INTRODUCTION
Stroke
is the third leading cause of death and an important cause of disability
worldwide1. Thrombosis and atherosclerosis are major contributors to
the development of ischemic stroke. Platelets play a critical role in
triggering arterial thrombosis and in promoting atherogenesis.2
Furthermore,
formation of platelet-leukocyte aggregates and leukocyte activation contribute
to vascular repair and microcirculatory disturbances in ischemic tissue.
Previously, in vitro, studies have demonstrated interaction of activated
platelets with monocytes and neutrophils.3 Binding of platelets via
P-selectin expressed on the surface of activated platelets to the leukocyte to
the leukocyte counter receptor P-selectin GP ligand-1 may alter leukocyte
recruitment and activation patterns4.
Platelet activation
is a crucial mechanism in arterial thrombogenesis and therefore in
the pathophysiology of ischemic stroke.5-8 Accordingly, antiplatelet
therapy plays a central role in secondary prevention of ischemic
stroke. Currently, decisions for antiplatelet drugs after stroke are
based on clinical grounds but not on platelet activation studies in
individual patients because of a lack of clinically applicable
methods to monitor platelet function5-8.
Two articles
recently showed that patients with acute cerebral ischemia have an
excess of circulating platelets that express P-selectin (CD62p) and
lysosome-associated membrane protein (CD63)9,10.
P-selectin is a
constituent of the membrane of
-granules and serves
as an adhesion receptor mediating the binding of platelets to
leukocytes. CD63 is part of platelet lysosomes and may protect the
plasma membrane from degradation by lysosomal enzymes. Both CD62P
and CD63 are expressed exclusively on platelet activation11,12.
The time course of
these platelet activation markers after stroke has not yet been
assessed. Whether their elevation during the subacute phase after
stroke could predict recurrent ischemic events and whether they may
be helpful in monitoring antiplatelet therapy are of interest9.
The aim of this
study was to assess the degree of platelet activation in human cerebral
ischemic stroke during acute stage and 90 days later. Furthermore, the current
study also tested the correlation between platelet activation markers and
clinical presentation of the studied patients.
SUBJECT AND
METHODS
This study was
undertaken on forty five patients (group I), recruited from the stroke unit of
the Neurology Department of Tanta University Hospital within 24 hours after
ischemic stroke and their clinical deficit lasted for at least 24 hours. Twenty
healthy age and sex matched controls (Group II) and twenty risk factor control
subjects (Group III) who were matched with patient in terms of age, sex,
hypertension, diabetes mellitus, smoking and hyperlipidaemia were enrolled in
the study.
Exclusion criteria
for all subjects were trauma, surgery, or acute organ ischemia
within the preceding 3 months (e.g. before the stroke); severe liver disease; renal failure;
cancer; chronic inflammatory diseases; and fever or acute
inflammatory or infectious conditions at the study entry.
Patients with an
infection after stroke or with a history of infection shortly before stroke
were excluded to avoid including the occurrence of platelet activation
attributable to parallel infection. However,
we systematically assessed symptoms of infection and body
temperature during the whole study period to recognize a possible
influence of infection on the variables. Also, we excluded patients suffered
from a new cerebral or cardiac ischemic event during the 90 days of
follow-up.
For healthy control
subjects, any vascular diseases or risk factors were additional exclusion
criteria. Subjects with a history of myocardial infarction or stroke were
excluded from the risk factor control group.
Vascular risk
factors were defined as follows: arterial hypertension, on antihypertensive
treatment or blood pressure
160/90 mm Hg at 2 readings
before stroke or >5 days after stroke; diabetes mellitus, on antidiabetic
treatment or elevated haemoglobin A1c or elevated blood glucose at 2 readings
before stroke or >5 days after stroke; hyperlipidemia, on lipid-lowering
medication or total cholesterol >220 mg/dL or triglycerides >180 mg/dL
before stroke; and carotid stenosis, diameter reduction of
50% of the internal
carotid artery on Duplex sonography13.
All stroke patients
underwent a cranial CT excluding cerebral haemorrhage.
