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EVALUATION OF PLATELET ACTIVATION BY FLOW CYTOMETRY, IN
PREECLAMPSIA
FREITAS, Letcia GonalvesMARTINS-FILHO, Olindo Assis
CARVALHO, Maria das Graas
SATHLER-AVELAR, Renato
DUSSE, Luci Maria SantAna
ABSTRACT
Preeclampsia (PE) is a serious complication of pregnancy associated with high
morbidity and mortality, maternal-fetal, whose etiology has not been
established. In pure form, it is characterized by the appearance, in normal
pregnant women after 20 weeks of pregnancy, hypertension and proteinuria.
The hemostatic and inflammatory changes associated with normal pregnancy
are more exacerbated in PE. Studies suggest that pronounced platelet
activation and aggregation contribute to the hypercoagulable state in this
disease. The aim of this study was to evaluate the expression of markers of
platelet activation in PE. Were evaluated 97 women, 35 of which were pregnant
women with PE (15 with severe-PEG and 20 mild-PEL), 31 normotensive
pregnant women (GN) and 31 nonpregnant women (CNG). Were evaluated the
mean fluorescence intensity (MFI) of biomarkers CD41a, CD61, CD42a, CD62P
and the ratio between them as well as the percentage of platelets CD62P +. The
IMF of CD62P and the percentage of CD62P+ platelet, in the three groups did
not differ (p = 0.67 and p = 0.38, respectively). After subdividing the group of
pregnant women with PE was not obtained difference (p = 0.69 for the IMF
CD62P and p = 0.54 for CD62P+ platelet). The MFI of CD41a, in pregnant
women with PE was lower than in CNG (p = 0.004) and GN compared to CNG
(p = 0.007). The MFI of CD61 on PEG group was lower than the CNG (p =
0.008). The MFI of CD41a and CD61 were lower in the PE group than in the
CNG, and the GN compared to CNG (p = 0.00 for both). Both were lower in
PEG and PEL compared to CNG (p = 0.00 for both). The MFI of CD42a, even
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after the division of the PE group did not differ (p = 0.44 and p = 0.57,
respectively). The ratios between the IMF of CD61/CD42a and CD41a/CD62P
were lower in NG than in the CNG (p = 0.002 and p = 0.007, respectively) and
in the group of PE and PEG compared to CNG (p = 0.001 for both). The reason
CD42a/CD41a MFI was greater in PE and PEG compared to CNG (p = 0.001
for both) and in GN compared to CNG (p = 0.002). The ratios of the MFI
CD61/CD41a, CD61/CD62P and CD42a/CD62P before (p = 0.29, p = .07 and p
= .73, respectively) and after (p = 0.42, p = 0,14, p = 0.69, respectively)
subdividing the PE group did not differ. Pregnancy is accompanied by changes
in expression of CD41a and CD61 on the platelet surface, and reduced platelet
counts.
Keywords: Preeclampsia, CD41a, CD61, CD42a, CD62P.
INTRODUCTION
Primary hemostasis and platelets
Platelets are cytoplasmic fragments of megakaryocytes. These are present
in the bone marrow and their maturation and differentiation depends on
thrombopoietin and interleukin (IL) IL-3, IL-6 and IL-11. The cytoplasmic
fragmentation of megakaryocytes occurs in the bone marrow and platelets are
released into the circulation, isolated or grouped (HARTWIG et al. 1995). For a
long time the platelets were considered mere onlookers on hemostasis (JURK &
KEHREL, 2005). Currently, it is clear that they play an essential role in this
process and have other important functions such as the maintenance of
vascular tone through the secretion of various substances including serotonin,thromboxane (TXA 2) and prostaglandin (HARTWIG et al. 1995).Thus, platelets
act as contributors and modulators of various disorders, including coronary
artery disease, deep vein thrombosis, myeloproliferative disorders, atrial
fibrillation, cancer, cerebrovascular accident (CVA), infection with human
immunodeficiency virus (HIV), kidney disease, diabetes, inflammatory bowel
disease and multiple sclerosis (HARTWING & ITALIAN-JR, 1995; GURNEY et
al., 2002; FARIAS & Bo, 2008; SHEREMATA et al., 2008; GILS Van et al.,2009; HESS & GRANT, 2011 ). The role of platelets in the hemostatic process
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includes their recruitment from the bloodstream to the subendothelial matrix
occurs when vascular damage and release of tissue factor (TF). When
activated, platelets quickly express glycoproteins (GP) surface that interact with
other platelet, vascular endothelial and inflammatory cells (Hartwig et al. 1995;
Lowenberg et al., 2010). A succession of events including contact, adhesion,
activation, secretion and aggregation culminates in the formation of platelet
plug, which temporarily prevents blood loss (JURK & KEHREL, 2005).
Subsequently, platelets also contribute to secondary hemostasis by activating
the coagulation cascade occurs in which the fibrin clot formation, plugging more
efficiently the injured site.
Platelet activation
Platelet activation occurs primarily through two mechanisms: release of
soluble substances (proteases, metabolites of arachidonic acid, peroxides,
platelet-activating factor and cathepsin G in pathological conditions) and by
direct contact with inflammatory cells. The activation results in an increase in
expression of adhesion molecules surface starting GPIIb/IIIa on platelets and
leukocytes CD11b/CD18. These leukocyte GP establish connections with
platelets (PANASLUK et al. 2005). P-selectin and GPIIb/IIIa are biomarkers of
platelet activation most commonly used in research. Besides these, the
lysosomal protein (CD63), glycoproteins p24 (CD9), GPIIa (CD29), GPIV
(CD36), GPIb (CD42b), GPIb (CD42c), GPIa (CD49b), GPIC (CD49f) and
adhesion molecule platelet-endothelial cell type 1 (PECAM-1 or CD31) have
been studied (Lazarus et al. 1995).
P-selectin (CD62P)
The P-selectin mediates platelet adherence of activated platelets to
neutrophils and monocytes. Monoclonal antibodies directed against these GP
only bind to platelets degranuladas and not those in the inactive state
(Michelson, 1996; Michelson et al. 2000). P-selectin is synthesized by
endothelial cells and megakaryocytes and then stored in platelet granules andWeibel-Palade bodies of endothelial cells (Gurney et al. 2002; YANG et al.
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2,009; Lowenberg et al. 2,010). When exposed on the platelet surface, P-
selectin average interactions between the endothelium, platelets and
leukocytes, and initially stabilizes the interaction GPIIb/IIIa-fibrinogen to the
platelet plug formation (Gurney et al. 2002; Theoret et al. 2006). However, this
biomarker is unstable and releases from the platelet surface rapidly, while still
functioning in plasma. The soluble P-selectin (sCD62P) is known to induce a
procoagulant state in monocytes and is considered a potent activator of T cells,
endothelium, platelets, essential for stabilizing an arterial thrombus (Jurk &
KEHREL, 2005).
Increased plasma levels of this biomarker has been observed in
thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, diabetes,
cancer, systemic inflammation, atherosclerosis, acute coronary syndrome, and
other cardiovascular events in smokers (GURNEY et al., 2002; Lowenberg et al.
, 2010). It is assumed that P-selectin presents important role as a connective
factor between inflammation and thrombosis. Moreover, their plasma levels are
elevated a few hours after the occurrence of venous thrombosis and remain
high for many months (Masopust et al., 2011).
Glycoprotein GPIIIa (CD61)
The GPIIIa is a transmembrane GP, 3 integrin expressed on platelets,
megakaryocytes, osteoclasts and endothelium. This GP is associated with
GPIIb to form the GPIIb/IIIa complex, which mediates platelet aggregation and
CD51 to form the CD51/CD61 complex, a vitronectin receptor. In addition, it
binds to fibrinogen, fibronectin, thrombospondin and von Willebrand factor
(vWF) to mediate the process of accession (di MINNO et al., 2009).
Glycoprotein IIb/IIIa (CD41a)
The GPIIb/IIIa was first described in the 1980s triggered by events in
platelet activation. This GP is activated after a signal initiated with intra-platelet
complex formation FvW-GPIb/IX/V and is able to bind to fibrinogen, fibronectin,
vWF, vitronectin and thrombospondin (Michelson, 1996; GURNEY et al. 2002;Delgado et al. 2003). The increased plasma levels of these GP was useful to
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evaluate the pro-thrombotic activity in various clinical conditions, including
coronary angioplasty, sickle cell disease in remission and during painful crisis
and hypertension caused by pregnancy, especially in preeclampsia-PE (Tomer,
2004). Clinically, platelet aggregation inhibitors that target GPIIb/IIIa are
effective to reduce thrombotic disorders during coronary intervention (Freedman
& LOSCALZO, 2002, Theoret et al. 2006).
GPIX glycoprotein (CD42a)
The GPIX form the complex GPIb/IX/V receptor for vWF, essential in the
process of platelet adhesion to subendothelial vascular when a injury occurs. In
contrast to antibodies that generally have higher affinity for platelet receptorswhen platelets are activated, CD42 has a higher affinity for platelets in an
inactive state. This probably occurs due to complex translocation to the
membrane connected to the channel system (Michelson, 1996).
A new theory of coagulation and the role of tissue factor and platelet
surface
The coagulation theory, proposed in the 1960s by MaFCarlane, Davieand Ratnoff, was revised and a new model was proposed by Hoffman & Monroe
in 2007. The model consists of three stages: initiation, amplification and
propagation, coordinated by FT (expressed in tissue cells, monocytes and
activated cells endotelials) and platelet surface. Then, when a blood vessel is
injured, a large number of platelets are recruited to the site in order to provide
sufficient area for a burst of thrombin generation and fibrin clot formation
effective in buffering the injury and ensuring hemostasis. The platelet surface is
specialized to coordinate the connection of the tenase and prothrombinase
complex (FXa, FVa).
