American Journal of Hematology 80:257–261 (2005)
Effect of Menopause on Platelet Activation Markers
Determined by Flow Cytometry
Tariq M. Roshan,1* Jamalludin Normah,1 Asia Rehman,2 and Lin Naing2
1
Department of Hematology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia.
2
School of Dental Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia
Pre-menopausal women have a lower risk of cardiovascular disease compared to postmenopausal women. Cardiovascular disease is more age dependent in women than in
men. The association of platelet activation and cardiovascular thrombotic events is well
established. Standardized techniques were used to evaluate platelet activation markers
by flow cytometry, using 3-color analysis (CD 61PerCP, CD 62P, and PAC-1) in 49 postmenopausal (mean ± SD age, 56.16 ± 33.51 years) and 42 pre-menopausal (age, 39.38 ±
7.07 years) women. Results of our study showed a significant increase in CD 62P in postmenopausal women as compared to the pre-menopausal group (2.66 ± 4.26% vs. 0.52 ±
2.71%, P < 0.001). Similarly, PAC-1 was significantly increased in post-menopausal
women (21.54 ± 2.48% vs. 3.70 ± 2.31%, P < 0.001). Furthermore, there was a significant
association of CD 62P with serum estradiol in both groups. PAC-1 was significantly
associated with age in both groups. The results suggest the role of platelets in the
increased incidence of thrombotic events and disease in post-menopausal women. Am.
J. Hematol. 80:257–261, 2005. ª 2005 Wiley-Liss, Inc.
Key words: platelet; platelet activation; flow cytometry; post-menopausal women
INTRODUCTION
Platelets play a central role in the formation of
thrombus and hence in cardiovascular events secondary to this complication. Cardiovascular events are a
major cause of mortality and morbidity in industrialized nations, and they are emerging as a major contributory factor to overall morbidity and mortality in
developing countries. In many instances of cardiovascular events occurring because of the aforementioned
factors, thrombogenesis is the important, single, most
common underlying pathological process. While
females are generally protected from cardiovascular
diseases before menopause, this population is at
higher risk of such events after menopause compared
to their male counterparts [1]. Platelet hyperactivity
and or circulating activated platelets have been
reported to be associated with many common clinical
conditions, including unstable angina [2], acute myocardial infarction [3], and stroke [4], as well as with
cigarette smoking [5]. Estrogen has an effect on
endothelial production of such vasoactive factors as
nitric oxide [6] and prostacyclin [7]. Nitric oxide and
prostacyclin, being potent vasorelaxants, also inhibit
ª 2005 Wiley-Liss, Inc.
platelet aggregation and adhesion. These mechanisms
are dependent on cyclic 3,5-guanosine monophosphate (cGMP) and cyclic adenosine monophosphate
(cAMP) [8,9]. cAMP and cGMP are antagonistic in
some tissues, including monocytes, but in platelets
they are synergistic. Increased intraplatelet levels
result in an increased calcium uptake into the dense
tubular system, which inhibits phosphatidyl inositol
metabolism. Thromboxane, which is largely a product
of activated platelets, causes platelet aggregation and
vasoconstriction and has been used as an index of
Contract grant sponsor: IRPA; Contract grant number: 304/
PPSP/6131213
*Correspondence to: Dr. Tariq M Roshan, Department of
Hematology, School of Medical Sciences, Universiti Sains
Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
E-mail: roshan@kb.usm.my
Received for publication 12 September 2004; Accepted 18 April 2005
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI: 10.1002/ajh.20472
258
Roshan et al.
platelet activation; its increased levels have been
shown after menopause [10]. There is also a positive
correlation found between thromboxane and the number of years after menopause [11]. Thromboxane levels
were also shown to be reduced after 1 year of hormone
replacement therapy, which indicates reduction in platelet activity [12]. Platelets undergo a dramatic shape
change with activation that precedes the release of
granules. When stimulated, alpha granules in platelets
coalesce in the center [13,14] and the membranes of
these granules fuse with the plasma membrane—
‘‘granule docking’’— and expose the contents [15]. PSelectin, a transmembrane protein, is incorporated in
the plasma membrane of platelets by this mechanism
and serves as a receptor for GP Ib [15]. P-Selectin
mediates interaction between the endothelium, platelets, and leukocytes [16]. With platelet activation,
there is a conformation change in GP IIb/IIIa
(aIIbb3), and these become neo-epitopes that can
bind to monoclonal antibody (PAC-1). PAC-1 is
directed against the fibrinogen-binding site exposed
by the conformational change in GP IIb/IIIa complex
of activated platelets [17]. The importance of GP IIb/
IIIa is undeniable, and these GPs have been a target in
cases of acute coronary syndrome [18]. However, little
work has been done on determining these glycoprotein
levels in health and disease [16].
