thrombosis in a well-characterized animal model of arterial thrombosis in the setting of diet-induced atherosclerosis.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animals
Sixty-six adult (7 to 10 years of age) female cynomolgus monkeys (Macaca fascicularis) were imported from Indonesia (Institut Pertanian Bogor, Bogor, Indonesia) and fed monkey chow (High Protein Monkey Chow, Ralston Purina Co) during a 3-month quarantine period. The monkeys were then fed their experimental diets for 27 to 30 months. All diets were moderately atherogenic and contained 0.28 mg cholesterol/kcal, with 40% calories from fat.
The premenopausal monkeys received the atherogenic diet either with (n=15) or without (n=13) a triphasic OC (Triphasil, Wyeth-Ayerst). On days 1 to 6 of the month, monkeys received ethinyl estradiol at 0.0020 mg/kg body wt and levonogestrel at 0.0033 mg/kg body wt. On days 7 to 11 of the month, monkeys received ethinyl estradiol at 0.0027 mg/kg body wt and levonorgestrel at 0.0050 mg/kg body wt. On days 12 to 21 of the month, monkeys received ethinyl estradiol at 0.0020 mg/kg body wt and levonorgestrel at 0.0083 mg/kg body wt. On days 22 to 28, monkeys received no hormone in their diets. These doses were calculated to approximate the dose of triphasic OCs given to women. Doses were adjusted for species differences in body weight and body metabolism.
Monkeys used for the postmenopausal hormone replacement part of the experiment were ovariectomized at the beginning of the treatment phase of the experiment and then received the atherogenic diet with (n=24) or without (n=14) HRT. The HRT consisted of CEE (Premarin, Wyeth-Ayerst) and MPA (Cycrin, Wyeth-Ayerst) added to the diet. The dose of both hormones was 0.04 mg CEE · kg body wt-1 · d-1 and 0.16 mg MPA · kg body wt-1 · d-1 added to the diet. These doses were chosen to approximate the daily doses of these hormones given to women (0.625 and 2.5 mg, respectively) and were calculated on the basis of body weight and metabolic rate differences between species.
Monkeys lived in social groups of 4 to 6 animals. All procedures were performed at the Comparative Medicine Clinical Research Center of the Bowman Gray School of Medicine in accordance with state and federal laws. Animal protocols were approved by the Bowman Gray Animal Care and Use Committee and conformed to guidelines set forth by the American Association for Accreditation of Laboratory Animal Care and by National Institutes of Health publication 86–23, Guide for the Care and Use of Laboratory Animals.
Blood Sample Analysis for Coagulation
Parameters
All blood samples were taken from monkeys after an overnight
fast. Monkeys were sedated with ketamine hydrochloride (10 to
15 mg/kg IM), and blood samples were taken from a femoral vein. Blood
samples were collected periodically at 3, 18, and 26 months after the
beginning of the atherogenic diet for the postmenopausal groups and at
21 and 28 months after the beginning of the atherogenic diet for the
premenopausal groups. Blood samples were drawn at various cycles of
hormonal treatments, with the exception of the last OC samples, which
were drawn at matched hormonal cycles. Blood samples were collected
into sodium citrate from the femoral vein after sedation with
ketamine hydrochloride (15 mg/kg). Blood analysis at
the time of collection consisted of platelet aggregation studies
with PRP with ADP (10-3 and
10-4 mol/L), manual platelet counts of PRP
and whole blood, and thrombin clotting times with PPP. PPP was frozen
for a later analysis of vWF antigen by ELISA (vWF antibody,
DACO) and protein C activity by a chromogenic assay
(Coamate, Chromogenix). vWF antigen and protein C activity are
expressed as a percent of a standard pool. The standard pool was made
from adult female cynomolgus monkeys unrelated to this study and not
receiving any hormone therapy. Blood samples were also collected before
and 12 months after the atherogenic diet for analysis of plasma
concentrations of Lp(a). Plasma Lp(a) concentrations were determined
with an ELISA for Lp(a) that was developed at Bowman Gray School of
Medicine Lipoprotein Core Laboratory.
Plasma Hormone Concentrations
Plasma concentrations of estradiol19 and
medroxyprogesterone20 were
measured as described previously. Plasma concentrations of
norgestrel were measured by ELISA methods in Dr Mark Wilson's
laboratory at Emory University.
Arterial Thrombosis
At the end of the dietary treatment phase of the experiment,
each monkey in the experiment was anesthetized with
ketamine hydrochloride (10 to 15 mg/kg body wt, IM) and
butorphanol (0.025 mg/kg body wt, IM). Additional doses of each agent
were given as needed to maintain anesthesia. Palpebral
reflex and response to pain (toe pinch) were used as criteria to
monitor the level of anesthesia. The ventral neck region
was shaved, and an incision was made to expose the left common
carotid.
The left carotid artery was dissected free of surrounding tissue over a 2-cm segment. A 5-mm Goldblatt clamp was placed proximally on the carotid artery, and a 20-MHz Doppler ultrasonic crystal was applied distally so that flow was not impaired. The Doppler crystal was energized by a range-gated pulsed Doppler unit, and the signal was range-gated to maximum clarity.