Evaluation of platelets by flow cytometry
Flow cytometry (FC) using monoclonal antibodies is presently the most
sensitive technique to detect the increased expression of surface molecules on
activated platelets (Harlow et al. 2002). The research involving platelet
evaluation using whole blood, washed platelets and platelet rich plasma (PRP).
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However, a major limitation of these studies is to activate in vitrothat may occur
during sample processing (Michelson, 1996).
The evaluation of platelet by FC presents broad clinical applicability,
including assessing hyperreactivity and/or activation of circulating platelets;
evaluate platelet hyporeactivity in neonates with intraventricular hemorrhage;
assess MPP in several clinical conditions, evaluate the platelet-monocyte
aggregate as sensitive markers of platelet activation in acute coronary
syndromes; predict risk of the occurrence of acute and subacute ischemic
events after angioplasty; assess platelet function in diseases such as acute
coronary syndrome, stroke and angioplasty; diagnose the disease pool stock;
determine the rate of thrombopoiesis; diagnose idiopathic thrombocytopenia
and purpura posttransfusion; diagnose diseases due to deficiency of GP platelet
surface; assess reticulated platelets; determine the flow of Ca2+ platelet;
diagnose neonatal thrombocytopenia alloimmune; evaluate alloimmunization in
patients multitransfused (about 15% are due to platelet antibodies); monitor
pharmacotherapy with antagonists of GPIIb/IIIa, heparin-induced
thrombocytopenia diagnose and monitor the quality of platelet concentrates
stored in blood banks (Lazarus et al. 1995; Michelson, 1996; Michelson et al. ,
2000; BROWN & WITTWER, 2000; CASTRO & Gourley, 2010).
Platelet changes during pregnancy
During normal pregnancy, occur significant hemostatic and inflammatory
changes in women, which favor a hypercoagulable state that seeks to protect
them from a potential postpartum hemorrhage (Robb et al., 2010). Thus, there
is an increase in the concentration of various procoagulant and reduction ofnatural anticoagulants (Perry & MARTIN, 1992).
The platelet count during pregnancy may remain unchanged (Ahmed et
al. 1993), due to a lower dilution effect (SEJENY et al. 1975, CUNNINGHAM &
Pritchard 1978; SILL et al. 1985) or increase (MOR et al. 1960). It is known that
during pregnancy the blood volume increases in the first quarter and at 34
weeks, when it reaches the maximum value, about 40 to 50% higher than in
nonpregnant women (CUNNINGHAM & PRITCHARD, 1978). This increase is
caused by the expansion of plasma volume, probably due to alterations in the
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renin-angiotensin-aldosterone system and eicosanoids, due to increased
concentrations of progesterone and estrogen (Mabie & Sibai, 1991). These
amendments are intended to enhance the ability of the kidneys to reabsorb
water and sodium (REMUZZI & RUGGENENTI, 1991). The hypervolemia
protects the mother and fetus from the harmful effects of the reduction in
venous return and cardiac output in the upright position during pregnancy and
blood loss during and after childbirth (CUNNINGHAM & PRITCHARD, 1978).
Studies suggest that occur during gestation platelet activation and
consumption, although its useful life does not change (Ahmed et al. 1993;
JANES & GOODALL, 1994; JANES et al. 1995). Pregnancy also provides
significant changes in the plasma membrane of platelets, such as increased
enzyme activity that catalyzes the hydrolysis of adenosine triphosphate
(ATPase), the decrease in membrane fluidity and the concentration of
cholesterol (RABINI et al. 1995).
A platelet hyperactivation, mediated by Ca2+ elevation is observed from
the 16th week of pregnancy and normalizes after six weeks of delivery.
Moreover, the susceptibility of platelets to the secretion of adenosine
triphosphate (ATP) and granules containing PF-4 and -thromboglobulin, is
progressive, suggesting an increase in turnover and activation of the platelets
(Morrison et al. 1985; Holthe et al. 2004).
Platelet changes in preeclampsia
Preeclampsia (PE), in pure form, is characterized by the appearance, in
normal pregnant women, hypertension and proteinuria after 20 weeks of
gestation. The PE is a serious complication of pregnancy and it is oftenassociated with considerable morbidity and mortality maternal-fetal (ACOG,
2002). According to the World Health Organization (2005), this disease is the
most common complication of pregnancy, occurring in 3-5% of pregnant women
worldwide. In developing countries, this percentage rises to 10%, due to
attendance often inadequate or absent, resulting in more than 60,000 maternal
deaths per year.
These disease has no cure, except for the removal of the placenta andmay develop into more serious conditions such as eclampsia, in which we
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observe a neurological disorder, manifested by seizures and coma may also
occur stroke and bleeding (Gibson et al. 1982), HELLP syndrome (haemolysis,
elevated liver enzyme activity, low platelet); hemostatic serious complications
(disseminated intravascular coagulation-DIC) and renal (Sibai et al., 1993a).
The ability of blood oxygenation in areas of slow flow in pregnant patients with
PE is reduced to 50%, contributing to prematurity, low fetal weight and
increased neonatal mortality (SIEKMANN & Heilmann, 1989).
Despite numerous studies, the etiology of PE remains unclear and there
is no specific therapy for the disease. The clinical management is restricted to
the administration of antihypertensive drugs and the prevention of eclampsia
and the anticonvulsant therapy with magnesium sulfate and administration of
corticosteroids to accelerate fetal lung maturation (Grill et al. 2009). Ongoing
studies evaluating a new drug that would act in the angiogenic balance by
altering placental receptors, especially the growth factor receptor vascular
endothelial 121 (VEGFR-121). It is recognized that this therapy contribute to
moderate manifestations of PE, while that prolong the gestation until full
development of the fetus (Li et al. 2007).
A better understanding of the role of platelets in the pathogenesis of PE
would allow other therapeutic possibilities, including the use of antiplatelets
(Kazmi et al., 2011). Platelets in patients with PE can release an excess amount
of TXA2, exacerbating the disease. The administration of low-dose aspirin was
tested in pre-eclamptic pregnancy, to reduce the biosynthesis of TXA2 without
compromising prostaglandin production, assuming the prevention of
vasoconstriction and the hemostatic problems in PE (Gibson et al., 1982).
Sibai et al. (1993b) conducted a large study of 3135 normotensive
pregnant women to see if treatment with aspirin was effective in reducing theincidence of PE and maternal-fetal morbidity. Observed a significant reduction
of disease incidence, and this occurred in 69 (4.6%) of patients who received a
daily dose of 60 mg of aspirin and 94 (6.3%) of those who received placebo.
There was no difference in the occurrence of complications in newborns in both
groups, although there was an increased risk of abrupt placenta.
A multicenter study involving 9364 pregnant women evaluated the
efficacy of aspirin, administered under similar conditions to the previous study.The authors concluded that this therapy is not associated with the incidence of
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intrauterine growth restriction or neonatal death. However, due to the risk of
maternal perinatal bleeding, this therapy is only justified in pregnant women at
high risk of early manifestation of the disease (CLASP, 1994).
Later, Sibai (1998) investigated alternatives to reduce the incidence and
severity of PE from the assessment made earlier randomized studies using
magnesium salts and zinc, dietary supplementation with fish oil and Ca 2+ and
low-dose aspirin. Concluded that these alternatives do not confer benefits or
they are minimal, which was later confirmed (NATIONAL HIGH BLOOD
PRESSURE EDUCATION PROGRAM WORKING GROUP ON HIGH BLOOD
PRESSURE IN PREGNANCY, 2000; ACOG, 2002; Sibai et al., 2005).
The platelet counts normally decreases in PE, but only about 18% of
patients develop thrombocytopenia (Perry & MARTIN, 1992; Ahmed et al. 1993;
JANES et al. 1995). Some mechanisms have been proposed to explain the
thrombocytopenia, as thrombin generation in the presence of circulating
immune and vascular injury, platelet aggregation and destruction mediated
immune mechanisms (PERRY & Martin, 1992).
Literature data show that hypertensive disorders in pregnancy occur with
platelet activation, increased levels of fibrinogen and adhesion molecules
derived from vascular endothelium (MACEY et al., 2010). Platelet activation
occurs in a conformational change of GPIIb/IIIa, located on the platelet surface,
favoring binding to fibrinogen, platelet aggregation and release the contents of
their granules. The degranulation contributes to placental and systemic vascular
changes, especially the release of vasoactive substances, mitogenic and
mioproliferativas such as TXA2, growth factors and platelet-derived
transformation and -thromboglobulin (JANES et al., 1995).
Although admittedly that the PE take place with inflammatory andcoagulation, platelet activation accompanied by the studies of literature are
controversial, with some showing an increase of markers of activation, while
others do not (Holthe et al., 2005; ACAR et al . Lok et al. 2,007; Robb et al.,
2010).
Changes in maternal platelet PE also can affect the newborn. A study in
pregnant women with PE and healthy pregnant women and their newborns,
showed an increased expression of CD62P, CD63, CD41, CD9 and CD36 inhealthy pregnant women and their newborns after in vitro activation of platelets
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with thrombin. Newborns of women with PE, however, showed lower expression
of CD62P, CD63, CD9 and CD36. Furthermore, platelets were less responsive
to activation in vitro. These findings suggest that PE influences the expression
of platelet surface GP in pregnant women and their newborns, which may have
altered platelet function, contributing to an additional risk of bleeding in
thrombocytopenic neonates (Huhne et al., 1996).
