EVALUATION OF PLATELET ACTIVATION BY FLOW CYTOMETRY, IN PREECLAMPSIA

FREITAS, Letcia Gonalves MARTINS-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

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

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 and Weibel-Palade bodies of endothelial cells (Gurney et al. 2002; YANG et al.

2,009; Lowenberg et al. 2,010). When exposed on the platelet surface, Pselectin 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

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 receptors when 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, Davie and 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).

However, a major limitation of these studies is to activate in vitro that 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 Ca 2+ 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 of natural 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

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 often associated 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 and may develop into more serious conditions such as eclampsia, in which we

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 TXA 2 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 the incidence 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

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 and coagulation, 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 in healthy pregnant women and their newborns after in vitro activation of platelets

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, Belo Horizonte, 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

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 semiquantitative 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 <100,000); i. Elevation of liver enzyme activity; j. Esquiscitos presence of blood in the film; k. Intrauterine growth restriction (IUGR).

The inclusion criteria for PEL group were: a. Systolic/diastolic less than 90 mmHg without exceeding 140 x 110 mmHg for diastolic blood pressure; b. Proteinuria and less than 300mg to 2g in 24-hour urine or greater than or equal to (+1) by semi-quantitative strips method in unic urine sample; c. Being in the third trimester of pregnancy.

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 mL of citrated blood were collected 4 ml of blood in ethylenediaminetetraacetic acid

(EDTA) to blood count. This was done using the electronic cell counter, CellDyn 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 5x10 6 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 discarded and the pellet was resuspended in 200L PBS. Table 1 Monoclonal antibodies labeled with fluorochromes used for analysis of
platelet glycoproteins.

Sample 1

Reagent/antibody PBS

Fluorocrom -

Volum 5L

Clone -

Phenotype target -

2 3 4 5

Anti-CD41a Anti-CD42a Anti-CD61 Anti-CD42a Anti-CD62P

PE FITC PE FITC PE

4L 4L 3L 4L 5L

HIP8 ALMA.16 VI-PL2 ALMA.16 AK-4

GPIIb/IIIa GPIX GPIIIa 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) versus SSC. 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 antiCD62P PE. Results were expressed as percentage of platelet CD42a+/CD62P+, corresponding to the upper right quadrant double-positive.

Plaquetas

IMF=197,5

FL2/Anti-CD41a PE FL2/Anti-CD41a PE

Plaquetas

SSC/Granulosidade

Nmero de plaquetas

IMF=978,6

FL2/Anti-CD61 PE

FL2/Anti-CD61 PE

Plaquetas IMF=410,8

FL1/Anti-CD42a FITC

FL1/Anti-CD42a FITC

FL2/Anti-CD62P PE

5,2% 94,8%

Plaquetas

FL1/Anti-CD42a FITC

FL1/Anti-CD42a FITC

Figure 2 Strategies 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.
+

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 test two 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.

RESULTS

The clinical characteristics of the three groups of participants in this study are presented in Tables 2 and 3. Table 2 Clinical characteristics of the study participants. PE (n=35) Idade (anos) IEP (meses) IMC (Kg/m2) IG (sem.) GPG (Kg) PA sist. (mmHg) PA diast. (mmHg)
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 de gestantes 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 tStudent). Os dados no-paramtricos so apresentados como medianaintervalo interquartil (KruskalWallis/Mann-Whitney/Correo de Bonferroni). 1 2 3 4 p : PE x GN x CNG; p : PE x GN; p : PE x CNG; p : GN x CNG.

GN (n=31)

CNG (n=31)

p1

p2

p3

p4

29 7 93 69 28 6 33 3 12 8 160 10

26 6 36 41 25 5 34 4 12 8 110 20

25 6 24 4 120 10

0,02* 0,01* 0,00*

0,06 0,16 0,04* 0,17 0,71 0,01*

0,01* 0,00* 0,00*

0,35 0,38 0,00*

100 10

70 20

80 10

0,00*

0,00*

0,00*

0,01*

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.

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 3 Clinical characteristics of the study participants after subdivision of the PE
group as severe and mild.

PEG (n=15)

PEL (n=20)

p1

p2

p3

p4

p5

p6

Idade (anos) IEP (meses) IMC (Kg/m2) IG (sem.) GPG (Kg) PA sist. (mmHg) PA diast. (mmHg)

31 6 127 66 26 5 33 3 14 5 160 20

28 8 54 52 30 6 34 3 11 10 160 10

0,02* 0,02* 0,00* 0,29 0,64 0,00*

0,16 0,02 0,03* 0,01*

0,02* 0,012** 0,86 0,00*

0,00* 0,37 0,00*

0,36 0,00* 0,00*

0,08 0,00* 0,00*

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: ganho de 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 no gestantes. *p0,05. **p0,017 (Correo de Bonferroni). 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). 1 2 3 4 5 6 p : PEG x PEL x GN x CNG; p : PEG x PEL; p : PEG x GN; p : PEG x CNG, p : PEL x GN, p : 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

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 4 Residue analysis for the variable "number of pregnancies" PEG groups, PEL,
NG and CNG.

Gestacional number Grups PEG PEL GN CNG 0 -1,7 -2,1* -2,8* 5,9* 1 -0,5 1,1 -1,0 0,4 2 1,7 0,4 2,9* -4,5*

*Localizao da diferena: Resduo 1,96 e Resduo -1,96. PEG: grupo de gestantes com PE grave; PEL: grupo de gestantes com PE leve; NG: grupo de gestantes normotensas; 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.

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 5 Platelet count and platelet activation markers obtained for PE groups (PEG
and PEL), GN and CNG. Parameters PE (n=35) 238,4 N de plaquetas x 10 /L IMF CD41a
3

PEG (n=15) 223,7 (94,9) 253,0 (182,5) 855,7 (323,0) 463,3 (170,0) 150,3 (55,9) 1,4 (2,0)

PEL (n=20) 249,4 (70,9) 297,3 (176,8) 940,0 (270,8) 433,0 (147,1) 157,8 (110,6) 1,2 (2,1)

GN (n=25) 219,3 (53,6) 239,9 (239,5) 888,0 (339,4) 439,0 (152,3) 154,1 (126,2) 1,2 (3,9)

CNG (n=25) 286,2 (67,0) 364,9 (426,1) 1330,6 (441,1) 486,2 (108,9) 163,1 (118,3) 1,6 (4,0)

(81,7) 278,3 (178,0)

IMF CD61

903,9 (292,3)

IMF CD42a

446,0 (155,6)

IMF CD62P

152,2 (99,4)

CD62P (%)

1,4 (2,1)

IMF: intensidade mdia de fluorescncia. # 3 Para N de plaquetas x 10 /L considerar n=31. Os dados paramtricos so representados como 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.