A schematic diagram of the steps in the injury/stenosis
procedure is given in Fig 1
. After a
30-minute period of stabilization following the surgical preparation,
the carotid artery was stenosed by closing the Goldblatt clamp
sufficiently to block reactive hyperemia. A 20-second total
occlusion was used to induce reactive hyperemia and confirm the
arterial blockade. A 30-minute period of observation for
CFR or PCF followed stenosis. CFR was characterized by a
gradual reduction in flow to zero followed by a sudden spontaneous
return of flow to the baseline level. PCF was characterized by a
gradual reduction in flow velocity, but, unlike CFR, there is no
spontaneous return of blood flow. If neither CFR nor PCF occurred, the
Goldblatt clamp was released after another 20-second occlusion to
confirm that reactive hyperemia had been blocked throughout the
period of observation.
|
After the stenosis was removed by opening the Goldblatt clamp and blood flow velocity stabilized, the carotid artery was injured in the area where the clamp was applied. Injury was induced by three occlusions of the artery with spring-loaded forceps (Castroviejo Needle Holder, J. Sklar, Inc). After 10 minutes of observation, the Goldblatt clamp was closed to block reactive hyperemia. If CFR or PCF occurred before or after the clamp was closed, flow velocity was monitored for 20 minutes and the experiment was stopped for that artery. If, within 30 minutes, neither CFR or PCF was seen, the clamp was reopened, and after stabilization of flow velocity, the pinch injury was repeated. If flow velocity remained unchanged, the Goldblatt clamp was tightened a third time for a final 30-minute period of observation. The results were expressed as the frequency of carotid arteries per treatment group that developed PCF, CRF, or both. At the end of the experiment, the artery segment was ligated, removed, and fixed with 4% paraformaldehyde.
Tissue Evaluation of Injury and Atherosclerosis
Transverse sections of the injury/stenosis site were
examined with light microscopy for the amount of arterial
injury and presence of thrombus. The cross sections were divided into
eight equal segments and analyzed for the presence or absence
of smooth muscle damage, medial hemorrhage, disruption of the
internal elastic lamina, and mural thrombus formation. Smooth muscle
damage was defined as pale-staining cytoplasm and contracted and
pyknotic nuclei. The amount of injury was expressed as the percentage
of segments with smooth muscle damage or medial hemorrhage.
Arterial cross sections stained with Verhoeff–van Gieson's stain were projected onto a digitizer and analyzed morphometrically (Imagepro, Media Cybernetics). Morphometric measurements included luminal area, intimal area, and medial area. The amount of atherosclerosis was expressed in two ways: the intimal area as a percentage of the area within the internal elastic lamina (percentage of luminal narrowing) or the intimal area as a percentage of the medial area. All sections were examined and measured blinded, without the observer knowing the treatment group.
Immunohistochemistry
Representative sections were evaluated
from all four groups for the presence of vWF. Sections were taken from
the injury/stenosis site from 16 premenopausal monkeys (8
receiving OCs and 8 not receiving OCs) and 23 postmenopausal monkeys
(12 receiving HRT and 11 not receiving HRT). These included sections
from 18 monkeys that had occlusive arterial thrombosis and
21 that had no alterations in flow velocity. Sections were
deparaffinized in xylene, rehydrated in graded alcohol, and
immunostained. Primary antibodies were localized with
appropriate biotinylated secondary antibody and tertiary avidin-biotin
complex stain (Vector Laboratories). Control slides were stained
without a primary antibody. Sections were counterstained with Mayer's
hematoxylin and examined by light microscopy.
Statistical Analysis
All measured variables were reported as mean±SEM.
Comparisons between means for all groups were made by an ANOVA and
Student's t test with a confidence level of 95%. The
Wilcoxon signed-rank test was used to compare paired data.
Nonparametric analyses were made with the
Kruskal-Wallis and the Mann-Whitney U tests with a
confidence level of 95%. The 
2 test for a
contingency table and Fisher's exact test were used to compare
frequency data and the Spearman's rank correlation to compare the
linear relationships with a 95% confidence level. Statistical
analyses were performed with a computer program (StatMost,
Dataxiom Software, Inc).
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Arterial Thrombosis
The incidence of arterial thrombosis after injury and stenosis is given in Table 1
and Fig 2. In the group receiving OCs, 2 had PCF
and 1 had equivocal changes in flow that were difficult to interpret as
CFR. There was no CFR or PCF in the other 12 monkeys in this group. The
incidence of arterial thrombosis was 14.2% (2 of 14) if
the one with equivocal flow reductions is excluded or 20% (3 of 15) if
it is scored as CFR. In the premenopausal group not receiving OCs, 4
monkeys had PCF (one of these was preceeded by CFR) and 3 had CFR. No
CFR or PCF was observed in the other 6 monkeys in this group. Thus, the
incidence of thrombosis was 53.9% (7 of 13) for this group. In the
group of postmenopausal monkeys receiving HRT, 7 had PCF (one of which
was preceded by CFR), 6 had CFR, and 11 had no CFR or PCF. The
incidence of arterial thrombosis was 54% (13/24). In the
group of postmenopausal monkeys not receiving HRT, 2 had PCF, 4 had
CFR, and 9 had no CFR or PCF, for an arterial thrombosis
incidence of 40% (6 of 15). The group of monkeys receiving OCs had a
lower incidence of arterial thrombosis than the
premenopausal group not receiving OCs. This rate was significant when
14.2% was compared with 53.9% (P=.033, Fisher's exact
test). If the equivocal one is included, the value is
P=.059. There was no significant difference in the incidence
of arterial thrombosis in the treated and untreated
postmenopausal groups (P=.18, Fisher's exact test). When
all four groups were compared using 14.2% for the OC group, the
dif-ference among groups was nearly significant (P=.084,
2 test for a contingency table). If 20% was
used, there was no significant difference among the groups
(P=.163, 2 test for a contingency
table).
|
|
Evaluation of Tissue Injury and Atherosclerosis
The amount of injury to the vessel wall as expressed by the
percentage of segments that had medial injury was similar among groups
(P=.97, ANOVA). The amount of damage to the media was
85.0±29% for the premenopausal group receiving OCs and 81.3±32% for
the group not receiving OCs. The postmenopausal group receiving HRT had
85.5±26% medial damage, and the group not receiving HRT had
85.3±22%.