By the use of monoclonal antibodies whole-blood
flow cytometry, it is possible to determine the activation state and or reactivity of circulating platelets.
Whole-blood flow cytometry has a number of advantages, including minimal manipulation of samples,
platelets being studied in a more physiological milieu,
thrombocytopenia not affecting the results, multiple
surface receptors being detected, and both reactivity
and activation being assessed with or without added
agonist. As there is beneficial effect of intraplatelet
levels of cGMP and endothelin-1 (ET-1) by estrogen,
loss of this effect after menopause should be seen on
platelet activation. No such study has been yet been
reported to observe platelet activation markers in
post-menopausal women.
MATERIALS AND METHODS
Study Design
A comparative cross-sectional study was designed
and approved by the Research and Ethics Committee,
University Sains Malaysia.
informed consent. Post-menopause was defined as
amenorrhea for the past 2 years [19]. Special care
was taken while recruiting subjects, and diagnosed
cases of diabetes mellitus, hypertension, hyperlipidemias, chronic medical illness, menstruation on the
day of study for pre-menopausal women, and previous thromboembolic phenomena were excluded
from the study. Also excluded from the study were
those volunteers who were taking aspirin, nonsteroidal anti-inflammatory drugs, and/or any other drugs
including antiplatelets 15 days prior to study day.
Volunteers who were taking oral contraceptive pills
and those who were taking any kind of hormonal
preparation, including hormonal replacement therapy, were not included in the study as effects of
hormone on intraplatelet cGMP, cAMP, and ET-1
have been shown [20]. Volunteers were called early in
the morning (8–10 AM), in batches. Volunteers were
also advised not to exercise strenuously before the
study as strenuous exercise may activate the platelets
[21].
Blood Sampling
Blood was taken from the volunteers in a highly
standardized manner. Volunteers were allowed to rest
for 10 min to attain basal body conditions upon
reporting to the laboratory. A butterfly cannula
(TOPÒ 21G3/40 ) was inserted into the antecubital
vein, with or without the use of tourniquet pressure
[22]. A three-way cannula was attached to the butterfly cannula to separate the first 8 mL and subsequent
2 mL of blood. No cuff or tourniquet was used while
drawing the blood. Sodium citrate, 3.8%, was used as
an anticoagulant for platelet activation studies.
Preparation and Staining of Platelets
for Activation Studies
Staining of the blood samples were done within 10
min of sample collection. A saturating concentration
of monoclonal antibodies (20 mL) was added to the
test sample, and negative controls were added with
prepared IgG1 and IgM. Fluorescein isothiocyanate
(FITC)-conjugated monoclonal antibodies to GP IIb/
IIIa (conformation dependent; PAC-1), Phycoerythrin (PE)-conjugated anti-CD 62P and Peridininchlorophyll (PerCP)-conjugated anti-CD61 were
from Becton Dickinson (San Jose, CA).
Analysis and Data Acquisition
Subject Recruitment
A total of 91 healthy volunteers, including 49 postmenopausal and 42 pre-menopausal women, were
recruited for this study after providing written
Three-color flow-cytometry was done, and positive
events for CD61 PerCP were acquired for platelets. Ten
thousand activation-independent events were acquired
for each sample. Dot plots and quadrant statistics were
Effect of Menopause on Platelet Activation Markers
used to calculate the percentage of positive activation
markers.
Statistical Analysis
For analysis in this current study, we used SPSS for
windows, version 11.0. Independent samples t-tests
were used to compare means of BMI, cholesterol,
CD 62P, and PAC-1 between the two groups.