It is important to recall the difficulty of diagnosing PE, established based
on blood pressure and proteinuria. Currently, no laboratory marker to submit a
favorable cost-effectiveness ratio was proposed for the diagnosis of this
disease. Understanding the role of platelets in the pathophysiology of PE which
results in the need for early termination of pregnancy, in most cases,
endangering the life of the mother and/or baby, is certainly of great value.
AIMS
The aim of this study was to evaluate the expression of platelet
membrane glycoproteins (P-selectin, GPIIb/IIIa, GPIIIa and GPIX) by flow
cytometry in samples from women with preeclampsia, and normotensive
nonpregnant women.
Material and methods
This research was previously approved by the Ethics Committee of the
Federal University of Minas Gerais, by the Ethics in Research of Santa Casa of
Belo Horizonte, the Research Center of Maternity Odete Valadares/Research
Ethics Committee of the Hospital Foundation the State of Minas Gerais, BeloHorizonte, the Ethics Committee in Research of Basic Health Unit Family
Guanabara/Betim and the Ethics Committee of the Hospital Municipal Odilon
Behrens, Belo Horizonte. The clarification of the objectives of the research was
done by researchers to all women in the study and signed a consent form was
obtained in all cases. A clinical record containing important data for analysis of
the results was completed in all cases.
Casuistry
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In this study, the assessment of platelet surface markers included 85
women, 35 women (41.18%) with PE, of which 15 (17.65%) had the severe
form-PEG of the disease and 20 (23.53%) to mild form-PEL, 25 (29.41%) and
25 normotensive pregnant women-GN (29.41%) non-pregnant women-CNG.
The inclusion criteria for PEG group were:
a. Systolic/diastolic blood pressure greater than or equal to 160 x 110
mmHg, measured in two steps with an interval of at least 2 hours and after rest;
b. Proteinuria greater than 2g in 24-hour urine or larger than (+2) by semi-
quantitative strips method in unic urine sample;
c. Being in the third trimester of pregnancy.
The presence of some of the clinical symptoms and clinical and laboratory
findings were also considered by the obstetric team to define the diagnosis of
severe PE as:
a. Epigastric pain in the upper abdomen;
b. Vision changes;
c. Exacerbation of deep tendon reflexes, and checked two reflexes (patellar
and upper limbs);
d. Headache;
e. Behavioral changes;
f. Dyspnea and signs of pulmonary congestion;
g. Urine volume of less than 500mL or 100mL in 24 hours in 4 hours (2
steps);
h. Thrombocytopenia (platelet count
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The inclusion criteria for GN group were:
a. Systolic / diastolic blood pressure at or below 120 x 80 mmHg and no
history of hypertension and PE;
b. No proteinuria;
c. Being in the third trimester of pregnancy.
The inclusion criteria for CNG group were:
a. Systolic/diastolic blood pressure at or below 120 x 80 mmHg and no
history of hypertension and PE;
b. No proteinuria;
Exclusion criteria common to all three groups were:
a. Obesity;
b. Presence of intercurrent diseases such as coagulation disorders,
cardiovascular diseases, autoimmune diseases, liver diseases, renal and
infectious, diabetes, cancer and chronic hypertension;
c. Labor advanced;
d. Presence of bleeding of any kind.
The diagnosis of PE was made by the obstetric team of the. The group
members CNG and GN were selected at the Hospital Municipal Odilon Behrens
and UBSF Guanabara/Betim. The women were matched according to age and
gestational age and belonged to the same social class.
Biological samples
Were collected 3.5 ml of blood of all women participating in the study.
The blood was collected in sodium citrate 3.2%, using vacuum system and
disposable material. Blood samples were processed within two hours after
venipuncture and the reading done immediately.
For the group of women with PE, the result of the blood test was
obtained from medical records. For groups GN and CNG, in addition to 3.5 mLof citrated blood were collected 4 ml of blood in ethylenediaminetetraacetic acid
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(EDTA) to blood count. This was done using the electronic cell counter, Cell-
Dyn 3500R (Abbott Diagnostics).
Method
The study of the expression of markers of platelet activation was
performed according to the original protocol defined by Tomer (2004), Noronha
et al. (2007) and Assinger et al. (2011), brief: The sample of citrated whole
blood previously centrifuged at 800rpm for 10 minutes to obtain PRP. To fix
platelets, 400L aliquots of PRP were dripped into 1.600L of fixative solution
(paraformaldehyde 10g/L sodium cacodylate at 10.2 g/L, sodium chloride 6.63
g/L. The solution pH was adjusted to 7.2 to 7.4) under vortexing and placed in a
4 to 8 C overnight. The platelets were washed with phosphate buffered solution
(0.015 M PBS, pH = 7.2) and centrifuged for 10 minutes at 2.200rpm. The
supernatant was discarded. The pellet was resuspended gently with PBS and
platelet count was performed using the Cell-Dyn hematology counter 3500R
(Abbott Diagnostics).
The fixed platelet suspension was adjusted to 5x106 platelets/mL.
Aliquots of 100mL of this suspension (500,000 platelets) were marked with
monoclonal antibodies (Pharmingen Becton Dickinson ) directed against
platelet GP as described in Table 1. The tubes contained an internal control for
self-fluorescence (background) in which the platelet suspension was incubated
in the absence of monoclonal antibodies. These samples were homogenised by
vortexing and then incubated at room temperature and under incubation for 30
minutes. After, the samples were washed with PBS, homogenized by vortexing,
and centrifuged for 10 minutes at 2.200rpm. The supernatant was discardedand the pellet was resuspended in 200L PBS.
Table 1Monoclonal antibodies labeled with fluorochromes used for analysis ofplatelet glycoproteins.
Sample Reagent/antibody Fluorocrom Volum ClonePhenotype
target
1 PBS - 5L - -
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2 Anti-CD41a PE 4L HIP8 GPIIb/IIIa
3 Anti-CD42a FITC 4L ALMA.16 GPIX
4 Anti-CD61 PE 3L VI-PL2 GPIIIa
5Anti-CD42a
Anti-CD62P
FITC
PE
4L
5L
ALMA.16
AK-4
GPIX
P-selectina
FITC: fluorescein isotiocianate; PE: ficoeritrin.
A total of 100,000 events were acquired per sample using the flow
cytometer FACScalibur (Becton Dickinson). The software CellQuest was
used for acquisition and data analysis.
The different strategies employed for analysis of platelet profile markers
are represented in Figure 1. The selective analysis of platelets was determined
by combination of fluorescence 1 (FL1) 2 or fluorescence (FL2) versusSSC.
After selecting the population of platelets were used one-dimensional
histograms of fluorescence to establish the mean fluorescence intensity (MFI)
for CD41a, CD42a, CD61 and CD61P. The percentage of CD62P+ platelet
population was defined from the combination anti-CD42a FITC versus anti-
CD62P PE. Results were expressed as percentage of platelet CD42a+/CD62P+,
corresponding to the upper right quadrant double-positive.
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Figure 2Strategies used for determining the mean fluorescence intensity of markers
of platelet activation and the percentage of platelets CD62P+. Figures obtained using the
Flow Jo software.
SSC/Granulosidade
FL2/Anti-CD41a PE
FL2/Anti-CD41a PE
FL2/Anti-CD61 PE
FL2/Anti-CD61 PE
FL1/Anti-CD42a FITC
FL1/Anti-CD42a FITC
FL1/Anti-CD42a FITCFL1/Anti-CD42a FITC
FL2
/Anti-CD62PPE
Nmerodeplaquetas
Plaquetas
Plaquetas
Plaquetas
Plaquetas
IMF=197,5
IMF=978,6
IMF=410,8
5,2%94,8%
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Defining the type of citrated blood sample
After completing the standardization of the technique to evaluate the
expression of markers of platelet activation and the establishment of strategy
analysis, were avalued the possible platelet activation in vitro at the time of
venipuncture. Thus, were assessed whether it would be necessary to dismiss a
small volume of blood (containing activators) before collecting the sample to be
used in the experiments.
Were collected two tubes of blood in sodium citrate (3.5 mL) of four
women, one pregnant woman with severe PE, a PE with mild and two
nonpregnant. The tubes were numbered as "1" and "2" in accordance with the
order of collecting and protocols defined been made to the two tubes in parallel.
The results showed no difference in the parameters evaluated (p> 0.05) in the
tubes "1" and "2". Thus, we chose to not despise a small volume of blood
before collection tube containing sodium citrate.
Statistical Analysis
The statistical analysis was performed using SPSS (version 13.0). Data
normality was tested by the Shapiro-Wilk method. To set up a blood sample
would be used in the first or second collection tube, were used the paired T-test
for normal data and Wilcoxon test for data that did not follow normality. For
continuous variables with normal distribution, comparison of means between
three or more groups was performed using ANOVA. When significant
differences were found in the ANOVA, were used the multiple comparison testtwo at Least Square Difference (LSD). The comparison of results between two
groups was made by Student's T-test. For continuous variables with non-normal
distribution, comparison of medians between three or more groups was
performed by Kruskal-Wallis. The comparison of results between two groups
was performed by Mann-Whitney test, with Bonferroni correction.
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RESULTS
The clinical characteristics of the three groups of participants in this study
are presented in Tables 2 and 3.
Table 2Clinical characteristics of the study participants.
PE (n=35) GN (n=31) CNG (n=31) p1 p2 p3 p4
Idade (anos)
IEP (meses)
29 7
93 69
26 6
36 41
25 6
-
0,02*
-
0,06
0,16
0,01*
-
0,35
-
IMC (Kg/m ) 28 6 25 5 24 4 0,01* 0,04* 0,00* 0,38
IG (sem.) 33 3 34 4 - - 0,17 - -
GPG (Kg) 12 8 12 8 - - 0,71 - -
PA sist.