Plaquetas (/L)
CD41a (IMF)
600000

* *

1500

* *
1000

400000

200000

500

0 PE GN CNG

0 PE GN CNG

CD61 (IMF)
3000

CD42a (IMF)
1000 p=0,44

*
2000

800

600 400

1000

200
0 PE GN CNG

0 PE GN CNG

CD62P (IMF)
800 p=0,67 600
10 15

CD62P (%)
p=0,38

400
5

200

0 PE GN CNG

0 PE GN CNG

*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.

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).

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)
1500
600000

CD41a (IMF) *

*
400000

*
1000

500
200000

0 PEG PEL GN CNG

0 PEG PEL GN CNG

CD61 (IMF)
2500
1000

CD42a (IMF)
p=0,57

*
2000 1500 1000 500 0 PEG PEL

* *

800 600 400 200 0

GN

CNG

PEG

PEL

GN

CNG

CD62P (IMF)
800 p=0,70

CD62P (%)
80 p=0,54

600

60

400

40

200

20

0 PEG PEL GN CNG

0 PEG PEL GN CNG

*p0,05 ou p0,008 (Correo de Bonferroni). Para os dados paramtricos foram realizados os testes ANOVA/LSD. Para os dados no-paramtricos foram realizados os testes Kruskal-Wallis/MannWhitney/Correo de Bonferroni.

Figure 4 Platelet 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 group compared 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 7 MFI ratios between the markers of platelet surface obtained for PE groups
(PEG and PEL) GN and CNG. Paameters IMF CD61/CD41a IMF CD61/CD42a IMF CD42a/CD41a IMF CD61/CD62P IMF CD42a/CD62P PE (n=35) 3,6 (2,3) 2,0 (0,9) 1,7 (2,0) 5,2 (2,6) 2,6 (2,0) PEG (n=15) 4,2 (12,1) 1,8 (0,70) 1,8 (8,0) 5,4 (2,5) 3,0 (1,6) PEL (n=20) 3,3 (1,9) 2,3 (0,7) 1,6 (1,0) 5,4 (2,1) 2,5 (1,6) GN (n=25) 3,1 (4,2) 1,9 (1,1) 1,7 (1,1) 4,8 (2,5) 2,5 (1,3) CNG (n=25) 2,8 (3,8) 2,7 (0,7) 1,2 (1,0) 7,6 (4,2) 2,8 (1,3)

IMF CD41a/CD62P

1,2 (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
40 p=0,29 30

CD61/CD42a
6

* *
4

20

2
10

0 PE GN CNG

0 PE GN CNG

CD42a/CD41a
20

CD61/CD62P
20 p=0,07 15

*
15

10

10

0 PE GN CNG

0 PE GN CNG

CD42a/CD62P
8 p=0,73 6

CD41a/CD62P
8

*
6

0 PE GN CNG

0 PE GN CNG

*p0,05 ou p0,017 (Correo de Bonferroni). Foram realizados os testes Kruskal-Wallis/Mann-Whitney/Correo de Bonferroni.

Figure 5 Ratio of the mean intensities of fluorescence CD61/CD41a, CD61/CD42a,

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
40 p=0,42 30

CD61/CD42a
6

*
4

20

2
10

0 PEG PEL GN CNG

0 PEG PEL GN CNG

CD42a/CD41a
20

CD61/CD62P
20 p=0,14 15

*
15

*
10

10

0 PEG PEL GN CNG

0 PEG PEL GN CNG

CD42a/CD62P
8 p=0,69 6
6 8

CD41a/CD62P *

4
4

0 PEG PEL GN CNG

0 PEG PEL GN CNG

Figure 6 Ratio of the mean intensities of fluorescence CD61/CD41a, CD61/CD42a,

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 comparison between 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

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 to fibrinogen, 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 the pathophysiology of PE, but the platelet count is not able to predict the

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.

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 the average 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.

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

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 of platelets 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).

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 of circulating 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

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 of platelets 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

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 the percentage 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 was made in MFI except for P-selectin, also expressed in percent. Only two studies

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 of agonist, 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

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 extreme values. 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 of platelets are activated in PE, but these are rapidly cleared from the circulation.

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 Pselectin 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, the extent 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 basal levels of CD62P, after stimulation with ADP, are high in healthy pregnant

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 pregnant women (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 circulating platelets can be evaluated in vitro in peripheral blood samples for FC.

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 no longer in the circulation, may also be used to explain these findings.

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 platelet activation 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 between platelets, leukocytes, endothelial cells, collagen and fibrinogen (Lowenberg et

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 the activation of coagulation proteins and the formation of fibrin clot. Physiologically,

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 nonpregnant 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 increased expression 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).

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 standardization of the technique of flow cytometry to evaluate platelet activation and the formation of platelet-leukocyte aggregates was determined, with the following advantages: Rapid implementation; Need for a small volume of blood; Good agreement between different analysts and Applicable in other studies to assess platelet activation. Pregnancy is accompanied by changes in expression of CD41a and CD61 on the platelet surface, and reduced platelet counts.

REFERENCES

ACAR, K.; SUCAK, A.; BEYAZIT, Y.; GENC, G.; HAZNEDAROGLU, I. C.; AKSU, S.; DANISMAN, N. Lack of platelet activation reflected by circulating soluble glycoprotein V in pre-eclampsia. Journal of Internacional Medical Research, v. 35, n. 5, p. 704-708, Sep.-Oct. 2007. ACOG practice bulletin. Diagnosis and management of preeclampsia and eclampsia. American College of Obstetricians and Gynecologists. Number 33, January 2002. ACOG Committee on Obstetric Practice. International Journal of Gynaecology and Obstetrics. v. 77, n. 1, p. 67-75, Apr 2002. AHMED, Y.; van IDDEKINGE, B.; PAUL, C.; SULLIVAN, H.F.; ELDER, M. G. Retrospective analysis of platelet numbers and volumes in normal pregnancy and in pre-eclampsia. British Journal of Obstetrics and Gynaecology. v. 100, n. 3, p. 216-220, Mar. 1993.