ANCOVA was used to compare CD 62P and PAC1 between pre- and post-menopausal women by
adjusting for BMI and cholesterol. P values < 0.05
were regarded as significant. Multiple linear regression was done to determine the association of platelet
activation markers with age, cholesterol, BMI, and
serum estradiol levels.
259
ple linear regression for activation markers showed
only age to be the main contributor for PAC-1, but
for CD 62P, serum estradiol was the major contributor. The results are shown in Tables III and IV.
DISCUSSION
To contribute to hemostasis, platelets need to be
activated. The assessment of inappropriate platelet
activity in vivo is one way to risk-stratify those who
are at risk of arterial thrombosis. Until recently, platelet aggregometry was regarded as the gold standard
for platelet function but with the availability of sensitive techniques like flow cytometry, reliable results
can be obtained. Some studies have been done to
look into the plasma measures of hemostasis and
factors which may predict the future onset of atheroRESULTS
thrombotic events [23], few of these studies have
Age of the healthy volunteers who responded for shown no correlation between platelet aggregation
this study was post-menopausal 56.16 ± 33.51 years and platelet count with the incidence of ischemic
and pre-menopausal 39.38 ± 7.07 years. Descriptive heart disease [24]. Activated platelet states have been
statistics of the study population are shown in Table I reported in different thrombotic events [2–4]. Gender
for both pre- and post-menopausal women, respec- difference in the development of coronary heart distively. The results showed significant difference (P < ease and its outcome are partly regulated by estrogen
0.001) of CD 62P, PAC-1, and cholesterol between and its receptors, and this protective effect of estrogen
the two groups while BMI was not significantly dif- on thrombogenesis is lost after menopause [25]. With
ferent (P ¼ 0.775) as seen in Table I. ANCOVA the loss of beneficial effect of estrogen, there is also a
results showed that CD 62P and PAC-1 were signifi- decrease in nitric oxide and PGI2 and an increase in
cantly different (P ¼ 0.025 and P < 0.001, respec- thromboxane apart from post-menopausal rise in
tively) between the two study populations after fibrinogen and plasminogen activator inhibitor. All
adjusting for BMI and cholesterol (Table II). Multi- of these events might result in platelet activation.
TABLE I. Comparison of Two Study Groups (42 Pre- and 49 Postmenopausal Women)*
Variable
Premenopausal group
mean (SD)
Age (year)
BMI (kg/m2)
Cholesterol (mmol/L)
Estradiol (pmol/L)
CD 62P (% positive cells)
PAC-1 (% positive cells)
39.38
25.09
5.53
381.15
0.52
3.70
(7.07)
(4.2)
(0.85)
(181.89)
(2.71)
(2.31)
Postmenopausal group
mean (SD)
56.16
25.35
6.41
36.38
2.66
21.54
(33.51)
(4.37)
(1.26)
(13.36)
(4.26)
(2.48)
a
t Stat. (df)
P value
(89)
(89)
(78)
(89)
(85)
(89)
<0.001
0.775
<0.001
<0.001
<0.001
<0.001
12.44
0.28
3.81
13.28
6.28
9.48
*Abbreviations: SD, standard deviation; BMI, body mass index.
a
Independent t-test.
TABLE II. Comparison of Markers Between 42 Pre- and 49 Post-menopausal women
Markers
CD62 (% positive)
PACI (% positive)
a
Premenopausal
Adj. meana (95% CI)
Postmenopausal
Adj. meana (95% CI)
F Stat.b (df)
P value
0.69 (0.41, 1.15)
4.39 (3.03, 6.35)
1.84 (1.13, 3.00)
17.11 (12.06, 24.53)
5.21 (1, 82)
19.05 (1, 82)
0.025
<0.001
Adjusted variable: BMI and cholesterol.
Analysis of covariance (ANCOVA).
b
b
260
Roshan et al.
TABLE III. Factors Associated With PAC-1 in the Whole Study Population
SLRa
Variable
Age (year)
Cholesterol (mmol/L)
BMI (kg/m2)
Serum estradiol (pmol/L)
bc
1.07
1.41
1.01
1.03
(95% CI)
(1.04,
(1.50,
(0.93,
(0.95,
1.09)
1.75)
1.04)
0.99)
MLRb
P value
<0.001
0.001
0.580
0.004
Adj. bd
1.00
1.06
1.02
0.80
(95% CI)
(1.12,
(0.86,
(0.96,
(0.95,
1.10)
1.30)
1.08)
1.01)
t-Stat.