(mmHg)
160 10 110 20 120 10 0,00* 0,01* 0,00* 0,00*
PA diast.
(mmHg)
100 10 70 20 80 10 0,00* 0,00* 0,00* 0,01*
IMC: ndice de massa corporal; IEP: intervalo entre partos; IG: idade gestacional; sem.: semanas; GPG:ganho de peso na gestao; PA: presso arterial; sist.: sistlica; diast.: diastlica; PE: grupo degestantes com PE; NG: grupo de gestantes normotensas; CNG: grupo de mulheres no gestantes.*p0,05. Os dados paramtricos so representados como mdiadesvio padro (ANOVA/LSD/Teste t-Student). Os dados no-paramtricos so apresentados como medianaintervalo interquartil (Kruskal-Wallis/Mann-Whitney/Correo de Bonferroni).p1: PE x GN x CNG; p2: PE x GN; p3: PE x CNG; p4: GN x CNG.
Clinical characteristics of group participants GN and PE were obtained
from medical records and antenatal card. Of the 67 women who participated in
the study, 56 (83.6%) made at least three visits for antenatal care. Data from
the CNG group participants were obtained during the interview.
The mean age of the members of the PE group was higher compared to
the CNG (p = 0.01) and the mean body mass index (BMI), as compared with
the GN (p = 0.04 ) and CNG (p = 0.00) group.
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There was no difference between the mean gestational age (GA) (p =
0.17) and the mean weight gain during pregnancy (WDP) (p = 0.71) and calving
interval (CI) (p = 0.16), compared to pregnant women with PE and GN.
The median values of systolic (p = 0.00) and diastolic blood pressure (p =
0.00) were different when comparing the three groups.
A similar analysis was performed after subdivision group severe PE
(PEG) and PE mild (PEL) as shown in Table 3.
Table 3Clinical characteristics of the study participants after subdivision of the PEgroup as severe and mild.
PEG (n=15) PEL (n=20) p p p p p p
Idade (anos)
IEP (meses)
31 6
127 66
28 8
54 52
0,02*
0,02*
0,16
0,02
0,02*
0,012**
0,00*
-
0,36
-
0,08
-
IMC (Kg/m2) 26 5 30 6 0,00* 0,03* 0,86 0,37 0,00* 0,00*
IG (sem.) 33 3 34 3 0,29 - - - - -
GPG (Kg) 14 5 11 10 0,64 - - - - -
PA sist.
(mmHg)
160 20 160 10 0,00* 0,01* 0,00* 0,00* 0,00* 0,00*
PA diast.
(mmHg)
110 20 100 11 0,00* 0,05* 0,00* 0,00* 0,00* 0,00*
IMC: ndice de massa corporal; IEP: intervalo entre partos; IG: idade gestacional; sem.: semanas; GPG: ganhode peso na gestao; PA: presso arterial; sist.: sistlica; diast.: diastlica. PEG: grupo de gestantes com PE
grave; PEL: grupo com PE leve; NG: grupo de gestantes normotensas; CNG: grupo de mulheres nogestantes.*p0,05. **p0,017 (Correo de Bonferroni). Os dados paramtricos so representados como mdiadesviopadro (ANOVA/LSD/Teste t-Student). Os dados no-paramtricos so apresentados como medianaintervalointerquartil (Kruskal-Wallis/Mann-Whitney/Correo de Bonferroni).p1: PEG x PEL x GN x CNG; p2: PEG x PEL; p3: PEG x GN; p4: PEG x CNG, p5: PEL x GN, p6: PEL x CNG.
The average age of the group members was superior in PEG than in GN
(p = 0.02) and CNG (p = 0.00) groups. The average BMI of women with severe
PE was lower than those with mild PE (p = 0.03), however, pregnant women
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with mild PE had lower mean BMI compared to the GN (p = 0.00) and CNG (p =
0.00).
There was no difference between the mean gestational age (p = 0.29)
and the mean GPG (p = 0.64), compared to pregnant women with severe or
mild PE and normotensive. The median IEP with severe group were higher than
in the GN group (p = 0.012). The median values of systolic (p = 0.00) and
diastolic (p = 0.00) were different when comparing the four groups.
Other clinical parameters were evaluated and tested for their association
with the occurrence of PE. Only the variable "number of pregnancies" was
significant (p = 0.00). Residual analysis is shown in Table 4.
Table 4Residue analysis for the variable "number of pregnancies" PEG groups, PEL,NG and CNG.
Grups
Gestacional number
0 1 2
PEG -1,7 -0,5 1,7
PEL -2,1* 1,1 0,4
GN -2,8* -1,0 2,9*
CNG 5,9* 0,4 -4,5*
*Localizao da diferena: Resduo 1,96 e Resduo -1,96.PEG: grupo de gestantes com PE grave; PEL: grupo degestantes com PE leve; NG: grupo de gestantesnormotensas; CNG: grupo de mulheres no gestantes.
We found greater number of nulliparous women in the group CNG.Regarding the occurrence of two or more pregnancies was higher in the group
NG and smaller in CNG group. No group stood out in the event of a pregnancy.
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Evaluation of platelet count and markers of platelet activation
In Table 5 are the results of platelet count and expression of biomarkers
of platelet activation obtained for pregnant women with PE (PEG and PEL), and
NG CNG.
Table 5Platelet count and platelet activation markers obtained for PE groups (PEGand PEL), GN and CNG.
Parameters PE (n=35) PEG (n=15) PEL (n=20) GN (n=25)#
CNG (n=25)#
N de plaquetas x
103/L
238,4
(81,7)
223,7
(94,9)
249,4
(70,9)
219,3
(53,6)
286,2
(67,0)
IMF CD41a 278,3
(178,0)
253,0
(182,5)
297,3
(176,8)
239,9
(239,5)
364,9
(426,1)
IMF CD61 903,9
(292,3)
855,7
(323,0)
940,0
(270,8)
888,0
(339,4)
1330,6
(441,1)
IMF CD42a 446,0
(155,6)
463,3
(170,0)
433,0
(147,1)
439,0
(152,3)
486,2
(108,9)
IMF CD62P 152,2
(99,4)
150,3
(55,9)
157,8
(110,6)
154,1
(126,2)
163,1
(118,3)
CD62P (%) 1,4
(2,1)
1,4
(2,0)
1,2
(2,1)
1,2
(3,9)
1,6
(4,0)
IMF: intensidade mdia de fluorescncia.#Para N de plaquetas x 103/L considerar n=31. Os dados paramtricos so representadoscomo mdia (desvio padro). Os dados no-paramtricos so apresentados como mediana(intervalo interquartil).
The Figures 3 and 4 illustrate the results for the platelet count and
expression of biomarkers of platelet activation in the PE, and GN CNG and after
subdivision of the group of women with PE in severe and mild.
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Figure 3 Platelet count, mean fluorescence intensity of CD41a, CD61, CD42a, CD62P
and percentage of CD62P+
platelets in the PE, GN and CNG.
A comparison of the mean platelet count showed a lower value in the
group of women with PE compared to CNG (p = 0.01) and in the GN group
compared to CNG (p = 0.00). There was no difference between the mean PE
and GN groups (p = 0.26).
Plaquetas (/L)
PE GN CNG0
200000
400000
600000*
*
CD41a (IMF)
PE GN CNG0
500
1000
1500
*
*
CD61 (IMF)
PE GN CNG0
1000
2000
3000
*
*
CD42a (IMF)
PE GN CNG0
200
400
600
800
1000
p=0,44
CD62P (IMF)
PE GN CNG0
200
400
600
800
p=0,67
CD62P (%)
PE GN CNG0
5
10
15
p=0,38
*p0,05 ou p0,017 (Correo de Bonferroni). Para os dados paramtricos foram realizados os testes ANOVA/LSD.Para os dados no-paramtricos foram realizados os testes Kruskal-Wallis/Mann-Whitney/Correo de Bonferroni.
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For the median MFI of CD41a and the average MFI of CD61 were
obtained in the group with lower values in PE compared to CNG (p = 0.00 for
both), and GN group compared to CNG (p = 0.01 and p = 0.00, respectively).
There was no difference between the groups GN and PE (p = 0.10 and p =
0.87, respectively).
A statistical comparison of the average MFI of CD42a and CD62P
medians of the IMF, as well as the percentage of CD62P+ platelets, the three
groups showed no significant difference (p = 0.44, p = 0.67 and p = 0.38 ,
respectively).
Plaquetas (/L)
PEG PEL GN CNG0
200000
400000
600000
* *
CD41a (IMF)
PEG PEL GN CNG0
500
1000
1500
*
*
CD61 (IMF)
PEG PEL GN CNG0
500
1000
1500
2000
2500
* **
CD42a (IMF)
PEG PEL GN CNG0
200
400
600
800
1000
p=0,57
CD62P (IMF)
PEG PEL GN CNG0
200
400
600
800
p=0,70
CD62P (%)
PEG PEL GN CNG0
20
40
60
80
p=0,54
*p0,05 ou p0,008 (Correo de Bonferroni). Para os dados paramtricos foram realizados os testesANOVA/LSD. Para os dados no-paramtricos foram realizados os testes Kruskal-Wallis/Mann-Whitne /Corre o de Bonferroni.
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Figure 4Platelet count, mean fluorescence intensity of CD41a, CD61, CD42a, CD62P
percentage of platelets and CD62P + for PEG, PEL, GN and CNG groups.