The amount of atherosclerosis in the carotid arteries
of the premenopausal and postmenopausal groups is given in Fig 3 as the percentage of luminal narrowing.
The difference among groups was nearly significant (P=.053,
Kruskal-Wallis test). The group of monkeys receiving OCs had less
atherosclerosis than the control group and the
postmenopausal groups. Pairwise comparisons revealed a nearly
significant difference between the OC group and the premenopausal group
not receiving OCs (P=.069, Mann-Whitney U test).
There was no significant difference between the treated and untreated
postmenopausal groups (P=.677, Mann-Whitney U
test). Similar results were seen when atherosclerosis
was expressed as a percentage of medial area.
|
To evaluate the relationship of thrombosis to the amount of
atherosclerosis, each group was divided into those that
formed occlusive thrombi and those that did not (Fig 4). The difference among these groups was
nearly significant (P=.055, Kruskal-Wallis test). Within the
HRT group, those that formed occlusive thrombi had a significantly
greater amount of atherosclerosis than those in this
group that did not (P=.016, Mann-Whitney U test),
and the difference in atherosclerosis was nearly
statistically significant within the OC group (P=.079,
Mann-Whitney U test). The OC group had both the lowest
incidence of thrombosis and the least amount of
atherosclerosis.
|
Combining all groups, the amount of atherosclerosis (luminal narrowing) was greater in those vessels that formed occlusive thrombi (37.6%) than in those that did not (16.2%) (P=.011, Mann-Whitney U test). In addition, the incidence of thrombosis, 60.7% (17 of 28) for those animals with the most atherosclerosis (upper half), was significantly greater than the incidence of 29% (8 of 28) for those with the least atherosclerosis (lower half) (P=.01 Fisher's exact test). With all groups combined, the occurrence of thrombosis (graded as 0=no flow alterations, 1=CFR, and 2=PCF) was significantly correlated to the amount of atherosclerosis (r=.338, P=.01, Spearman's rank correlation).
Immunohistochemistry
A section of an injured stenosed vessel
immunostained for vWF is shown in Fig 5. In normal vessels, vWF is confined to
the endothelium and subendothelium. In
the stenosed and injured vessels, the luminal surfaces that were
denuded of endothelium often lacked staining for vWF.
Staining for vWF was usually associated with areas that contained
platelets. The damaged media accumulated vWF only in areas of
medial hemorrhage that were platelet rich. Thrombi had a
mixed staining pattern for vWF, with areas rich in red blood cells
lacking vWF staining and areas rich in platelets staining heavily.
Staining for vWF was not associated with uninjured plaque. There were
no distinctive differences in the staining pattern among the
groups.
|
Coagulation Parameters
The plasma concentrations of vWF antigen for the
premenopausal and postmenopausal groups are given in Table 2 for 26 to 28 months after the
atherogenic diet was begun. At this time point, the vWF antigen was
significantly different among groups (P=.002, Kruskal-Wallis
test). The OC group had lower vWF concentrations than the premenopausal
group not receiving OCs (P=.018, Mann-Whitney U
test). Although the HRT group had lower vWF concentrations than the
untreated postmenopausal group, this difference was not significant
(P=.21, Mann-Whitney U test). When all groups
were combined, plasma vWF concentrations were not correlated with the
amount of atherosclerosis (r=-.06,
P=.67, Spearman's rank correlation). High concentrations of
plasma vWF were not associated with arterial thrombosis:
the plasma vWF concentrations were similar for those that formed
occlusive thrombi and those that did not (103.6 versus 102.2%,
P=.42, Mann-Whitney U test).
|
There were no differences in plasma Lp(a) concentrations among the
groups (P=.67, Kruskal-Wallis test) and no changes with 1
year of atherogenic diet feeding (P=.30, t test;
P=.47, Mann-Whitney U test) (Table 2
). There were
no significant correlations between Lp(a) concentration and
atherosclerosis (r=.168, P=.216,
Spearman's rank correlation), nor was there a difference in Lp(a)
levels between monkeys that formed occlusive thrombi and those that did
not (36.3 versus 36.0 mg/dL, P=.97, Mann-Whitney
U test).
Platelet aggregation to ADP was observed in all monkeys.
There was a significant difference among the groups at the last time
point in the mean slope of the aggregation curve (P<.01,
Kruskal-Wallis test) (Table 2). No differences in the mean slope of the
aggregation curves were observed between the premenopausal monkeys
receiving OCs and the premenopausal group not receiving OCs
(P=1.00, Mann-Whitney U test). No differences
were observed between the treated and untreated postmenopausal groups
(P=.22, Mann-Whitney U test). There was a
significant difference in the slope of the aggregation curve between
the premenopausal and postmenopausal animals (3.8 versus 6.2,
P=.00, Mann-Whitney U test). There was no
significant difference in platelet counts among groups at the final
time point (P=.187, Kruskal-Wallis test) (Table 2) and no
significant differences in slope or platelet count between those
that formed occlusive thrombi and those that did not (P=.62
and P=.25, respectively, Mann-Whitney U
test).