2.92
0.65
0.70
1.72
P value
0.006
0.520
0.490
0.090
a
Simple linear regression.
Multiple linear regression (R2 ¼ 0.24); the model reasonably fits well, model assumptions are met; and there is no interaction between
independent variables and no multicollinearity problem.
c
Crude regression coefficient.
d
Adjusted regression coefficient.
b
TABLE IV. Factors Associated With CD 62P in the Whole Study Population
SLRa
Variable
Age (year)
Cholesterol (mmol/L)
BMI (kg/m2)
Serum estradiol (pmol/L)
bc
1.07
1.53
1.04
1.05
(95% CI)
(1.04,
(1.19,
(0.88,
(0.91,
1.09)
1.97)
1.02)
0.98)
MLRb
P value
Adj. bd
<0.001
0.001
0.210
0.001
1.05
1.20
1.00
1.06
(95% CI)
(0.98,
(0.87,
(0.87,
(0.91,
1.13)
1.63)
1.09)
0.97)
t-Stat.
P value
1.56
1.22
0.06
3.3
0.130
0.230
0.960
0.002
a
Simple linear regression.
Multiple linear regression (R2 ¼ 0.27); the model reasonably fits well, model assumptions are met, and there is no interaction between
independent variables and no multicollinearity problem.
c
Crude regression coefficient.
d
Adjusted regression coefficient.
b
The present study looked directly into the effect on
platelet activation after menopause and is the extension of the above observation. When we studied our
volunteers, there was no clinical evidence of cardiovascular and thrombotic disease; however, increased platelet activation markers were noted in postmenopausal women. As shown in Table I, there was
a significant difference in serum estradiol levels of both
groups, and this might have result in platelet activation
in the post-menopausal group. However, all postmenopausal women were healthy, and this observation
explains that it is not only platelet activation leads to
thrombotic complications; are other confounding factors or events might also increase the incidence of
thrombosis in post-menopausal women.
A simple linear regression model of the study population showed significant association of both activation markers (PAC-1 and CD 62P) with cholesterol
(P ¼ 0.001) as shown in Tables III and IV. Due to the
effect of cholesterol on platelet activation and the
significant difference between the two study populations, multiple linear regression was done, after which
cholesterol lost its significant association with PAC-1
and CD 62P (P ¼ 0.50 and P ¼ 0.23, respectively). A
significant effect of the serum estradiol level on PAC-
1 and CD 62P was observed, as shown by SLR in
Tables III and IV; however, the multiple linear regression model showed that age remained the most significant factor for PAC-1, and serum estradiol levels
remained the most significant contributor for CD 62P
in both groups. It remains unexplained, however, why
serum estradiol was not a significant contributor to
PAC-1 in the adjusted model in our population.
In our study, we have also shown that CD 62P
levels were lower as compared to PAC-1 in both
pre- and post-menopausal groups. This can be
explained by the stability of the CD markers. Activation-dependent increases in platelet surface P-selectin
is not reversible over time in vitro [26]. This proves
that, at least in our study, platelets were activated in
vitro. However, this is not the case in vivo, where
platelets lose their surface P-selectin rapidly [27,28].
Due to this fact, a number of authors cannot recommended these markers as the ideal markers for circulating degranulated platelets [29] unless platelets are
continuously activated or they are drawn within 5
min of activation.
Although the reader must draw their own conclusions, we believe that, although there is significant
difference in platelet activation markers in pre- and
Effect of Menopause on Platelet Activation Markers
post-menopausal women, platelet activation cannot
be taken as sole marker for increased thrombotic
risk in post-menopausal women. Taking our results
as ‘‘pre-event’’ data, we recommend a follow-up study
to show the relative risk of thrombosis in the cases
that show higher platelet activation markers.
14.
15.
16.
ACKNOWLEDGMENTS
The authors thank to IRPA for the research grant
under which this study was performed.
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