It was observed a reduction in mean platelet count in the PEG groupcompared to CNG (p = 0.01). There was no difference between the groups
means PEL and PEG (p = 0.28), PEG, PEL and GN (p = 0.84 and p = 0.13,
respectively) and between groups PEL and CNG (p = 0, 66).
For the median MFI of CD41a expression was observed in the lowest
PEG group compared to CNG (p = 0.00). The comparison between both
groups: PEG and PEL (p = 0.48), PEG and GN (p = 0.69), PEL and GN (p =
0.73), PEL and CNG (p = 0.10) no difference.The average MFI of CD61 was lower in the PEG and PEL groups
compared to CNG (p = 0.00 for both). The comparison between both groups:
PEG and PEL (p = 0.49), PEG and GN (p = 0.79), PEL and GN (p = 0.63) did
not differ.
The comparison of the average MFI of CD42a, the medians of CD62P
and the percentage of CD62P+ platelets in the four groups did not differ (p =
0.57, p = 0.70 and p = 0.54, respectively).The Table 6 shows the ratios between the IMF markers of platelet
surface obtained for pregnant women with PE (PEG and PEL), GN and CNG.
Table 7MFI ratios between the markers of platelet surface obtained for PE groups(PEG and PEL) GN and CNG.
Paameters PE (n=35) PEG (n=15) PEL (n=20) GN (n=25) CNG (n=25)
IMF CD61/CD41a 3,6(2,3)
4,2(12,1)
3,3(1,9)
3,1(4,2)
2,8(3,8)
IMF CD61/CD42a2,0
(0,9)1,8
(0,70)2,3
(0,7)1,9
(1,1)2,7
(0,7)
IMF CD42a/CD41a1,7
(2,0)1,8
(8,0)1,6
(1,0)1,7
(1,1)1,2
(1,0)
IMF CD61/CD62P5,2
(2,6)5,4
(2,5)5,4
(2,1)4,8
(2,5)7,6
(4,2)
IMF CD42a/CD62P2,6
(2,0)3,0
(1,6)2,5
(1,6)2,5
(1,3)2,8
(1,3)
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IMF CD41a/CD62P1,2
(1,5)1,6
(1,3)1,7
(1,2)1,4
(1,0)2,6
(1,6)
IMF: intensidade mdia de fluorescncia.Os dados so apresentados como mediana (intervalo interquartil).
Figures 5 and 6 illustrate the ratios of MFIs markers of platelet surface, in
the PE, GN and CNG and after subdivision of the group of women with PE
(PEG and PEL), respectively.
CD61/CD41a
PE GN CNG0
10
20
30
40
p=0,29
CD61/CD42a
PE GN CNG0
2
4
6
**
CD42a/CD41a
PE GN CNG0
5
10
15
20
*
*
CD61/CD62P
PE GN CNG0
5
10
15
20
p=0,07
CD42a/CD62P
PE GN CNG0
2
4
6
8
p=0,73
CD41a/CD62P
PE GN CNG0
2
4
6
8
*
*
*p0,05 ou p0,017 (Correo de Bonferroni). Foram realizados os testes Kruskal-Wallis/Mann-Whitney/Correode Bonferroni.
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Figure 5Ratio of the mean intensities of fluorescence CD61/CD41a, CD61/CD42a,
CD42a/CD41a, CD61/CD62P, CD42a/CD62P CD41a/CD62P and PE groups, GN and CNG.
The median ratio between the IMF the CD61/CD42a and CD41a/CD62P
were lower in the PE group compared to CNG (p = 0.00 and p = 0.015,
respectively) and GN compared to CNG (p = 0.00 and p = 0.01). The
comparison of medians between groups PE and GN showed no significant
difference (p = 0.57 and p = 0.56, respectively).
For the median ratio of MFI values obtained CD42a/CD41a were higher
in the PE group compared to CNG (p = 0.00) and in the GN group compared to
CNG (p = 0.00). The comparison of medians between groups PE and GN
showed no significant difference (p = 0.57).
The median ratios of the IMF CD61/CD41a, CD61/CD62P and
CD42a/CD62P, in the three groups showed no significant difference (p = 0.29, p
= 0.07 and p = 0.73, respectively ).
CD61/CD41a
PEG PEL GN CNG0
10
20
30
40
p=0,42
CD61/CD42a
PEG PEL GN CNG0
2
4
6
* *
CD42a/CD41a
PEG PEL GN CNG0
5
10
15
20
*
*
CD61/CD62P
PEG PEL GN CNG0
5
10
15
20
p=0,14
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Figure 6 Ratio of the mean intensities of fluorescence CD61/CD41a, CD61/CD42a,
CD42a/CD41a, CD61/CD62P, CD42a/CD62P CD41a/CD62P and PEG groups, PEL, GN and
CNG.
The median ratio of CD61/CD42a MFI was lower in the PEG group
compared to CNG (p = 0.00). The comparison between both groups: PEG and
PEL (p = 0.10), PEG and NG (p = 0.67), PEL and GN (p = 0.23), PEL and CNG
(p = 0.10) not significantly different.
However, for the median ratio of CD42a/CD41a MFI values were
obtained in the upper PEG group compared to CNG (p = 0.00). The comparisonbetween both groups: PEG and PEL (p = 0.10), PEG and GN (p = 0.67), PEL
and GN (p = 0.23), PEL and CNG (p = 0.10) not significantly different.
The median values of the ratio of the CD41a/CD62P MFI was higher in
the CNG group compared to GN (p = 0.01) as previously described. The
comparison between both groups: PEG and PEL (p = 0.67), PEG and GN (p =
0.94), PEG and CNG (p = 0.10), PEL and GN (p = 0.41), PEL and CNG (p =
0.06) showed no significant difference.The median ratios of the CD61/CD41a, CD61/CD62P and CD42a/CD62P
IMF in four groups showed no significant difference (p = 0.42, p = 0.14 and p =
0.69, respectively).
DISCUSSION
The PE is a complex disease whose etiology is not yet fully known. The
development of this disease is associated with an aberrant trophoblastic
CD42a/CD62P
PEG PEL GN CNG0
2
4
6
8
p=0,69
CD41a/CD62P
PEG PEL GN CNG0
2
4
6
8*
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invasion in early pregnancy, which may support the hypothesis that platelets are
activated at an early stage of pregnancy. There is evidence that platelet
activation precedes the onset of clinical symptoms (FALCONER et al., 1987;
VANTROYEN & VANSTRAELEN, 2002).
Although some help tests are part of the monitoring of pregnant women
with suspected PE, the diagnosis is mainly based on clinical data, blood
pressure measurement and determination of proteinuria (GRILL et al., 2009).
The blood pressure is known to be subject to postural changes and emotional.
Proteinuria is routinely detected by dipstick or measured quantitatively in urine
sample or isolated 24 hours, tests also have limitations. Thus, a major limitation
to studies involving the PE is the difficulty of diagnosis, which can lead to
erroneous conclusions and justify the variability of results found in literature.
The new understanding of the process of coagulation and platelet
surface highlights the FT as essential to trigger the cascade sequence of
reactions that culminates in the formation of fibrin clot. Coat et al. (1992) found
that the platelet membrane of pregnant women with PE presents an abnormal
lipid composition. It is known that platelet activation is associated with the
conformational change of GPIIb/IIIa, located on the platelet surface, favoring
binding to fibrinogen, platelet aggregation and release the contents of their
granules. Thus, platelet activation could contribute to placental and systemic
vascular changes observed in PE, especially the release of vasoactive
substances, mitogenic and mioproliferativas (JANES et al., 1995).
Some studies about platelet activation in patients with PE (in the
presence and absence of proteinuria) and normotensive and nonpregnant
women concluded that pregnant women with PE and proteinuria had evidence
of platelet activation and degranulation, increased platelets attached tofibrinogen, increased expression of CD63 (marker of platelet activation) and
plasma levels of-thromboglobulin. There was a moderate correlation between
the ratio of the fibrinogen-bound platelets and CD63 expression in all groups
(JANES & GOODALL, 1994; JANES et al. 1995).
Similar investigation conducted by Harlow et al. (2002) revealed that
there was increased expression of CD62P and CD63 in the group with PE.
These researchers proposed that platelet activation is an important factor in thepathophysiology of PE, but the platelet count is not able to predict the
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occurrence of the disease in all pregnant women. Bagamery et al. (2005) also
evaluated the levels of CD63, however concluded that there is greater platelet
activation in pregnant women with PE.
However, the in vitro platelet hyperactivity in PE is seemingly
contradictory, since aggregation is reduced compared to various agonists,
reflecting the depletion of platelets resulting from continuous activation. The
reduced secretion of ATP also confirms the hypothesis hyperstimulation
(Louden et al. 1991). If the platelet hyperfunction is cause or effect of PE still
requires further studies.
Lok et al. (2007) evaluated platelet activation from the release of platelet
microparticle (MPP) expressing P-selectin and the plasma concentration of
sCD62P. They found an increased concentration of sCD62P in PE, although it
did not differ from normotensive pregnant women, although the number of MPP
expressing P-selectin is higher in PE.
In further studies, Lok et al. (2008) investigated whether levels of MPP
are associated with the severity of PE. They observed that during normal
pregnancy the number of circulating initially reduces MPP, normalizing later.
The number of MPP derived from placenta samples increased gradually
throughout pregnancy in normal and PE decreased due to thrombocytopenia.
There was also an increased number of monocyte-derived microparticles in PE,
which could reflect the activation of these cells resulting from systemic
inflammatory state. Admitted that the release of inflammatory mediators from
activated platelets trigger inflammatory responses in endothelial cells and
monocytes.