There were no differences among the groups for protein C plasma
concentrations (P=.33, Kruskal-Wallis test) (Table 2). There
was no significant correlation of protein C to
atherosclerosis (r=-.20, P=.22,
Spearman's rank correlation) and no difference in protein C in animals
that formed occlusive thrombi versus those that did not (111.7 versus
112.8% standard pool, P=.55, Mann-Whitney U
test). The clotting times were within the normal range for all
monkeys.
Plasma Hormone Concentrations
Plasma concentrations of estradiol in the OC and HRT groups of
animals were 356±23 and 320±30 pmol/L, respectively. Plasma
medroxyprogesterone concentrations in the
HRT-treated monkeys were 61±15 pmol/L. Plasma levonorgestrel
concentrations were 55±10 pmol/L.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The major finding of this study was that current OC and HRT regimens given to nonhuman primates do not increase the susceptibility of the artery wall to develop an injury-induced thrombus in this model. In fact, arteries from OC-treated animals had a reduced incidence of arterial thrombosis after injury.
OCs and Thrombosis
Since the introduction of OCs, there has been controversy
regarding their use and the association with thrombotic
cardiovascular events. Several retrospective and
prospective studies have linked OC use to increased thrombotic
complications.1 2 3 Other studies have disagreed,
indicating that the increased risk could be explained by factors such
as smoking.8 9 As low-dose OCs became widely
used, studies indicated a lower thrombotic
risk.21 22 23 It is now generally accepted that the
use of low-dose OCs decreases the thrombotic risk compared with
higher-dose compounds. This finding is particularly true for deep vein
thrombosis22 24 but may not be as well defined
for arterial events such as stroke and myocardial
infarction. Studies have shown that both low- and high-dose OCs
increase levels of several coagulation factors (II, VII, VIII, IX, and
X; fibrinogen; and soluble fibrin). These studies have been previously
reviewed.5 6 7 It has been suggested that the
increase in coagulation factors may produce a "hypercoagulation
state." However, there is no direct evidence that this results in
thrombosis in vivo. Any changes in coagulation may be balanced by
changes in fibrinolytic activity. OCs have been reported to increase
factor XII–dependent
fibrinolysis.25 In addition, if
such a hypercoagulable state resulted in vascular thrombosis, then
markers of in vivo clotting activity would be expected to increase.
However, OCs have not been shown to increase thrombin–antithrombin III
complexes or prothrombin 1 and 2, indicators of elevated levels of
factor Xa generation.26
This study focused on arterial thrombotic effects of OCs and HRT rather than coagulation parameters or venous thrombosis. In this model, OC administration did not increase the incidence of arterial thrombosis and, in fact, may have lowered the incidence.
When all groups were considered, those with the most atherosclerosis had the highest incidence of arterial thrombosis. The OC group had both the lowest incidence of thrombosis and the least amount of atherosclerosis. The reduced atherosclerosis seen in the OC group could indicate a slowing of atherogenesis and a more stable plaque, resulting in less thrombosis. A previous study examining the effects of contraceptives on atherosclerosis showed that OCs of similar formulation, although not identical to those in this present study, were not associated with increased coronary plaque size; in fact, at the intermediate total cholesterol and HDL cholesterol levels, these OCs were associated with decreased plaque size.27 In that study, it was suggested that the ethinyl estradiol in the OCs neutralized the atherogenic influence of decreased HDL caused by the contraceptive progestin levonorgestrel through estrogenic influences on cellular proliferation and metabolism in arterial tissue.
HRT and Thrombosis
In this study, HRT had no apparent effect on the incidence
of arterial thrombosis in the postmenopausal monkeys. Much
of the concern for HRT and thrombosis comes from the use of OCs. The
studies examining HRT and thrombosis are limited and show no evidence
of increased thromboembolism in women taking
HRT.28 29 Platelet adhesion to fibronectin,
collagen I, and collagen III has been shown to be increased in women
taking OCs, but women receiving HRT had platelet adhesion similar
to that of premenopausal women, postmenopausal women not receiving HRT,
and men.30 Recently, it has been shown that HRT
does not alter the plasma levels of prothrombin fragment 1 and 2 or
thrombin-antithrombin III complex,31 indicating
that HRT does not increase in vivo clotting activity. In addition, it
has been shown that transdermal estradiol does not alter platelet
numbers, plasma concentrations of a number of coagulation factors, or
fibrinolytic activity.32
HRT is associated with a reduction of coronary heart disease partially because of its beneficial effects on lipoproteins.12 13 14 In this study, the amount of atherosclerosis in the injured/stenosed vessels and incidence of thrombosis were similar in both postmenopausal groups. Thus, the beneficial effects of HRT in postmenopausal women may involve mechanisms not detectable with this model.
Coagulation Parameters
This study did not attempt to measure all the coagulation factors
that have been shown to change with estrogen and/or progestin intake.