ALVAREZ, D. F.; HELM, K.; DEGREGORI, J.; ROEDERER, M.; MAJKA, S. Publishing flow cytometry data. American Journal of Physiology - Lung Cellular and Molecular Physiology. v. 298, n.2, p. L127130, Feb. 2010. AMERICAN COLLEGE OF OBSTETRICIANS AND GYNECOLOGISTS. Practice bulletin: clinical management guidelines for obstetrician-gynecologists. Diagnosis and management of preeclampsia and eclampsia. Obstetrics and Gynecology. n. 33, Jan. 2002. ARAKAWA, K.; YASUDA, S.; HAO, H.; KATAOKA, Y.; MORII, I.; KASAHARA, Y.; KAWAMURA, A.; ISHIBASHI-UEDA, H.; MIYAZAKI, S. Significant association between neutrophil aggregation in aspirated thrombus and myocardial damage in patients with ST-segment elevation acute myocardial infarction. Circulation Journal. v. 73, p. 139-144, 2009. AREFIEVA, T. I.; PROVATOROV, S. I.; SAMKO, A. N.; KRASNIKOVA, T. I.; RESINK T. J.; ERNE, P.; TKACHUK, V. A.; CHAZOV, E. I. Monocyte integrin expression and monocyte-platelet complex formation in human with coronary restenosis. Clinical and Experimental Pharmacology and Physiology. v. 28, n. 10, p. 804-808, Oct. 2001. ASHMAN, N.; MACEY, G. M.; FAN, L. S.; AZAM, U.; YAQOOB, M. M. Increased platelet-monocyte aggregates and cardiovascular disease in endstage renal failure patients. Nephrology Dialysis Transplantation, v. 18, p. 2088-2096, 2003. ASSINGER, A; BUCHBERGER, E; LAKY, M; ESFANDEYARI, A; BROSTJAN, C; VOLF I. Periodontopathogens induce soluble P-selectin release by endothelial cells and platelets. Thrombosis Research. v. 127, p. 20-26, 2011. BACAL, N. S.; FAULHABER, M. H. W. Aplicao prtica em citometria de fluxo.1. ed. So Paulo: Ateneu, 2003. BAGAMERY, K.; KVELL, K.; GRAHAM, J. Different platelet activation levels in non-pregnant, normotensive pregnant, pregnancy-induced hypertensive and pre-eclamptic women. A pilot study of flow cytometric analysis. European Journal of Obstetrics & Gynecology and Reproductive Biology, v. 121, p. 117-123, 2005. BLANN, A. D.; DRAPER, Z. Platelet activation as a marker of heart attack. Clinical Chimica Acta. v. 412, n. 11-12, p. 841-842, May 2011. BONNAR, J.; MCNICOL, G. P.; DOUGLAS, A. S. Coagulation and fibrinolytic mechanisms during and after normal childbirth. British Medical Journal, v. 2, n. 5703, p. 200-203, 1970. BREMME, K.; BLOMBCK, M. Hemostatic abnormalities may predict chronic hypertension after preeclampsia. Gynecology and Obstetric Investigation. v. 41, n. 1, p. 20-26, 1996.

BROWN, M.; WITTWER, C. Flow Cytometry: Principles and Clinical Applications in Hematology. Clinical Chemistry. v. 46, n. 8 Pt 2, p. 12211229, 2000. BURTIS, C. A.; ASHWOOD, E. R.; BRUNS, D. E. Tietz fundamentos de qumica clnica. 6. ed. Rio de Janeiro: Elsevier, 2008. CANZONERI, B. J.; LEWIS, D. F.; GROOME, L.; WANG, Y. Increased neutrophil numbers account for leukocytosis in women with preeclampsia. American Journal of Perinatology. v. 26, n. 10, p. 729-732. Nov. 2009. CARON, C.; GOUDEMAND, J.; MAREY, A.; BEAGUE, D.; DUCROUX, G; DROUVIN, F. Are haemostatic and fibrinolytic parameters predictors of preeclampsia in pregnancy-associated hypertension? Thrombosis and Haemostasis. v. 66, n. 4, p.410-414, 1991. CASTRO, C.; GOURLEY, M. Diagnostic Testing and Interpretation of Tests for Autoimmunity. Journal of Allergy Clinical Immunology. v. 125, n. 2, p. S238 S247, Feb. 2010. CEYHAN, T.; BEYAN, C.; BAER, .; KAPTAN, K.; GNGR, S.; IFRAN, A. The effect of pre-eclampsia on complete blood count, platelet count and mean platelet volume. Annals of Hematology, v. 85, p. 320-322, 2006. CHAPPELL, L. C; SHENNAN, A. H. Assessment of proteinuria in pregnancy: urinary spot protein:creatinine ratio can reliably rule out proteinuria in pregnancy. British Medical Journal. v. 336, n. 7651, p. 968-969, May 2008. CHU, S. G.; BECKER, R. C.; BERGER, P. B.; BHATT, D. L.; EIKELBOOM, J. W.; KONKLE, B.; MOHLER, E. R.; BERGER, J. S. Mean platelet volume as a predictor of cardiovascular risk: a meta-analysis. Journal of Thrombosis and Haemostasis. v. 8, n. 1, p. 148-156, Jan. 2010. CHU, A. J. Tissue factor, blood coagulation, and beyond: an overview. International Journal of Inflammation. v. 2011, p. 1-30, 2011. CLASP (Collaborative Low-dose Aspirin Study in Pregnancy) Collaborative Group.CLASP: a randomised trial of low-dose aspirin for the prevention and treatment of pre-eclampsia among 9364 pregnant women. The Lancet. v. 343, p. 619-629, Mar. 1994. COATA, G.; FRUSCA, T.; BARANZELLI, D.; COSMI, E. V.; Di RENZO, G. C.; ANCESCHI, M. M. Abnormal platelet lipid membrane composition in pregnancy induced hypertension. Journal of Perinatal Medicine. v. 20, n. 2, p. 123-127, 1992. CUNNINGHAM, F. G.; PRITCHARD, J. A. Hematologic considerations of pregnancy-induced hypertension. Seminars in Perinatology. v. 2, n. 1, p. 2938, Jan. 1978. CUNNINGHAM, F. G. Williams obstetricia. 20. ed. Rio de Janeiro: Guanabara Koogan, 2000.