Paradoxically, Robb et al. (2010) evaluated several markers of platelet
activation in PE and did not receive sharp expression of PECAM-1 (CD31),CD61, CD42a, CD62P and CD63. There was an increased expression of
sCD62P throughout normal pregnancy, as in PE and subsequent reduction in
the postpartum period. The CD31 marker was the best that was associated with
PE. They concluded that platelet activation is increased during pregnancy, but
is not altered in PE. However, it did not exclude the contribution of platelets to
the development of PE, assuming the increased activation in the maternal-fetal
interface.
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Holthe et al. (2004) determined the state of platelet activation (from the
markers CD61, CD42a, CD62P, and CD63 ligand activated fibrinogen receptor
type 1-PAC-1) in patients with PE, normotensive and nonpregnant women at
baseline and after activation in vitro. Observed that in normal pregnancy or PE
is greater platelet activation, and that the increased expression of CD63 is more
significant in PE.
In subsequent studies, Holthe et al. (2005) evaluated microparticles,
platelet-platelet aggregates and expression of P-selectin. Observed that in PE
(in the presence or absence of agonist) is smaller proportion of microparticles
than in normotensive pregnant women because they are more easily removed
from circulation. The microparticles and platelets in PE expressing P-selectin in
larger quantity, which determines a procoagulant state. Because P-selectin
mediates binding between platelets, leukocytes and endothelium, their
presence is associated with thrombogenicity (Holthe et al. 2005; LOK et al.
2007).
Acar et al. (2007) investigated the association between the PE and the
expression of soluble GPV, considered a biomarker of platelet activation. They
found no difference between women with PE and normotensive, although the
platelet count was lower in the group with PE. They proposed that there is likely
a subpopulation of pregnant women with PE in which platelet activation, if it
occurs, is a secondary event. The primary cause of hypercoagulability would
stimulate the occurrence of inflammation, endothelial dysfunction and
overactive leukocytes.
It is known that the PE preferably covers the ends of childbearing age
(Gibson et al. 1982; HAWFIELD & Freedman, 2009; ZHONG et al., 2010). The
analysis of the age of the members of the study (Table 3) revealed that theaverage age of the group of nonpregnant women was lower than that of the
group with PE (p = 0.01) and the group with severe PE (p = 0.00 ). The average
age of pregnant women with severe PE was higher compared to normotensive
pregnant women (p = 0.02) (Table 4). Knowing that platelet function does not
vary in the age of the study participants (minimum 19 and maximum: 36 years),
it can be inferred that the age difference observed in the study groups did not
affect the results.
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A comparison of PE pregnancy (PEG separately and PEL) and
normotensive pregnancy showed no difference between the mean gestational
age (p = 0.17 and p = 0.30, respectively). There was also no difference between
the median and normotensive pregnant women with PE compared to EPO (p =
0.16). However, the medians of EPO in severe PE group were higher than in
normotensive pregnant women (p = 0.012), which at first suggests a period
longer calving favors the occurrence of PE, being more evident in severe cases
disease (Young et al., 2010).
The average BMI of the patients with PE was higher than that of
normotensive pregnant women (p = 0.04), although included in the group of
"over-weight". Pregnant women with PE also had higher mean BMI for
nonpregnant women (p = 0.00), as was expected. The average BMI of women
with severe PE was lower than that of women with mild PE (p = 0.03).
Interestingly, women with mild PE had lower mean BMI compared to GN groups
(p = 0.00) and CNG (p = 0.00). There was no difference in the median of GPG
between the groups PE (p = 0.71), PEG and PEL (p = 0.64) compared to
normotensive pregnant women.
The presence of edema was observed, in varying degrees, in 16 patients
(48%) with PE, and +1 in 04 pregnant women (12%), +2 in 10 (30%), +3 in 01
(3%) and +4 01 (3%). However, no association was found between the intensity
of edema and severity of PE (p = 0.25). Edema was once considered a
parameter for diagnosis of PE. However, because it is a very common finding in
pregnancy, this is no longer a diagnostic criterion.
Platelet Markers
Care in the pre-analytical
The evaluation of in vitro platelet is complex, whereas the habitat of
platelets is the circulating blood. Once the blood is collected, platelets tend to
become activated and aggregate together. The ideal way to collect the blood
sample for evaluating platelets in vitro to avoid or minimize platelet activation, is
discussed. Van Ierssel et al. (2010) propose that the ideal is to disregard the
first few milliliters of blood leaving the vessel, in order to discard the tissue
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factor released by puncturing the tissues and the vessel wall with the needle
collection, which could induce platelet activation. Moreover, van Berrs et al.
(2009) prioritize shorter tourniquet, preventing platelet activation, microparticle
formation and hemolysis area dammed by garrote.
In the present study, after careful evaluation of interferences in the act of
collecting platelet activation, the choice was made to collect the pipe sodium
citrate (used for evaluating platelet in vitro) through accurate puncture, in which
the blood vessel is reached immediately and the tourniquet is released as soon
as the blood begins to flow. The tubes used had the inner walls coated with
silicone, to prevent platelet activation induced by negative charge on the glass.
The tubes were centrifuged at maximum two hours after collection at low speed
(800rpm) for 10 minutes in order to pellet the erythrocytes and leukocytes and
platelets remain in suspension in the plasma. Immediately after centrifugation,
PRP was removed and added to the fixative solution.
Platelet count
The change in the number of circulating platelets during pregnancy is
controversial. Ahmed et al. (1993) showed that the number of platelets is not
changed during pregnancy. However, other studies have shown a decrease in
circulating platelets due to a dilution effect (SEJENY et al. 1975,
CUNNINGHAM & Pritchard 1978; SILL et al. 1 985) and one showed an
increase of platelet counts (MOR et al., 1960).
In the present study, the comparison of the mean platelet count showed
a lower value in pregnant women with PE compared to the group of
nonpregnant women (p = 0.01). The comparison of the average number ofplatelets in the group of normotensive pregnant women compared to
nonpregnant women showed a reduction in platelet count in normotensive
pregnant women (p = 0.00). No difference was observed comparing the mean
platelet count of pregnant women with normotensive and PE (p = 0.26) (Figure
15). To subdivide the group of women with PE (severe and mild) also observed
a reduction in mean platelet count in the group with severe PE compared to
nonpregnant women (p = 0.01) (Figure 16).
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Several studies have shown a reduction in the number of circulating
platelets in pregnant women with PE compared with normotensive patients
(REDMAN et al., 1978; GILES & INGLIS, 1981; Neiger et al., 1992, Fitzgerald
et al., 1996; JREMO et al. 2000; Edelstam et al. 2001; Holthe et al. 2004;
Holthe et al. 2005; LOK et al. 2007). Redman et al. (1978) and Holthe et al.
(2004) admitted that the constant platelet activation in PE may result in an
increase in platelet consumption, which may exceed the capacity of production
of the bone marrow, resulting in reduced number of circulating platelets.
McCrae (2010) reported that a thrombocytopenia occurs in 50% of cases of PE,
however, Fitzgerald et al. (1996), admitted that thrombocytopenia is a less
common finding, occurring between 11 and 29% of pregnant women. Fallahian
& Nabaie (2005) found that the average number of platelets is significantly
reduced 3-6 weeks before delivery in pre-eclamptic pregnancy, but still within
the reference range. At delivery there is a significant reduction. Admitted even if
a mild or subclinical thrombocytopenia during the second half of pregnancy may
precede the PE, this parameter may be useful as a predictor of the disease.
However, Laskin et al. (2011) recognized that the platelet count is less sensitive
to aid the diagnosis of PE and should not be used in isolation, although it is a
simple, quick and inexpensive.
Platelet count less than 100 x 103/L is a sign of serious illness. If
delivery is not made, these levels are still decreasing and there is an increased
risk of maternal hemorrhage. It is unclear if the newborn may also develop
thrombocytopenia (NATIONAL HIGH BLOOD PRESSURE EDUCATION
PROGRAM WORKING GROUP ON HIGH BLOOD PRESSURE IN
PREGNANCY, 2000). Several mechanisms have been proposed to explain the
thrombocytopenia. Among them, the generation of thrombin in the presence ofcirculating immune and vascular injury; result of platelet aggregation, and the
resulting destruction mediated by immunological mechanisms (PERRY &
Martin, 1992).
In the present study, no reduction was obtained in the platelet count in
women with PE compared to normotensive. However, 07 patients (20%) with
PE, and 05 (33.3%) and severe PE 02 (10%) PE light, showed values below the
limit of the reference range (150 x 103
/L) whereas 04 (11.4%) had very low
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levels (99, 102, 112 and 118 x 103/L). Of these, two progressed to HELLP
syndrome.
In concordance with the results obtained in this study Janes & Goodall
(1994), Konijnenberg et al. (1997a), Makuyana et al. (2002) and Santos & Son
(2004) also found no difference in platelet counts between women with PE and
normotensive pregnant women.
McCrae (2010) reported that a possible explanation for the change in
platelet count in studies involving pregnant women with PE, normotensive
pregnant women and nonpregnant women would thrombocytopenia from other
causes such as hypersplenism, autoimmune diseases and several platelet
changes. However, in this study these variables were considered as exclusion
criteria and probably did not affect the evaluation of this parameter.
A comparison of the mean platelet count of the patients with mild and
severe forms of PE in this study showed no difference (p = 0.23). In agreement
with this result, Neiger et al. (1992) and Ceyhan et al. (2006) also reported that
the platelet count did not differ in these groups.
Contrary to the results of this study, stn et al. (2007), Canzoneri et al.
(2009) and McCrae (2010), found a reduction of platelet count in the group with
severe regarding shape and light in normotensive pregnant women, suggesting
a correlation between the platelet count and severity of PE. McCrae (2010)
admitted that the intensity of thrombocytopenia is directly associated with
disease severity.