Two parameters that have previously been shown to be
important in this injury/stenosis model are vWF and
Lp(a).33 34 In this model, the absence of vWF
prevents arterial thrombosis, whereas high levels of Lp(a)
have been shown to be associated with an increase in
arterial thrombosis.34 In this study,
neither the plasma concentrations of vWF nor those of Lp(a) were
associated with occlusive arterial thrombosis. In the
previous studies, vWF was absent, whereas in this study, even the
lowest levels would be sufficient to support arterial
thrombosis.33 The formation of
neointima during atherogenesis has been associated with a
local increase in vWF in the vessel wall.35 VWF
was clearly present in the damaged arterial wall and
most likely serves as an adhesive protein to anchor the forming
thrombus. The concentration of vWF was lower in the OC group. Although
plasma vWF concentrations increase during
pregnancy,3 it is unclear why they should be
lower in the OC group in this study.
In this study, high plasma concentrations of Lp(a) were not associated with occlusive arterial thrombi. The previous study examining the effect of plasma Lp(a) was done in the absence of atherosclerosis, which seems to be an important contributor to the arterial thrombotic process. In addition, other studies have shown a decrease in Lp(a) with HRT17 18 and a correlation of Lp(a) to the amount of atherosclerosis.36 Neither of these was observed in this study. This finding could reflect that the last Lp(a) measurement was done at 1-year after diet feeding rather than at the termination of the study.
Studies looking at platelet function and hormonal status have had varied results. HRT has been associated with decreased platelet aggregation, and OCs have been associated with increased platelet adhesion.30 37 This study demonstrated an increased slope of the aggregation curve in the postmenopausal monkeys and an increase in platelet count in the HRT group, again showing an effect of hormonal status on platelets. However, these changes did not appear to affect arterial thrombosis.
Arterial thrombosis is dependent on multiple factors influenced by various components, including the vessel wall, coagulation factors, platelets, and the fibrinolytic system. Therefore, it is unlikely that any one factor alone in the complicated setting of atherosclerosis will account for all changes in arterial thrombosis.
Relationship of OCs, HRT, and Coronary Heart
Disease
Coronary heart disease results when myocardial blood
flow is reduced by occlusion of the coronary arteries. This
condition can occur when atherosclerotic plaque grows into the lumen
and creates a physical blockage to blood flow. Atherosclerotic arteries
are prone to spasm,38 which can also reduce
coronary blood flow. Finally, a thrombus can form in a segment
of a coronary artery, creating a physical blockage of the
artery. The pathogenesis of coronary artery thrombosis is
unclear but may be related to areas of atherosclerotic plaque that
rupture, exposing underlying thrombogenic material to circulating
blood.39 40
There is ample evidence that estrogens reduce the risk of coronary heart disease, possibly by inhibiting the progression of atherosclerotic plaque27 or by protecting atherosclerotic arteries against vasospasm.41 Although progestins may ameliorate some of these beneficial affects of estrogens on atherogenesis27 and vasospasm,41 the net result seems to be that exogenously administered hormones at new lower doses reduces the risk of coronary heart disease in women.
The effect of exogenously administered hormones on risk of thrombosis has been unclear. It seems logical that these hormones must not have an overwhelmingly harmful effect on thrombosis risk or they would be associated with a more decisive increased risk of coronary heart disease. The results of the present experiment suggest that these hormones do not increase the risk of thrombosis once the plaque is disrupted. The present study does not address, however, whether or not exogenously administered hormones reduce the risk of the plaque rupturing.
This study was not designed to examine the mechanism by which OCs or HRT regimens affect thrombosis. Circulating coagulation factors measured in this experiment were not greatly affected by treatment or associated with the formation of occlusive arterial thrombi. The amount of atherosclerotic plaque seemed to play an important role in regulating the incidence of thrombosis. If exogenously administered sex hormones do reduce thrombosis risk, they may do so by affecting atherogenesis or having other effects on the artery wall that directly or indirectly act on thrombotic/thrombolysis regulation.
Cynomolgus Monkey Model
Cynomolgus monkey females were used because they have
reproductive characteristics similar to those of
women.42 43 44 45 These include a 28-day menstrual
cycle, the occurrence of natural menopause, and cyclic changes in
plasma concentrations of estradiol, progesterone, follicle-stimulating
hormone, and luteinizing hormone that are similar both qualitatively
and quantitatively to those of women. Like premenopausal women,
premenopausal female cynomolgus monkeys have significantly higher
plasma concentrations of HDL cholesterol than their male
counterparts, and sex differences in the extent of coronary
artery atherosclerosis are like those of human beings.
Delivery of OCs in an experimental diet based on caloric requirements
and body weight has proved effective in previous studies with female
cynomolgus monkeys in preventing ovulation as determined by menstrual
cycles and plasma hormonal
concentrations.19 27 46 Postmenopausal cynomolgus
monkeys, like women, have reduced plasma concentrations of HDL
cholesterol and increased amounts of coronary
atherosclerosis compared with premenopausal females fed
the same atherosclerotic diet for the same length of
time.47 Thus, both premenopausal and
postmenopausal (ovariectomized) female cynomolgus monkeys share with
women many of the same risk factors for the development of
coronary artery disease. Furthermore, estrogen replacement
therapy inhibits progression of coronary artery
atherosclerosis,47 suggesting
that, like postmenopausal women, ovariectomized cynomolgus females
respond similarly to hormone treatment.
In this cynomolgus monkey model, a combination of arterial stenosis and injury was used to induce occlusive arterial thrombus formation measured as blood flow reductions. It has been shown in pigs that a combination of both injury and stenosis is necessary to induce occlusive arterial thrombosis.48 Evidence indicates that reductions in blood flow are due to the interactions of platelets with the vessel wall and with each other, leading to vascular occlusion.49 The technique, developed originally in dogs, has been used in other species, including monkeys.34 50 Although this model does not require the presence of atherosclerotic plaque and does not directly mimic what happens during plaque rupture, it, like other injury thrombosis models, causes intimal injury and exposes subintimal components to circulating blood in a setting of altered blood flow.