da COSTA MARTINS, P.; van DEN BERK, N.; ULFMAN, L. H.; KOENDERMAN, L.; HORDIJK, P. L.; ZWAGINGA, J.J. Platelet-monocyte complexes support monocyte adhesion to endothelium by enhancing secondary tethering and cluster formation. Arteriosclerosis, Thrombosis, and Vascular Biology. v. 24, p. 193-199, 2004. DELGADO, A. V.; ALEXANDER, S. L.; MCMANUS, A. T.; PUSATERI, A. E. Antibodies against human cell receptors, CD36, CD41a, and CD62P crossreact with porcine platelets. Cytometry B Clinical Cytometry. v. 56, n. 1, p. 62-67, Nov. 2003. de TUTE, R. M. Flow cytometry and its use in the diagnosis and management of mature lymphoid malignancies. Histopathology. v. 58, n. 1, p. 90105, Jan. 2011. di MINNO, G.; COPPOLA, A.; di MINNO, A. N. D.; POON, M. C. Glan zmanns thrombasthenia (defective platelet integrin IIb-3): proposals for management between evidence and open issues. Thrombosis and Haemostasis. v. 102, n. 6, p. 1157-1164, Dec. 2009. DUNDAR, O.; YORUK, P.; TUTUNCU, L.; ERIKCI, A. A.; MUHCU, M.; ERGUR, A. R.; ATAY, V.; MUNGEN, E. Longitudinal study of platelet size changes in gestation and predictive power of elevated MPV in development of preeclampsia. Prenatal Diagnosis. v. 28, p. 1052-1056, Sep. 2008. EDELSTAM, G.; LOWBEER, C.; KRAL, G.; GUSTAFSSON, S. A.; VENGE, P. New reference values for routine blood samples and human neutrophilic lipocalin during thirdtrimester pregnancy. Scandinavian Journal of Clinical and Laboratory Investigation. v. 61, p. 583592, 2001. ENDLER, G.; KLIMESCH, A.; SUNDER-PLASSMANN, H.; SCHILLINGER, M.; EXNER, M.; MANNHALTER, C.; JORDANOVA, N.; CHRIST, G.; THALHAMMER, R.; HUBER, K.; SUNDER-PLASSMANN, R. Mean platelet volume is an independent risk factor for myocardial infarction but not for coronary artery disease. British Journal of Haematology. v. 117, n. 2, p. 399404, May 2002. FALCONER, J.; PINEO, G.; BLAHEY, W.; BROWEN, T.; DOCKSTEADER, B.; JADUSINGH, I. Essential thrombocythemia associated with recurrent abortions and fetal growth retardation. American Journal of Hematology. v. 25, n. 3, p. 345-347, 1987. FALLAHIAN, M.; NABAIE, F. Subclinical thrombocytopenia and preeclampsia. International Journal of Gynaecology and Obstetrics. v. 89, p. 47-48, 2005. FARIAS, M. G.; B, S.D.; Determinao do intervalo de referncia para o volume plaquetrio mdio (VPM) utilizando o analisador hematolgico Pentra 120 ABX; Revista Brasileira de Anlises Clnicas. v. 40, n. 1, p. 39-41, 2008. FENG, R.; SHIMAZAKI, C.; INABA, T.; TAKAHASHI, R.; HIRAI, H.; KIKUTA, T.; SUMIKUMA, T.; YAMAGATA, N.; ASHIHARA, E.; FUJITA, N.; NAKAGAWA, M. CD34+/CD41a+ cells best predict platelet recovery after autologous peripheral

blood stem cell transplantation. Bone Marrow Transplantation. v. 21, n. 12, p. 1217-1222, 1998. FITZGERALD, M. P.; FLORO, C.; SIEGEL, J.; HERNANDEZ, E. Laboratory findings in hypertensive disorder of pregnancy. Journal of The National Medical Association. v. 88, n. 12, 1996. FLETCHER, A. P.; ALKJAERSIG, N. K.; BURSTEIN, R. The influence of pregnancy upon blood coagulation and plasma fibrinolytic enzyme function. American Jounal of Obstetrics and Gynecology. v. 134, p. 743-751, 1979. FREEDMAN, J.; E.; LOSCALZO, J. Platelet-monocyte aggregates bridging thrombosis and inflammation. Circulation. v. 105, p. 2130-2132, 2002. FURIE, B.; FURIE, B. C. Mecanisms of thrombus formation. The New England Journal of Medicine. V. 359, n. 9, p. 938-949, 2008. GATTI, L.; TENCONI, P. M.; GUAMERI, D.; BERTULESSI, C.; OSSOLA, M. W.; BOSCO, P.; GIANOTTI, G. A. Hemostatic parameters and platelet activation by flow-cytometry in normal pregnancy: a longitudinal study. International Journal of Clinical and Laboratory Research. v. 24, n. 4, p. 217-219, 1994. GERVASI, M. T.; CHALWORAPONGSA, T.; PACORA, P.; NACCASHA, N.; YOON, B. H.; MAYMON, E.; ROMERO, R. Phenotypic and metabolic characteristics of monocytes and granulocytes in preeclampsia. American Journal of Obstetrics and Gynecology. v. 185, n. 4, p. 792-797, Oct. 2001. GIBSON, B.; HUNTER, D.; NEAME, P. B.; KELTON, J. G. Thrombocytopenia in preeclampsia and eclampsia. Seminars in Thrombosis and Hemostasis. v. 8, n. 3, p. 234-247, Jul. 1982. GILABERT, J.; ESTELLS, A.; AZNAR, J.; ESPAA, F.; ANDRS, C.; SANTOS, T.; VALLS, J. Contribution of platelets to increased plasminogen activator inhibitor type 1 in severe preeclampsia. Thrombosis and Haemostasis. v. 63, n. 3, p. 361-366, 1990. GILES, C. The platelet count and mean platelet volume. British Journal of Haematology. v. 48, n. 1, p. 31-37, May 1981. GILES, C.; INGLIS, T. C. M. Thrombocytopenia and macrothrombocytosis in gestacional hypertension. British Journal of Haematology. v. 88, p. 11151119, Nov. 1981. GKALIAGKOUSI, E.; CORRIGALL, V.; BECKER, S.; WINTER, P.; SHAH, A.; ZAMBOULIS, C.; RITTER, J.; FERRO, A. Decreased platelet nitric oxide contributes to increased circulating monocyte-platelet aggregates in hypertension. European Heart Journal. v.30, p. 3048-3054, 2009. GRILL, S.; RUSTERHOLZ, C.; ZANETTI-DLLENBACH, R.; TERCANLI, S.; HOLZGREVE, W.; HAHN, S.; LAPAIRE, O. Potential markers of preeclampsia: a review. Reproductive Biology and Endocrinology. v. 7, n. 70, Jul. 2009.