Comparison of platelet count of pregnant women with severe and mild
compared to normotensive pregnant women showed no difference (p = 0.84
and p = 0.13, respectively). Similar results were obtained by Ceyhan et al.
(2006). However, stn et al. (2007) observed a reduction in the number ofplatelets in pregnant women with severe PE compared with normotensive
patients (p = 0.00). For the mild form of the disease, these researchers found
no difference and proposed that the change in the number of platelets in this
case could be subclinical.
Comparison of platelet count in normotensive pregnant and non-pregnant
women in the present study revealed that this was lower in normotensive
pregnant women, which is consistent with several studies (SEJENY et al., 1975;GIBSON et al., 1982 ; Holthe et al. 2004; Holthe et al. 2005; CEYHAN et al. and
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2006 McCrae, 2010). However, Giles & Inglis (1981) found no difference
between these groups.
A limitation of this study concerning the platelet count, that is, the group
with PE, was obtained in three different cell counters. Eleven women with PE
(31.4%) were selected in the maternity Odete Valadares, 18 (51.5%) in the
maternity Hilda Brando of Santa Casa de Belo Horizonte and 06 (17.1%) in the
Maternity Hospital Municipal Odilon Behrens. Diso addition, the platelet count
for the control groups (GN and CNG) was performed at another counter
hematological laboratory. Although the four clinical laboratories are embedded
in programs of quality control, we can not rule out differences in the accuracy of
platelet count between the automated counters used.
Markers expression of platelet activation
The importance of platelets in the pathophysiology of PE stems from the
fact that this disease is a deficiency in the production of PG and TXA2
biosynthesis excessive. There is also activation and aggregation of platelets
followed by a stimulation of the coagulation cascade resulting in the formation of
thrombi in placental vessels and vital organs maternal (CLASP, 1994;
NATIONAL HIGH BLOOD PRESSURE EDUCATION PROGRAM WORKING
GROUP ON HIGH BLOOD PRESSURE IN PREGNANCY , 2000; ACOG, 2002;
Sibai et al. 2005; YOUNG et al. 2,010; Kazmi et al. May 2011).
Markers of platelet surface
In the FC method, the data can be analyzed in two ways: on thepercentage of particles that express antigens on the surface, or by mean
fluorescence intensity (MFI) of the particle population that expresses this
antigen. The first strategy is useful when you know that the biomarker is
restricted to a subpulation and second, when dealing with constituent
biomarkers. The MFI values enable the distinction of which are at the extremes
of the range of linearity (Konijnenberg et al. 1997a, b).
The expression of surface markers results of the present study wasmade in MFI except for P-selectin, also expressed in percent. Only two studies
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evaluated the expression of P-selectin by MFI (STAR et al. Konijnenberg et al.
1997). The markers of platelet activation were evaluated to P-selectin (CD62P),
GPIIb/IIIa (CD41a), GPIIIa (CD61) and GPIX (CD42a).
P-selectin (CD62P)
A statistical comparison of the medians of the MFI and CD62P
percentage of CD62P+ platelets in the three groups of women evaluated in this
study showed no significant difference (p = 0.67 and p = 0.38, respectively)
(Figure 15). After subdividing the group of pregnant women with PE as severe
and mild neither difference was obtained comparing the four groups (p = 0.70
for the IMF CD62P and p = 0.54 for platelet CD62P+) (Figure 16).
Experiments in vitro suggest that the maximal expression of P-selectin
requires stimulation with agonists such as thrombin (JANES & GOODALL,
1994). It should be noted that in this study, the expression of P-selectin was
investigated without prior platelet activation in vitro. This decision was made
starting from the premise that platelets would already be activated in vivo in PE,
as suggested by the literature (CLASP, 1994; NATIONAL HIGH BLOOD
PRESSURE EDUCATION PROGRAM WORKING GROUP ON HIGH BLOOD
PRESSURE IN PREGNANCY, 2000; Yoneyama et al . 2001; ACOG, 2002;
Sibai et al. 2005; MACEY et al. and YOUNG et al. 2,010; Kazmi et al. May
2011).
Star et al. (1997) evaluated the expression of P-selectin in normotensive
pregnant and nonpregnant women, before and after activation of platelets with
thrombin and TXA2 analogues. In the absence of agonist, no difference was
observed between the two groups, as in the present study. In the presence ofagonist, platelet activation was lower in normotensive pregnant women
compared to nonpregnant women.
In agreement with the results obtained in this study, Gatti et al. (1994),
Harlow et al. (2002) and Macey et al. (2010) found no difference in the
expression of P-selectin between normotensive pregnant and nonpregnant
women. Harlow et al. (2002) found that, on average, 0.61 1.15% of platelets in
normotensive pregnant women are CD62P+
, unlike nonpregnant women (0.43
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1.13%), but this difference was not significant. These researchers found no
correlation between the intensity of platelet activation and severity of PE.
The results of this study are in agreement with those observed by Robb
et al. (2010). These researchers reported no difference in the percentage of
platelets expressing P-selectin among pregnant women with PE and
normotensive pregnant women. They concluded that platelet activation
increases during pregnancy, but is not affected by the disease.
Contrary to the results of this study, Goodall & Janes (1994),
Konijnenberg et al. (1997a), Harlow et al. (2002), Holthe et al. (2004) and
Macey et al. (2010) found an increase in platelet activation in patients with PE
compared to normotensive pregnant women and nonpregnant women.
Yoneyama et al. (2001) and Tomer (2004) also obtained conflicting results
when evaluating pregnant patients with PE and normotensive pregnant women.
Janes & Goodall (1994) evaluated the platelets before and after in vitro
stimulation with ADP and epinephrine, but the employee was the CD63 marker.
At baseline, these researchers observed a significantly reduced expression of
CD63 (about 0.25% of platelets) in nonpregnant women compared to
normotensive pregnant women (0.53%). They also reported that pregnant
women with PE had higher levels of this GP (0.65%) compared to
normotensive.
Konijnenberg et al. (1997a) found a higher percentage of platelets
expressing P-selectin in the group of pregnant women with PE (4.0%, 1.0 to
20.2) compared to normotensive pregnant women (0%, -1.0 to 5, 8). This
occurred in 04 patients (40%) normotensive and to a greater extent, 10 (100%)
in pre-eclamptic pregnancy. However, a critical analysis of this study reveals
that the results did not show uniform distribution in the two groups, with extremevalues. The expression of P-selectin showed great variation even among
healthy controls. Thus, one might question the definition of the percentage of
CD62P+ platelets considered indicative of the presence of increased platelet
activation in a given population. These researchers concluded that platelets are
more activated during pregnancy, and this activation is most evident in PE.
However, they admitted that only a subpopulation of platelets appears to be
activated. They admitted, though, that it is possible that a greater number ofplatelets are activated in PE, but these are rapidly cleared from the circulation.
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They also underlined that this subpopulation of platelets is activated (even
modestly) only if the GP membrane have been previously activated by thrombin
in vitro.
In parallel to the percentage Konijnenberg et al. (1997a), we investigated
the expression of P-selectin in MFI. Reported that the average MFI for P-
selectin was 10.3, ranging from 7.5 to 11.5, in normotensive pregnant women
and 11.3, ranging from 8.0 to 15.3, in patients with PE. There was great
variability in the data groups, but there was no significant difference between
them. They concluded that this parameter is limited to indicate the presence of
platelet activation in normal pregnancy and in PE. Finally, admitted that other
mechanisms, different platelet activation may also be involved in the
pathophysiology of PE. It should be noted that these researchers present the
results only after stimulation with agonists.
Harlow et al. (2002) compared the expression of P-selectin in
normotensive pregnant women and pre-eclamptic and concluded that this was
higher in pre-eclamptic (1.35 1.22% versus 0.61 1.15%, p = 0,02). However,
the increased expression of this marker was observed in 53% of pregnant
women with PE and 25% of normotensive pregnant women, which shows that
platelet activation is not exclusive and does not occur in all pregnant women
with PE. Janes et al. (1995) using CD63 obtained results similar to those
reported by Harlow et al. (2002). These researchers concluded that it is unlikely
that platelet activation is a constant event in PE and their in vitro evaluation as a
predictor of disease has limited value.
Macey et al. (2010) obtained a percentage of CD62P+ platelets higher in
women with PE (0.8 2.6%) compared to normotensive pregnant women (0.3
0.5%), suggesting that platelets are activated in this disease. However, theextent to which platelets are activated in normal pregnancy and in PE remains
uncertain.
Yoneyama et al. (2001) obtained an increase in the percentage of
CD62P+ platelets in pregnant women with PE (7.8 1.2%) compared to
normotensive pregnant women (4.7 0.7%), differing from the results obtained
in the present study.
Goodall & Janes (1994) and Holthe et al. (2004) reported that the basallevels of CD62P, after stimulation with ADP, are high in healthy pregnant
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women compared to nonpregnant women. Robb et al. (2011) also observed this
increase, even at baseline.
It should be noted that most studies in the literature using whole blood
samples with the justification that would easily activated platelets in the steps of
centrifugation, separation and attachment. Centrifugation also result in loss of
platelets, which would be even more significant for those of larger, more
activated (JANES et al. 1995). In this study, we chose to use the PRP fixed in
paraformaldehyde and sodium cacodylate to minimize possible interference of
erythrocytes in the analysis by FC. The possible activation during manipulation
of the sample was minimized by fast attachment of platelets after separation of
the PRP.