The origin of
cerebral ischemia was classified according to the Trial of Org 10172 in Acute
Stroke Treatment (TOAST) criteria.14
Stroke severity was assessed with the National Institutes of Health Stroke
Scale (NIHSS)15
on days 1, 14, and 90 after ischemia.
In stroke patients,
blood samples were taken within 24 hours and on days 14, and 90 after ischemia.
Measurements were performed once in both control groups. Serum and citrated
plasma samples were collected at each venipunctue and were stored at -80oC
until measurements. Blood was obtained under minimal tourniquet pressure from
the antecubital vein using a sterile 22-gauge needle and syringe in a single
attempt. The first 3 mL blood was used to separate serum, and the following 3
mL was drawn into a Vacutainer tubes containing 3.8% buffered sodium citrate. Mixtures of blood and sodium citrate were
centrifuged for 15 min at 1.500 revolutions per minute at room temperature. The
supernatant platelet-rich plasma was used to assess the platelet activity. The
time between blood collection and antibody labelling was standardized to within
1 h.
Platelet activity
was determined with respect to alpha granule degranulation (surface expression
of P selectin or CD62p antigen). A Fluorescein isothiocyanate conjugated
(FITC)–labeled antibodies to glycoprotein IIIa (CD61; Becton Dickinson) was
used as an activation-independent marker of platelets. Phycoethrin (PE)–labeled
antibodies (Immunotech) against CD62 or CD63 were used to assess platelet
activation. The percentage of platelets expressing CD62p or CD63 was defined as
the fraction exhibiting specific binding (i.e., CD62p or CD63) minus
that exhibiting nonspecific binding (i.e. the percentage defined with
the IgG-PE conjugate) from among the 10,000 platelets sorted.16,17
Statistical Analysis
The data are
presented as means and percentiles. Student-T test was used to compare
different groups and to test significance. AOVA test also was used.
P<0.05 was considered significant.
RESULTS
Forty five cerebral ischemic stroke patients (group I), twenty healthy
controls (group II) and twenty risk controls (group III) were included in this study. The mean age of
group I was 60.3 (SD±13.6) years , group
II was 59.4(SD, ±14.2) years and
61.5 years (SD, ±13.2) for group III
.There was no significant difference between the three groups regarding age
(P=0.51) and sex p=0.41) (Table 1).
The distribution of
hypertension, diabetes mellitus, smoking, or cardiac disease did not vary among
group I and group III subjects and the difference was non-significant (Table
2). There were no risk factors at all among group II.
The percentage of
platelets expressing CD62p was significantly higher in group I on day one after ischemia (mean,
3.1%) than that in both group II (mean, 1.4% ) and group III (mean 1.6%;) subjects (P<0.001). The
expression of CD62p on platelets was decreased over time after stroke. The
difference between the three groups was no longer significant on days 14 and 90
after stroke (Figure 1).
CD63 expression on
platelets was significantly higher on day one among group I patients (mean,
3.1%) compared with group II and III subjects (mean1.5% and 1.4% respectively)
(P<0.001). The percentage of platelets expressing CD63 decrease on the 14 day and 90 day after stroke in group I patients but
still higher than in group II and III. There was non significant difference
when compared with group II and III subjects (Figure 1).
The most frequent
etiology of ischemic stroke among patients
was large artery atherosclerosis 17 patients (37.7%), then 15 (33.3%)
were due to undetermined aetiology, 8 (11.1%) from small artery disease and 5
patients (11.1%) had cardioembolic source of their ischemic stroke (Table 3).
The mean of National Institutes of Health Stroke Scale
(NIHSS) score for the group I
patients at admission was 6 (1 to 27),
after 14 days was 3 (0 to 5 ) and after
90 days was 1 (0-18).
CD63 expression on
day one was influenced by the stroke pathophysiological mechanism where
cardioembolic stroke patients had a statistically significant higher difference
[(median 3.25% ) with p value 0.012 ] compared to other stroke mechanisms. On
the other hand, CD62p expression was not influenced by the stroke
pathophysiology (Table 3)
Stroke severity
was not correlated with CD62P and CD63 expression at any time point (Table 4).
Vascular risk
factors did not influence the results for CD62p and CD63 among group I and
group III subjects (Table 5).