GURNEY, D.; LIP, G. Y.; BLANN, A. D. A reliable plasma marker of platelet activation: does it exist? American Journal of Hematology. v. 70, n. 2, p. 139144, Jun. 2002. HARDING S. A.; DIN, J. N.; SARMA, J.; JESSOP, A.; WEATHERALL, M.; FOX, K. A. A. NEWBY, D. E. Flow cytometric analysis of circulating platelet-monocyte aggregates in wholw blood: Methodological considerations. Thrombosis and Haemostasis. v. 98, p. 451-456, 2007. HARLOW, F. H.; BROWN, M. A.; BRIGHTON, T. A.; SMITH, S. L.; TRICKETT, A. E.; KWAN, Y-L; DAVIS, G. K.. Platelet activation in the hypertensive disorders of pregnancy. American Journal of Obstetrics and Gynecology. v. 187, n. 3, p. 688-695, Sep. 2002. HARTWIG, J. H.; ITALIANO-JR, J. E. Life of the blood platelet. In HANDIN R. I.; LUX S. E.; STOSSEL T. P. Blood: principles, practice of hematology. Philadelphia: J. B. Lippincott, 1995. Cap. 33, p. 959-1080. HAUKKMAAA, L.; SALMINEN, M. S.; LAIVUORI H et al. Risc for subsequent coronary artery disease after preeclampsia. American Journal of Cardiology. v. 93, p. 805-808, 2004. HAWFIELD, A.; FREEDMAN, B. I. Pre-eclampsia: the pivotal role of the placenta in its pathophysiology and markers for early detection. Therapeutic Advances in Cardiovascular Disease. v. 3, n. 1, p. 65-73, Feb. 2009. HEILMANN, L.; SIEKMANN, U. Hemodynamic and hemorheological profiles in women with proteinuric hypertension of pregnancy and in pregnant controls. Archives of Gynecology and Obstetrics. v. 246, n. 3. p. 159-168, 1989. HESS, K.; GRANT, P. J. Inflammation and thrombosis in diabetes. Thrombosis and Haemostasis. v. 105, sup 1, p. S43-54, May 2011. HIGGINS, J. R.; WALSHE, J. J.; DARLING, M. R. N.; NORRIS, L.; BONNAR, J. Hemostasis in the uteroplacental and peripheral circulations in normotensive and preeclamptic pregnancies. American Journal of Obstetrics and Gynecology. v. 179, p. 520-526, 1998. HIRAFUJI, M.; SHINODA, H. Platelet-leukocyte interaction in adhesion to endothelial cells induced by platelet-activating factor in vitro. British Journal of Pharmacology, v. 103, p. 1333-1338, 1991. HIRAKATA, V. N.; CAMEY, S. A. Anlise de concordncia entre mtodos de Bland-Altman. Revista do Hospital das Clnicas de Porto Alegre. v. 29, n. 3, p. 261-268, 2009. HOFFMAN, M.; MONROE, D. M. Coagulation 2006: a modern view of hemostasis. Hematology Oncology Clinics of North America. v. 21, n. 1, p. 1-11, Feb. 2007. HOLTHE, M. R.; STAFF, A. C.; BERGE L. N.; LYBERG T. Different levels of platelet activation in preeclamptic, normotensive pregnant, and nonpregnant

women. American Journal of Obstetrics and Gynecology. v. 190, p. 11281134, 2004. HOLTHE, M. R.; LYBERG, T.; STAFF, A. C.; BERGE, L. N. Leukocyte-platelet interaction in pregnancies complicated with preeclampsia. Platelets. v. 16, n. 2, p. 9197, Mar. 2005. HOWARTH, S.; MARSHALL, L. R.; BARR, A. L.; EVANS, S.; PONTRE, M.; RYAN, N. Platelet indices during normal pregnancy and pre-eclampsia. Britsh Journal of Biomedical Science. v. 56, n. 1, p. 20-22, 1999. HU, H.; LI N., YNGEN, M.; OSTENSON, C. G.; WALLEN, N. H.; HJEMDAHL, P. Enhanced leukocyte-platelet cross-talk in type 1 diabetes mellitus: Relationship to microangiopathy. Journal of Thrombosis and Haemostasis. v. 2, n.1, p. 58-64, Jan. 2004. JANES, S. L.; GOODALL, A. H. Flow cytometric detection of circulating activated platelets and platelet hyper-responsiveness in pre-eclampsia and pregnancy. Clinical Science. v. 86, n. 6, p. 731-739, Jun. 1994. JANES, S. L.; KYLE, P. M.; REDMAN, C.; GOODALL, A. H. Flow cytometric detection of activated platelets in pregnant women prior to the development of pre-eclampsia. Thrombosis and Haemostasis. v. 74, n. 4, p. 1059-1063, Oct. 1995. JREMO, P.; LINDAHL, T. L.; LENNMARKEN, C.; FORSGREN, H. The use of platelet ensity and volume measurements to estimate the severity of preeclampsia. European Journal of Clinical Investigation. v. 30, n. 12, p. 11131118, 2000. JAROSZESKI, M. J.; RADCLIFF, G. Fundamentals of flow cytometry. Molecular Biotechnology, v. 11, n. 1, p. 37-53, 1999. JIN, A. Y.; TUOR, U. I.; RUSHFORTH, D.; KAUR, J.; MULLER, R. N.; PETTERSON, J. L.; BOUTRY, S.; BARBER, P. A. Reduced blood brain barrier breakdown in P-selectin deficient mice following transient ischemic stroke: a future therapeutic target for treatment of stroke. BioMed Center Neuroscience, v. 11, n. 12, 2010. JOSEPH, J. E.; HARRISON, P.; MACKIE, I. J.; ISENBERG, D. A.; MACHIN, S. J. Increased circulating plateletleucocyte complexes and platelet activation in patients with antiphospholipid syndrome, systemic lupus erythematosus and rheumatoid arthritis. British Journal of Haematology. v. 115, p, 451-459, 2001. JURK, K.; KEHREL, B. E. Platelets: physiology and biochemistry. Seminars of Thrombosis and Hemostasis. v. 31, n. 4, p. 381-392, 2005. KAGEYAMA, K.; NAKAJIMA, Y.; SHIBASAKI, M.; HASHIMOTO, S.; MIZOBE, T. Increased platelet, leukocyte, and endothelial cell activity are associated with increased coagulability in patients after total knee arthroplasty. Journal of Thrombosis and Haemostasis. v. 5, n. 4, p. 738-745, 2007.