It should also be noted that P-selectin is unstable and releases from the
platelet surface rapidly, while the plasma remains in a soluble and functional but
not detectable by FC. It is known that platelets which have lost the P-selectin
may remain activated even in the circulation, although not express this more GP
(Michelson, 1996; Konijnenberg et al. 1997; Michelson et al. 2000).
GPIIb/IIIa (CD41a)
In the present study, values below the median MFI of CD41a, in pregnant
women with PE compared to nonpregnant women (p = 0.00) and in
normotensive pregnant women compared to nonpregnant women (p = 0.01) (
Figure 15). Making up the subdivision of the group of women with EP, we
observed a reduction in the median MFI of CD41a in pregnant women with
severe PE compared to nonpregnant women (p = 0.00). There was no
difference between the medians of the groups PE and normotensive pregnantwomen (p = 0.10), severe PE and mild PE (p = 0.48), severe PE and
normotensive pregnant women (p = 0.69), and mild PE pregnant normotensive
(p = 0.73), mild PE and nonpregnant women (p = 0.10) (Figure 16)
Admittedly, circulating platelets are at different stages of activation.
Platelets can be activated as a result of vascular lesions with release of FT and
activation process can be reversible or with such intensity that culminates in
platelet aggregation in injured site. Obviously, only those who still circulatingplatelets can be evaluated in vitro in peripheral blood samples for FC.
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Konijnenberg et al. (1997b) proposed that is understandable that most of the
platelets are identified in PE still in the native state, not activated, since the
activated already be entrapped in platelet aggregation and no longer in the
circulation.
The results of this study, which was obtained from the smaller MFI
CD41a in the PE and severe PE could be justified by the premise Konijnenberg
et al. (1997b), previously cited.
Only three other studies that evaluated the GPIIb/IIIa inhibitors during
pregnancy were found in the literature. Contrary to the results of this work, Star
et al. (1997) and Sheu et al. (2002) found no difference in the expression of
GPIIb/IIIa when compared normotensive pregnant and nonpregnant women.
Khne et al. (1996) in turn, also found no difference in expression of GP in
patients with PE compared to normotensive pregnant women.
It should be noted that the interaction antibody-GPIIb/IIIa can be affected
by in vitro manipulation of the sample, or, more commonly, by the use of
fasteners and platelet inhibitors, which may mask the expected levels of platelet
activation (JANES et al., 1994). Differences in pre-analytical stage could explain
the discrepancy between our results and those found in the literature.
GPIIIa (CD61)
The average MFI of CD61 was lower in pregnant women with PE
compared to non-pregnant women and in normotensive pregnant women
compared to nonpregnant women (p = 0.00 for both) (Figure 15). The average
MFI of CD61 was lower in the groups with severe and mild PE compared to
nonpregnant women (p = 0.00 for both). The comparison between both groups:mild PE and severe PE (p = 0.49), severe PE and normotensive pregnant
women (p = 0.79), mild PE and normotensive pregnant women (p = 0.63)
showed no significant difference ( Figure 16). These results were similar to
those obtained for CD41a (GPIIb/IIIa) that was expected since both recognize
the same glycoprotein complex, confirming that these two biomarkers are
equivalent. The hypothesis that the platelets are identified in the FC less active,
since that would already be trapped in the activated platelet aggregate and nolonger in the circulation, may also be used to explain these findings.
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Paradoxically the results obtained in the present study, Holthe et al.
(2004) observed that the density of CD61 on the platelet surface levels at
baseline and after stimulation with agonist was higher in PE compared to
normotensive pregnant women and nonpregnant women. No difference was
found between normotensive pregnant and nonpregnant women. These
researchers proposed that platelets are activated during pregnancy and more
intensely in PE, highlighting the importance of platelets in the pathogenesis of
this disease.
GPIX (CD42a)
A comparison of mean MFI of CD42a the three groups (Figure 15) and
after division of the PE group showed no difference (p = 0.44 and p = 0.57,
respectively) (Figure 16).
In accordance with the results of this study, Star et al. (1997) and Holthe
et al. (2004) also observed no difference in the expression of CD42a between
normotensive pregnant women and non-pregnant, even after activation with
agonist. Conversely, Holthe et al. (2004) had higher expression GPiX in
pregnant women with PE compared to normotensive pregnant women.
The setting is a variable that needs to be controlled because the affinity
of binding to the antibody-dependent activation can be reduced compared to
platelets unfixed. An argument in favor of immediate fixation is that platelet
activation is time dependent in vitro to various platelet surface antigens such as
CD42, CD41 and CD62P (Michelson, 1996). Unfortunately, for most markers
exocytosis, the reactions are reversible and suffer interference from sample
handling, fixing and use of inhibitors, which can mask the levels of plateletactivation expected (JANES & GOODALL, 1994). Possibly, this may be one
reason for the discrepancy between studies.
Ratio biomarkers platelet surface
The platelet GP superficies have important functions in the process of
hemostasis and coagulation, especially with regard to the interaction betweenplatelets, leukocytes, endothelial cells, collagen and fibrinogen (Lowenberg et
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al., 2010). Like most markers of platelet activation in this study is constitutive,
we chose to evaluate the existence of the predominance of GP determined from
the ratio between these groups of pregnant women with PE, normotensive
pregnant women and nonpregnant women.
The median ratio of MFI CD61/CD42a (GPIIIa and GPIX) and
CD41a/CD62P (GPIIb/IIIa and P-selectin) revealed significantly lower in the
group of normotensive pregnant women compared to nonpregnant women (p =
0.00 p = 0.01). The comparison of medians between groups PE and
normotensive pregnant women showed no significant difference (p = 0.57 and p
= 0.56, respectively) (Figure 17). For the reason CD61/CD42a, there was a
median lower in the group of PE and PE severe compared to nonpregnant
women (p = 0.00 for both) (Figure 18).
Analysis of these results suggest that in pregnant women, both
normotensive and preeclamptic less likely expressing platelet GPIIIa and,
therefore, less amount of GPIIb/IIIa complex in the circulation. This result allows
further inferred that in pregnancy the platelets are mainly aggregated and
probably adhered on vascular sites.
It is known that fibrinogen levels are elevated in normal pregnancy,
integrating a number of haemostatic abnormalities resulting in a
hypercoagulable state in this physiological condition. Hypercoagulability is
increasing throughout gestation and aims to prepare the woman's body for the
hemostatic challenge associated with the delivery of the placenta and
simultaneous rupture of numerous blood vessels. The formation of clots in the
extremities of injured vasculature of the uterine wall is initiated promptly,
preventing excessive bleeding after childbirth (STIRLING et al., 1984; WANG et
al., 2002).One hypothesis to explain the activation of the GPIIb/IIIa receptors and
consequently the aggregation of platelets could be an increased plasma
fibrinogen in pregnancy (Bonnar et al. 1970). The levels are also significantly
elevated thrombin, altering the interactions of cells endotelias and increasing
the permeability of the endothelium. The formed thrombin activates platelets,
leukocytes and endothelial cells (Wang et al. 2002). Hoffman & Monroe (2007)
admitted that platelet aggregates are retained in small vessels or determine theactivation of coagulation proteins and the formation of fibrin clot. Physiologically,
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a discrete fibrin deposition in small capillaries placenta is admitted as a way to
prevent rupture of the same and ensure the integrity of placental vascularization
(FLETCHER et al., 1979; CARON et al., 1990).
Assessing the median ratio of MFI CD42a/CD41a (GPIX and GPIIb/IIIa)
was higher than in group PE and severe PE compared to the group of non-
pregnant women and the group of normotensive pregnant women compared to
nonpregnant women ( p = 0.00 for both) (Figures 17 and 18).
The reasoning for the interpretation of this result is similar to that done for
CD61/CD42a and CD41a/CD62P. However, the GPIIb/IIIa is now in the
denominator, resulting in a numerically higher ratio. The predominance of
GPIIb/IIIa is understandable since it is the first to be platelet glycoprotein and is
expressed in larger quantities. This glycoprotein binds to a multitude of
adhesive molecules, is fundamental in the process of platelet aggregation
(Lowenberg et al., 2010).
A statistical comparison of the ratios of median MFI of CD61/CD41a
(GPIIIa and GPIIb/IIIa) of CD61/CD62P (GPIIIa and P-selectin) and
CD42a/CD62P (GPIX and P-selectin) before (p = 0,29, p = 0.07 and p = 0.73,
respectively) and after (p = 0.42, p = 0.14 and p = 0.69, respectively) the
subdivision of the PE group, showed no difference between groups assessed
(Figures 17 and 18). No studies in the literature that compared the ratios of
these glycoproteins.
CONCLUSION
The results obtained in this study did not demonstrate an increasedexpression of markers of platelet activation in PE. However, one can not rule
out a role of this activation in the pathophysiology of the disease, since it is
possible to admit the exacerbation of platelet activation in the maternal-fetal
interface.
Although studies involving the uterine circulation are not very common,
due to the difficulty of obtaining samples for laboratory evaluation, it has been
reported a pronounced activation of coagulation in this location (Higgins et al.,1998).
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It should be noted that the discrepancies of the results of studies in the
literature concerning the expression of markers of platelet activation may result
from methodological differences. Although the assessment of markers of
platelet activation is increased in the study of PE, there is still no consensus on
the optimal method for assessing these parameters, including the definition of
anticoagulant to be used in the collection of blood sample, the sample type
(whole blood or PRP), the specification of antibodies and the use or not of
fixative in processing the sample. Another important aspect refers to the wide
variability in the general population, the expression of markers of platelet
activation, which hinders the establishment of cut-off for these parameters.
Certainly, advances in research of these markers by FC and standardization of
the pre-analytical and analytical methods will contribute to greater
understanding of platelet activation in the pathophysiology of PE.
The standardizatio