Experimental Considerations
The present report examines only one section of the carotid
artery at the injury/stenosis site and may not reflect the
overall amount of atherosclerosis in these monkeys.
When all animals were considered, there was a significant correlation
of atherosclerosis and thrombosis. This finding is not
necessarily surprising, because arterial thrombosis is
usually associated with the rupture of plaque. Under the experimental
conditions of this study, external injury to the vessel wall and
stenosis were used to initiate the thrombotic events. Thus,
interpretations about atherosclerosis and thrombosis in
this study should be done cautiously, and conclusions about plaque
stability cannot be determined.
The lower incidence of arterial thrombosis in the OC
group should also be interpreted cautiously, because the conditions of
injury and stenosis were chosen to enhance thrombotic effects.
Generally, OCs are taken by young women who have limited
arterial disease and are at low risk of
arterial thrombosis anyway. It was originally anticipated
from previous studies with monkeys and other species that the overall
incidence of flow reductions would be higher and that qualitative
differences in flow reductions (CFR verses PCF) may be observed.
Qualitative differences were not observed, and the overall incidence of
thrombosis in this study allowed for detection of differences between
groups of 
25% at a confidence level of 95%.
Conclusions
It is generally thought that administration of exogenous sex
hormones (whether as OCs or HRT) increases the risk of thrombotic
disease. Much of the concern is founded in much higher doses of
estrogens and progestins prescribed in the 1960s and 1970s.
Additionally, risk of venous thrombosis and arterial
thrombosis often were not differentiated. Concern about the thrombotic
effects of sex hormones has promoted the concept of stopping all forms
of exogenous estrogen/progestin therapies in women at increased risk of
thrombosis. The actual risk of the newer OC and HRT regimens on
arterial thrombosis has yet to be delineated. Therefore,
the goal of the present study was to use a well-established
nonhuman primate model of atherosclerosis to examine
the effects of present day OC and HRT regimens on incidence of
arterial thrombosis after arterial injury.
Results indicate that neither triphasic OCs nor HRT (CEE plus MPA)
increases the risk of arterial thrombosis and that
arterial thrombosis may be particularly dependent on the
amount of atherosclerosis present. These data may
provide some reassurance that current OC and HRT regimens do not
adversely affect the risk of arterial thrombosis in
women.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported by NIH grants HL-49085, HL-46409, and P01–45666.
Received February 21, 1997; accepted September 23, 1997.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Stadel BV. Oral contraceptives and cardiovascular disease. N Engl J Med. 1981;305:612–618, 672–677.[Medline] [Order article via Infotrieve]
2. Helmrich SP, Rosenberg L, Kaufman DW, Strom B, Shapiro S. Venous thromboembolism in relation to oral contraceptive use. Obstet Gynecol. 1987;69:91–95.[Medline] [Order article via Infotrieve]
3. Hannaford PC, Croft PR, Kay CR. Oral contraception and stroke: evidence from the Royal College of General Practitioners' Oral Contraception Study. Stroke. 1994;25:935–942.[Abstract]
4. Meade TW, Greenberg G, Thompson SG. Progestogens and cardiovascular reactions associated with oral contraceptives and a comparison of the safety of 50- and 30-mg oestrogen preparations. BMJ. 1980;1:1157–1161.
5. Bonnar J. Coagulation effects of oral contraception. Am J Obstet Gynecol. 1987;157:1042–1048.[Medline] [Order article via Infotrieve]
6. Beller FK, Ebert C. Effects of oral contraceptives on blood coagulation: a review. Obstet Gynecol Surv. 1985;40:425–435.[Medline] [Order article via Infotrieve]
7. Notelovitz M. Oral contraception and coagulation. Clin Obstet Gynecol. 1985;28:73–83.[Medline] [Order article via Infotrieve]
8. Layde PM, Beral V. Further analyses of mortality in oral contraceptive users: Royal College of General Practitioners' Oral Contraceptive Study. Lancet. 1981;1:541–546.[Medline] [Order article via Infotrieve]
9. Croft P, Hannaford PC. Risk factors for acute myocardial infarction in women: evidence from the Royal College of General Practitioners' Oral Contraceptive Study. BMJ. 1989;298:165–168.
10. Pabinger I, Schneider B, the GTH Study Group on Natural Inhibitors. Thrombotic risk of women with hereditary antithrombin III-, protein C- and protein S-deficiency. Thromb Haemost. 1994;71:548–552.[Medline] [Order article via Infotrieve]
11.
Barrett-Connor E, Bush TL. Estrogen and
coronary heart disease in women. JAMA. 1991;265:1861–1867.
12.
Criqui MH, Suarez L, Barrett-Connor E, Mc Phillips J,
Wingard DL, Garland C. Postmenopausal estrogen use and mortality:
results from a prospective study in a defined, homogeneous community.
Am J Epidemiol. 1988;128:606–614.
13.
The Writing Group for the PEPI Trial. Effects of
estrogen or estrogen/progestin regimens on heart disease risk factors
in postmenopausal women: the Postmenopausal Estrogen/Progestin
Interventions (PEPI) trial. JAMA. 1995;273:199–208.
14. Knopp RH. The effects of postmenopausal estrogen therapy on the incidence of atherosclerotic vascular disease. Obstet Gynecol. 1988;72(suppl):23S–30S.