KAITO, K.; OTSUBO, H.; USUI, N.; YOSHIDA, M.; TANNO, E. K.; MATSUMOTO, K.; HIRATA, R.; DOMITSU, K.; KOBAYASHI, M. Platelet size deviation width, platelet large cell ratio, and mean platelet volume have sufficient sensivity and speificity in the diagnosis of immune thrombocytopenia. British Journal of Haematology, n. 128, p. 698-702, 2005. KASHANIAN, M.; BARADARAN, H. R.; BAHASADRI, S.; ALIMOHAMMADI, R. Risk Factors for Pre-Eclampsia: A Study in Tehran, Iran. Archives of Iranian Medicine. v. 14, n. 6, p. 412-415, Nov. 2011. KAZMI, R. S.; COOPER, A. J.; LWALEED, B. A. Platelet function in preeclampsia. Seminars of Thrombosis and Hemostasis. v. 37, n. 2, p. 131-136, Mar 2011. KEY, N. S.; MACKMAN, N. Tissue factor and its measurement in whole blood, plasma, and microparticles. Seminars in Thrombosis and Hemostasis. v. 36, n. 8, p. 865-875, 2010. KHALID, S. K.; WOJDYLA, D.; SAY, L.; GLMEZOGLU, A. M.; van LOOK, P. F. WHO analysis of causes of maternal death: a systematic. Lancet. v. 367, n. 9516, p. 10661074, Apr. 2006. KIM, S. W.; LIM, Y. A. Establishment of reference values for platelet activation markers by flow cytometry. The Korean Journal of Laboratory Medicine. v. 26, n. 5, p. 323-328, Oct. 2006. KONIJNENBERG, A.; STOKKERS, E. W.; van der POST, J. A.; SCHAAP, M. C.; BOER K.; BLEKER O. P.; STURK A. Extensive platelet activation in preeclampsia compared with normal pregnancy: enhanced expression of cell adhesion molecules. American Journal of Obstetrics and Gynecology. v. 176, n. 2, p.461-469, Feb. 1997a. KONIJNENBERG, A.; van der POST, J. A.; MOL, B. W.; SCHAAP, M. C.; LAZAROV, R.; BLEKER, O. P.; BOER, K.; STURK, A. Can flow cytometric detection of platelet activation early in pregnancy predict the occurrence of preeclampsia? A prospective study. American Journal of Obstetrics and Gynecology. v. 177, n. 2, p.434-442, Aug. 1997b. KHNE, T.; RYAN, G.; BLANCHETTE, V.; SEMPLE, J. W.; HORNSTEIN, A.; MODY, M.; CHANG, W.; McWHIRTER, L.; FREEDMAN, J. Platelet-Surface Glycoproteins in Healthy and Preeclamptic Mothers and Their Newborn Infants. Pediatric Research. v. 40, n. 6, p. 876-880, Dec. 1996. LAIN, K. Y.; MARKOVIC, N.; NESS, R. B.; ROBERTS, J. M. Effect of smoking on uric acid and other metabolic markers throughout normal pregnancy. The Journal of Endocrinology & Metabolism. v. 90, p. 5743-5746, 2005. LASKIN, S.; PAYNE, B.; HUTCHEON, J. A.; QU, Z.; DOUGLAS, M. J.; FORD, J.; LEE, T.; MAGEE, L. A.; von DADELSZEN, P. The role of platelet counts in the assessment of inpatient women with preeclampsia. Journal of Obstetrics and Gynecology of Canada. v. 33, n. 9, p. 900-908, 2011.

LAZARUS, A. H.; WRIGHT, J. F.; BLANCHETTE, V.; FREEDMAN, J. Analysis of platelets by flow cytometry. Transfusion Science. v. 16, n. 4, p. 353-361, Dec. 1995. LINDHEIMER, M. D.; SPARGO, B.H.; KATZ, A. Renal biopsy in pregnancy hypertension. Journal of Reproductive Medicine. v.15, p.189-194, 1975. LI, Z.; ZHANG, Y.; YING, M. A. J.; KAPOUN, A. M.; SHAO, Q.; KERR, I.; LAM, A.; O'YOUNG, G.; SANNAJUST, F.; STATHIS, P.; SCHREINER, G.; KARUMANCHI, S. A.; PROTTER, A. A.; POLLITT, N. S. Recombinant vascular endothelial growth factor 121 attenuates hypertension and improves kidney damage in a rat model of preeclampsia. Hypertension.v. 50, p. 686692, 2007. LOK, C. A.; NIEUWLAND, R.; STURK, A.; HAU, C. M.; BOER, K.; VANBAVEL, E.; VANDERPOST, J. A. Microparticle-associated P-selectin reflects platelet activation in preeclampsia. Platelets. v. 18, n. 1, p. 68-72, Feb. 2007. LOK, C. A.; van DER POST, J. A.; SARGENT, I. L.; HAU, C. M.; STURK, A.; BOER, K.; NIEUWLAND, R. Changes in microparticle numbers and cellular origin during pregnancy and preeclampsia. Hypertension and pregnancy. v. 27, n. 4, p. 344-360, 2008. LORENZI, T. F. Fisiologia das clulas do sangue e hemostasia. In: LORENZI, T. F. Manual de hematologia: propedutica e clnica. 4. ed. Rio de Janeiro: Guanabara Koogan, 2006. Cap. 2, p. 152-194. LOUDEN, K. A.; BROUGHTON, P. F.; HEPTINSTALL, S.; FOX, S. C.; MITCHELL, J. R.; SYMONDS, E. M. Platelet reactivity and serum thromboxane B2 production in whole blood in gestational hypertension and pre-eclampsia. British Journal of Obstetrics and Gynaecology. v. 98, n. 12, p. 1239-1244, Dec. 1991. LWENBERG, E. C.; MEIJERS, J. C. M.; LEVI, M. Platelet-vessel wall interaction in health and disease. The Netherlands Journal of Medicine, v. 68, n. 6, Jun. 2010. MACEY, M.; MCCARTHY, D.; AZAM, U.; MILNE, T.; GOLLEDGE, P.; NEWLAND, A. Ethylenediaminetetraacetic acid plus citrate-theophyllineadenosine-dipyridamole (EDTA-CTAD): a novel anticoagulant for the flow cytometric assessment of platelet and neutrophil activation ex vivo in whole blood. Cytometry B Clinical Cytometry. v. 52, n.1 p. 30-40, Jan. 2003. MACEY, M. G.; BEVAN, S.; ALAM, S.; VERGHESE, L.; AGRAWAL, S.; BESKI, S.; THURAISINGHAM, R.; MACCALLUM, P. K.; Platelet activation and endogenous thrombin potential in pre-eclampsia. Thrombosis Research, v. 125, p. 76-81, 2010. MACEY, M. G.; ENNIKS, N.; BEVAN, S. Flow cytometric analysis of microparticle phenotype and their role in thrombin generation. Cytometry B Clinical Cytometry. v. 80, n.1, p. 5763, Jan. 2011.