On
day 90 after stroke, CD62p and CD63 expression did not differ
between patients treated with aspirin, clopidogrel, or anticoagulants
(P>0.05 for all) (Table 6).
DISCUSSION
There is a
well-recognized need for clinically applicable methods to monitor
platelet function in the treatment and prevention of ischemic
stroke. Platelet activation in the acute and chronic stages after
stroke has been demonstrated with methods such as aggregometry,
assessment of plasma levels of platelet release products, and
urinary excretion of thromboxane metabolites. None of these methods
proved to be a useful tool in daily clinical practice partly because
of insufficient reproducibility of results and partly because of the
expense of time and equipment required18.
The
main methodical problem is artifactual in vitro activation of
platelets during blood sampling and processing. Furthermore, measurements
of release products may have a low sensitivity because of dilution
effects in plasma. Flow cytometric analysis of platelet function has
the advantage that it can use fixation procedures that block
metabolic processes in cells within milliseconds but leave
activation-dependent neoantigens on the cell surface preserved.
Therefore, the possibility of artifactual cell activation is reduced
to the venipuncture in this technique. We found good reproducibility
of the assessment of both platelet activation markers with a low
intratest variability and a low to moderate intertest variability in
healthy subjects. Therefore, an important prerequisite for clinical
application is fulfilled by this method19.
In accordance with
previous reports11,13, the current study found a significant
increase in CD62p and CD63 expression within 24 hours after cerebral
ischemia.
The differential
expression pattern of CD62p and CD63 over time is an interesting
finding that may be explained as follows. A recent report has shown
that increased expression of CD62p or CD63 after stroke was not
associated with an increased proportion of circulating platelets
with shape change.9 The shape change is a very early and
reversible event during platelet activation and a very sensitive
marker of instantaneous platelet activation. This indicates that
platelets after stroke are not particularly activated during their
passage in peripheral venous blood. Thus, CD62p and CD63 are memory
markers of platelet activation, and the increased expression of
secretion-dependent antigens after stroke indicates that platelets
had previously undergone activation. In a baboon model, degranulated
platelets rapidly lost surface CD62p but continued to circulate and
function.20 A similar mechanism may be operative in
humans, and the degranulated platelets may not lose CD63, which
could explain the decline in CD62p expression parallel to
continuously elevated CD63 expression after acute stroke. Platelets
possess a lifespan of about 7 to 10 days. Therefore, increased CD63
expression in subacute stroke indicates continuously ongoing
platelet activation. Altogether, in the subacute stage after
ischemic stroke, CD63 is a more sensitive marker of platelet
activation than CD62p, most likely because of shedding of CD62p.13
This study
revealed that vascular risk factors and clinical stroke severity did not
significantly modify CD62p and CD63 expression, and stroke patients
had persistently higher CD63 expression than subjects with vascular
risk factors. The simultaneous presence of multiple risk factors was
not associated with higher platelet activation. This indicates that
platelet activation markers may confer relevant information
independently from and in addition to clinical risk factor
assessment.
Regarding stroke pathophysiology, the findings of the current study did
not support the previous observation that atherothrombotic stroke is
associated with a particularly high platelet activation.21
However, our study was not designed to investigate the influence of
different stroke origins on platelet activation, and results have to
be viewed with caution because of the small numbers of subjects in
subgroups. Moreover, we did not find a significant influence of
medication for secondary prevention on platelet activation markers.
The fact that medication with platelet inhibitors was not associated
with lower antigen expression by platelets is in line with previous
results showing that aspirin does not modify the excretion of a-granules.22,23
Several studies have shown that fibrinogen, C-reactive protein (CRP),
and leukocyte count are increased after ischemic stroke.
Furthermore, all 3 parameters were shown to be independently
associated with the risk of first-ever and recurrent vascular events24,25,
although the specificity of the link with vascular events was questioned
recently.(26) The role of inflammatory parameters, mainly CRP, as
an important predictive index for recurrent ischemic events after
stroke was recently established.24,25 An interesting question is
whether the expression of platelet neoantigens, mainly of CD63, may
provide additional information on future vascular events after
stroke and thus may guide therapeutic decisions. This question needs
to be answered in larger prospective studies.
[Disclosure: Authors
report no conflict of interest]
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