15. Sullivan JM, vander Zwaag R, Lemp GF, Hughes JP, Maddock V, Kroetz FW, Ramanathan KB, Mirvis DM. Postmenopausal estrogen use and coronary atherosclerosis. Ann Intern Med. 1988;108:358–363.
16. Wagner JD, Clarkson TB, St Clair RW, Schwenke DC, Shively CA, Adams MR. Estrogen and progesterone replacement therapy reduces low density lipoprotein accumulation in the coronary arteries of surgically postmenopausal cynomolgus monkeys. J Clin Invest. 1991;88:1995–2002.
17. Van der Mooren MJ, Demacker NM, Thomas CMG, Borm GF, Rolland R. A 2-year study on the beneficial effects of 17B-oestradiol-dydrogesterone therapy on serum lipoproteins and Lp(a) in postmenopausal women: no additional unfavorable effects of dydrogesterone. Eur J Obstet Gynecol Reprod Biol. 1993;52:117–123.[Medline] [Order article via Infotrieve]
18.
Kim CJ, Jang HC, Cho DH, Min YK. Effects of hormone
replacement therapy on lipoprotein(a) and lipids in postmenopausal
women. Arterioscler Thromb. 1994;14:275–281.
19. Koritnik DR, Clarkson TB, Adams MR. Cynomolgus macaques as models for evaluating the effects of contraceptive steroids on plasma lipoproteins and coronary artery atherosclerosis. In: Gregoire AT, Blye R, eds. Contraceptive Steroids: Pharmacology and Safety. New York, NY: Plenum; 1986:303–319.
20.
Cornette JC, Kirton KT, Duncan GW. Measurement of
medroxyprogesterone acetate (Provera) by
radioimmunoassay. J Clin Endocrinol Metab. 1971;33:459–466.
21. Mishell DR. Use of oral contraceptives in women of older reproductive age. Am J Obstet Gynecol. 1988;158:1652–1657.[Medline] [Order article via Infotrieve]
22.
Gerstman BB, Piper JM, Tomita DK, Ferguson WJ, Stadel
BV, Lundin FE. Oral contraceptive estrogen dose and the risk of deep
venous thromboembolic disease. Am J Epidemiol. 1991;133:32–37.
23. Lidegaard O. Oral contraception and risk of a cerebral thromboembolic attack: results of a case control study. BMJ. 1993;306:956–966.
24. Böttiger LE, Boman G, Eklund G, Westerholm B. Oral contraceptives and thromboembolic disease: effects of lowering estrogen content. Lancet. 1980;1:1097–1101.[Medline] [Order article via Infotrieve]
25. Campbell SJ, Mackie IJ, Robinson GE, Machin SJ. Contact factor mediated fibrinolysis is increased by the combined oral contraceptive pill. Br J Obstet Gynecol. 1993;100:79–84.[Medline] [Order article via Infotrieve]
26. Saleh AA, Brockbank N, Dorey LG, Ozawa T, Dombrowski MP, Bottoms SF, Cotton DB, Mammen EF. TAT complexes and prothrombin fragment 1+2 in oral contraceptive users. Thromb Res. 1994;73:137–142.[Medline] [Order article via Infotrieve]
27. Adams MR, Clarkson TB, Koritnik DR, Nash HA. Contraceptive steroids and coronary artery atherosclerosis in cynomolgus macaques. Fertil Steril. 1987;47:1010–1018.[Medline] [Order article via Infotrieve]
28. Devor M, Barrett-Connor E, Renvall M, Feigal D, Ramsdell J. Estrogen replacement therapy and the risk of venous thrombosis. Am J Med. 1992;92:275–282.[Medline] [Order article via Infotrieve]
29. Thrombotic Risk Factors (THRIFT) Consensus Group. Risk of and prophylaxis for venous thromboembolism in hospital patients. BMJ. 1992;305:567–574.
30. Dores GM, Miller ME, Thorpe SL. Platelet adhesion at low shear rate: study of a normal population. Thromb Res. 1993;69:173–184.[Medline] [Order article via Infotrieve]
31. Saleh AA, Dorey LG, Dombrowski MP, Ginsburg KA, Hirokawa S, Kowalczk C, Hirata J, Bottoms S, Cotton DB, Mammen EF. Thrombosis and hormone replacement therapy in postmenopausal women. Am J Obstet Gynecol. 1993;169:1554–1557.[Medline] [Order article via Infotrieve]
32. Fox J, George J, Newton JR, Parsons AD, Stuart GK, Stuart J, Sturdee DW. Effect of transdermal oestradiol on the haemostatic balance of menopausal women. Maturitas. 1993;18:55–64.[Medline] [Order article via Infotrieve]
33.
Nichols TC, Bellinger DA, Johnson TA, Lamb MA, Griggs
TR. Von Willebrand's disease prevents occlusive thrombosis in stenosed
and injured porcine coronary arteries. Circ Res. 1986;59:15–26.
34.
Williams JK, Bellinger DA, Nichols TC, Griggs TR, Bumol
TF, Fouts RL, Clarkson TB. Occlusive arterial thrombosis in
cynomolgus monkeys with varying plasma concentrations of
lipoprotein(a). Arterioscler Thromb. 1993;13:548–554.
35.
Kockx MM, De Meyer GRY, Andries LJ, Bult H, Jacob WA,
Herman AG. The endothelium during cuff-induced
neointima formation in the rabbit carotid artery.