MAKUYANA, D.; MAHOMED, K.; SHUKUSHO, F. D.; MAJOKO, F. Liver and kidney function tests in normal and pre-eclamptic gestation: a comparison with non-gestational reference values. Central African Journal of Medicine. v. 48, p. 5559, 2002. MARTINEZ, E. Z.; LOUZADA-NETO, F.; PEREIRA, B. B. A curva ROC para testes diagnsticos. Cadernos Sade Coletiva. v. 11, n. 1, p. 7-31, 2003. MARQUARDT, L.; RUF, A.; MANSMANN, U.; WINTER, R.; SCHULER, M.; BUGGLE, F.; MAYER, H.; GRAU, A. J. Course of platelet activation markers after ischemic stroke. Stroke. v. 33, n. 11, p. 2570-2574, Nov. 2002. MASOPUST, J.; MAL, R.; ANDRS, C.; VALI, M.; BAANT, J.; HOSK, L. Markers of thrombogenesis are activated in unmedicated patients with acute psychosis: a matched case control study. BMC Psychiatry. v. 11, n. 2, Jan. 2011. MCCRAE, K. R. Thrombocytopenia in pregnancy. Hematology/The Education Program of the American Society of Hematology. v. 2010; p. 397-402, 2010. MEADS, C. A.; CNOSSEN, J. S.; MEHER, S.; JUAREZ-GARCIA, A.; ter RIET, G.; DULEY, L.; ROBERTS, T. E.; MOL, B. W.; van der POST, J. A.; LEEFLANG, M. M.; BARTON, P. M.; HYDE, C. J.; GUPTA, J. K.; KAN, K. S. Methods of prediction and prevention of pre-eclampsia: systematic reviews of accuracy and effectiveness literature with economic modeling. Health Technology Assessment. v. 12, n. 6, p. 1-270, Mar. 2008. MELLEMBAKKEN, J. R.; SOLUM, N. O.; UELAND, T.; VIDEM, V.; AUKRUST, P. Increased concentrations of soluble CD40 ligand, RANTES and GRO-alpha in preeclampsia--possible role of platelet activation. Thrombosis Haemostasis. v. 86, n. 5, p. 1272-1276, Nov. 2001. MICHELSON, A. D. Flow cytometry: a clinical test of platelet function. Blood. v. 87, n.12, p. 4925-4936, Jun. 1996. MICHELSON, A. D.; BARNARD, M. R.; KRUEGER, L. A.; FRELINGER, A. L.; MARK, I. Furman evaluation of platelet function by flow cytometry. Methods. v. 21, n. 3, p. 259-270, Jul. 2000. MORRISON, R.; CRAWFORD, J.; MACPHESON, M.; HEPTINSTALL, S. Platelet behavior in normal pregnancy, pregnancy complicated by essential hypertension and pregnancy-induced hypertension. Thrombosis and Haemostasis. v. 54, n. 3, p. 607-611, 1985. MOR, A.; YANG, W.; SCHWARZ, A.; JONES, W. C. Platelet counts in pregnancy and labor. Obstetrics and Gynecology. v. 16, p. 338-342, 1960. NEIGER, R.; CONTAG, S. A.; COUSTAN, D. R. Preeclampsia effect on platelet count. American Journal of Perinatology. v. 9, p.378380, 1992.

NORONHA, J. F.; COSTA, F. F.; SAAD, S. T.; LORAND-METZE, I. G.; GROTTO, H. Z. Evaluation of reticulated platelets in patients with sickle cell diseases. Thrombosis Research. v. 121, n. 2, p. 259-267, May 2007. OSTERUD, B.; BJORKLID, E. Sources of tissue factor. Seminars of thrombosis and hemostasis. v. 32, n. 1, p. 11-23, 2006. PANASLUK, A.; ZAK, J.; KASPRZCKA, E.; JANICKA, K.; PROKOPOWICZ, D. Blood platelet and monocyte activations and relation to stages of liver cirrhosis. World Journal of Gastroenterology. v. 11, n. 18, p. 2754-2758, 2005. PASAOGLU, H.; BULDUK, G.; OGUS, E.; PASAOGLU, A.; ONALAN, G. Nitric oxide, lipid peroxides, and uric acid levels in pr-eclampsia and eclampsia. The Tohoku Journal of Experimental Medicine. v. 202, p. 87-92, 2004. PERRY, K. G. Jr.; MARTIN, J. N. Jr. Abnormal hemostasis and coagulopathy in preeclampsia and eclampsia. Clinical Obstetrics and Gynecology. V. 35, n. 2, p. 338-350, Jun. 1992. PITKIN, R. M.; WITTE, D. L. Platelet and leukocyte counts in pregnancy. Journal of the American Medical Association. v. 242, n. 24, p. 2696-2698, Dec. 1979. RABINI, R. A.; SALVOLINI, E.; STAFFOLANI, R. PUGNALONI, A.; SIMONELLI, L.; BIAGINI, G.; CESTER, N.; GARZETTI, G. G.; MAZZANTI, L. Biochemical-morphological modifications of platelet membranes during pregnancy-induced hypertension. Experimental and Molecular Pathology. v. 63, n. 3, p. 175-185, 1995. RAMSAY, J. E.; STEWART, F.; GREEN, I. A.; SATTAR, N. Microvascular dysfunction: a link between pre-eclampsia and maternal coronary heart disease. British Journal of Obstetrics and Gynaecology. v. 110, p. 1029-1031, 2003. REDMAN, C. W. G.; BONNAR, J.; BEILIN, L. Early platelet consumption in preeclampsia. British Medical Journal. v. 25, p. 467-469, Feb. 1978. REMUZZI, G.; RUGGENENTI, P. Prevention and treatment of pregnancyassociated hypertension: what have we learned in the last 10 years? American Journal of Kidney Disease. v. 18, n. 3, p. 285-305, Sep. 1991. Report of the National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy. National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy. American Journal of Ebstetrics and Gynecology. v. 183, n. 1, p. S1-22, Jul. 2000. REZENDE, J. D. Obstetrcia. Rio de Janeiro: Guanabara Koogan, 2005. ROBB, A. O.; DIN, J. N.; MILLS, N. L.; SMITH, I. B.; BLOMBERG, A.; ZIKRY, M. N.; RAFTIS, J. B.; NEWBY, D. E.; DENISON, F. C. The influence of the menstrual cycle, normal pregnancy and pre-eclampsia on platelet activation. The Journal of Thrombosis and Haemostasis, v. 103, n. 2, p. 372-378, Feb. 2010.