Arterioscler Thromb. 1993;13:1874–1884.
36.
Budde T, Fechtrup C, Bosenberg E, Vielhauer C, Enbergs
A, Schulte H, Assmann G, Breithardt G. Plasma Lp(a) levels correlate
with number, severity, and length-extension of coronary lesions
in male patients undergoing coronary arteriography for
clinically suspected coronary atherosclerosis.
Arterioscler Thromb. 1994;14:1730–1736.
37. Bar J, Tepper R, Fuchs J, Pardo Y, Goldberger S, Ovadia J. The effect of estrogen replacement therapy on platelet aggregation and adenosine triphosphate release in postmenopausal women. Obstet Gynecol. 1993;81:261–264.[Medline] [Order article via Infotrieve]
38. Roberts W, Curry R, Isner J. Sudden death in Prinzmetal's angina with coronary spasm documented by arteriography: analysis of three necropsy cases. Am J Cardiol. 1982;50:203–210.[Medline] [Order article via Infotrieve]
39. Davies M. A macro and micro view of coronary vascular insult in ischemic heart disease. Circulation. 1990;82(suppl II):II-38-II-46.
40.
Davies M, Thomas A. Plaque fissuring: the cause of
acute myocardial infarction, sudden ischemic death and
crescendo angina. Br Heart J. 1985;53:363–373.
41.
Williams JK, Adams MR, Klopfenstein HS. Estrogen
modulates responses of atherosclerotic coronary arteries.
Circulation. 1990;81:1680–1687.
42.
Mahoney CJ. A study of the menstrual cycle in
Macaca irus with special reference to the detection of
ovulation. J Reprod Fertil. 1970;21:153–163.
43. MacDonald GJ. Reproductive patterns of three species of macaques. Fertil Steril. 1971;22:373–377.[Medline] [Order article via Infotrieve]
44.
Jewett DA, Dukelow WR. Serial observations of
follicular morphology near ovulation in Macaca fascicularis.
J Reprod Fertil. 1972;31:287–290.
45. Hamm TE, Adams MR, Kaplan JR, Clarkson TB, Bullock BC. Effects of gender and social behavior on the development of coronary artery atherosclerosis in cynomolgus macaques. Atherosclerosis. 1983;48:221–233.[Medline] [Order article via Infotrieve]
46. Clarkson TB, Shively CA, Morgan TM, Koritnik DR, Adams MR, Kaplin JR. Oral contraceptives and coronary artery atherosclerosis of cynomolgus monkeys. Obstet Gynecol. 1990;75:217–222.[Medline] [Order article via Infotrieve]
47.
Adams MR, Kaplan JR, Manuck SB, Koritnik DR, Parks JS,
Wolfe MS, Clarkson TB. Inhibition of coronary artery
atherosclerosis by 17-beta estradiol in ovariectomized
monkeys: lack of an effect of added progesterone.
Arteriosclerosis. 1990;10:1051–1057.
48. Nichols TC, Bellinger DA, Reddick RL, Read MS, Koch GG, Brinkhous KM, Griggs TR. Role of von Willebrand factor in arterial thrombosis: studies in normal and von Willebrand disease pigs. Circulation. 1991;83(suppl IV):IV-56-IV-64.
49.
Folts JD, Gallagher K, Rowe GG. Blood flow reductions
in stenosed canine coronary arteries: vasospasm or platelet
aggregation? Circulation. 1982;65:248–255.
50.
Coller BS, Folts JD, Scudder LE, Smith SR.
Antithrombotic effect of a monoclonal antibody to the platelet
glycoprotein IIb/IIIa receptor in an experimental animal
model. Blood. 1986;68:783–786.
This article has been cited by other articles:
 |
|
 |
|
  V. L. Ballard and J. M. Edelberg Harnessing Hormonal Signaling for Cardioprotection Sci. Aging Knowl. Environ., December 21, 2005; 2005(51): re6 - re6. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Hultgren, P. Olofsson, and E. Wahlberg Reproductive History in Women with Lower Limb Ischemia Angiology, July 1, 2004; 55(4): 373 - 383. [Abstract] [PDF]
|
|
|
|
 |
|
 C. K. Derian, B. P. Damiano, M. F. Addo, A. L. Darrow, M. R. D'Andrea, M. Nedelman, H.-C. Zhang, B. E. Maryanoff, and P. Andrade-Gordon Blockade of the Thrombin Receptor Protease-Activated Receptor-1 with a Small-Molecule Antagonist Prevents Thrombus Formation and Vascular Occlusion in Nonhuman Primates J. Pharmacol. Exp. Ther., February 1, 2003; 304(2): 855 - 861. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Zitzmann, R. Junker, A. Kamischke, and E. Nieschlag Contraceptive Steroids Influence the Hemostatic Activation State in Healthy Men J Androl, July 1, 2002; 23(4): 503 - 511. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Brighouse Hormone replacement therapy (HRT) and anaesthesia Br. J. Anaesth., May 1, 2001; 86(5): 709 - 716. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. M. C. Rosano, C. M. Webb, S. Chierchia, G. Luigi Morgani, M. Gabraele, P. M. Sarrel, D. de Ziegler, and P. Collins Natural progesterone, but not medroxyprogesterone acetate, enhances the beneficial effect of estrogen on exercise-induced myocardial ischemia in postmenopausal women J. Am. Coll. Cardiol., December 1, 2000; 36(7): 2154 - 2159. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||