ROBERTS, J. M.; TAYLOR, R. N.; MUSCI, T. J.; ROGGERS, G. M.; HUBEL, C. A.; MCLAUGHLIN, M. K. Pr-eclmpsia: an endothelial cell disorder. American Journal of Obstetric and Gynecology. v. 161, n. 5, p. 1200-1204, Nov. 1989. ROBERTS, J. M.; REDMAN, C. W. G. Pre-eclampsia: more than pregnancyinduced hypertension. Lancet. v. 341, n. 8858, p. 1447-1454, Jun. 1993. SACKS, G. P.; STUDENA, K.; SARGENT, K.; REDMAN, C. W. Normal pregnancy and preeclampsia both produce inflammatory changes in peripheral blood leukocytes akin to those of sepsis. American Journal of Obstetric and Gynecology. v. 179, n. 1, p. 80-88, Jul. 1998). SANTOS, E. V.; FILHO, J. M. Plaquetograma em gestantes normais e com precmpsia. Revista Brasileira de Ginecologia e Obstetrcia. v. 26, n. 3, p. 201-206, 2004. SATHLER-AVELAR, R.; VITELLI-AVELAR, D. M.; MASSARA, R. L.; de LANA, M.; PINTO DIAS, J. C.; TEIXEIRA-CARVALHO, A.; ELI-SANTOS, S. M.; MARTINS-FILHO, O. A. Etiological treatment during early chronic indeterminate Chagas disease incites an activated status on innate and adaptive immunity associated with a type 1-modulated cytokine pattern. Microbes and Infection. v. 10, n. 2, p. 103-113, Feb. 2008. SBRANA, S.; BUFFA, M.; BEVILACQUA, S.; SPILLER, D.; PARRI, M. S.; GIANETTI, J.; De FILIPPIS, R.; CLERICO, A. Granulocyte and monocyte platelet adhesion index in coronary and peripheral blood after extracorporeal circulation and reperfusion. Cytometry B Clinical Cytometry. v. 72, n. 3, p. 215222, May 2007. SEJENY, S. A.; EASTHAM, R. D.; BAKER, S. R. Platelet counts during normal pregnancy. Journal of Clinical Pathology. v. 28, n. 10, p. 812-813, 1975. SHEU, J. R.; HSIAO, G.; SHEN, M. Y.; LIN, W. Y.; TZENG, C. R. The hyperaggregability of platelets from normal pregnancy is mediated through thromboxane A2 and cyclic AMP pathways. Clinical Laboratory and Haematology. v. 24, p. 121-129, 2002. SHEREMATA, W. A.; WENCHE,. J. Y.; HORSTMAN, L. L.; AHN, Y. S.; ALEXANDER, S.; MINAGAR, A. Evidence of platelet activation in multiple sclerosis. Journal of Neuroinflammation, v. 5, n. 27, 2008. SHOJI, T.; KOYAMA, H.; FUKUMOTO, S.; MAENO, T.; YOKOYAMA, H.; SHINOHARA, K.; EMOTO, M.; SHOJI, T.; INABA, M.; NISHIZAWA, Y. Plateletmonocyte aggregates are independently associated with occurrence of carotid plaques in type 2 diabetic patients. Journal of Atherosclerosis and Thrombosis. v. 12, n. 6, p. 344-352, 2005. SIBAI, B. M.; MABIE, W. C. Hemodynamics of preeclampsia. Clinics in Perinatology. v. 18, n. 4, p. 727-747, Dec. 1991. SIBAI, B. M.; RAMADAN, N. K; USTA, I. Maternal morbidity and mortality in 442 pregnacies with hemolysis, elevated liver enzymes, and low platelets HELLP

syndrome. American Journal of Obstetrics and Gynecology. v. 169, n. 4, p.1000-1006, Oct 1993a. SIBAI, B. M.; CARITIS, S. N.; THOM, E.; KLEBANOFF, M.; MCNELLIS, D.; ROCCO, L.; PAUL, R. H.; ROMERO, R.; WITTER, F.; ROSEN, M. et al. Prevention of preeclampsia with low-dose aspirin in healthy, nulliparous pregnant women. The National Institute of Child Health and Human Development Network of Maternal-Fetal Medicine Units. The New England Journal of Medicine. v. 329, n. 17, p. 1213-1218, Oct. 1993b. SIBAI, B.; DEKKER, G.; KUPFERMINC, M. Pre-eclampsia. Lancet. v. 365, p. 785-799, 2005. SIBAI, B. M. Prevention of preeclampsia: a big disappointment. American Journal of Obstetrics and Gynecology. v. 179, n. 5, 1998. SILJANDER, P. R. Platelet-derived microparticles an updated perspective. Thrombosis Research. v. 127 Suppl. 2, p. S3033, Jan. 2011. SILL, P. R.; LIND, T.; WALKER, W. Platelet values during normal pregnancy. British Journal of Obstetrics and Gynaecology. v. 92, n. 5, p. 480-483, 1985. SLOAND, J. A.; SLOAND, E. M. Studies on platelet membrane glycoproteins and platelet function during hemodialysis. Journal of the American Society of Nephrology. v. 8, n. 5, p. 799-803, May 1997. STAR, J.; ROSENE, K.; FERLAND, J.; DILEONE, G.; HOGAN, J.; KESTIN, A. Flow cyrometric analysis of platelet activation throught normal gestation. Obstetrics and Gynecology. v. 90, n. 4 Pt 1, p. 562-568, Oct. 1997. STIRLING, Y.; WOOLF, L.; NORTH, W. R.; SEGHETCHIAN, M. J.; MEADE, T. W. Haemostasis in normal pregnancy. Thrombosis and Haemostasis, v. 52, n. 2, p. 176-182, 1984. THANGARATINAM, S.; ISMAIL, K. M. K.; COOMARASAMY, A.; KHAN, K. S. Accuracy of serum uric acid in predicting complications of pr-eclampsia: a systematic review. A International Journal of Obstetrics and Gynecology. v. 113, n. 4, p. 369-378, Apr. 2006. THORT, J. F.; CHAHROUR, W.; YACOUB, D.; MERHI, Y. Recombinant Pselectin glycoprotein-ligand-1 delays thrombin-induced platelet aggregation: a new role for P-selectin early aggregation. British Journal of Pharmacology. v. 148, n. 3, p. 299-305, 2006. TOMER, A. Platelet activation as a marker for in vivo prothrombotic activity: detection by flow cytometry. Journal of Biological Regulators & Homeostatic Agents. v. 18, n. 2, p.172-177, Apr.- Jun. 2004. TROGSTAD, L.; MAGNUS, P.; STOLTENBERG, C. Pre-eclampsia: Risk factors and causal models. Best Practice & Research Clinical Obstetrics and Gynecology. v. 25, n. 3, p. 329-342, Jun. 2011.

TURGUT, B.; TURGUT, N.; YAHYA, .; TEKGNDZ, E.; PAMUK, E. G.; MUZAFFER, D. Differences in plateletleukocyte aggregates among subtypes of acute cerebral ischemia. Journal of the Neurological Sciences. v. 305, n. 1-2, p. 126-130, Jun. 2001. STN, Y. E.; DOGAN, K.; TRKOGLU, I.; STN, Y.; MEYDANLI, M. M.; KAFKASLI, A. Evaluation of hemoglobin and platelet levels in mild, moderate and severe preeclampsia. Perinatal Journal. V. 15, n. 3, p. 93-98, Dec, 2007. UZAN, J.; CARBONNEL, M.; PICONNE, O.; ASMAR, R.; AYOUBI, J. M. Preeclampsia: pathophysiology, diagnosis, and management. Journal of Vascular Health and Risk Management. v. 7, p. 467-474, 2011. van BEERS, E. J.; SCHAAP, M. C.; BERCKMANS, R. J. et al. Circulating erythrocyte-derived microparticles are associated with coagulation activation in sickle cell disease. Haematologica. v. 94, p. 15131519, 2009. van BLADEL, E. R.; ROEST, M.; de GROOT, P. G.; SCHUTGENS, R. E. Upregulation of platelet activation in hemophilia A. Haematologica. v. 96, n. 6, p. 888-895, Mar. 2011. van GILS, J. M.; ZWAGINGA, J. J.; HORDIJK, P. L. Molecular andfunctional interactions among monocytes, platelets, and endothelial cells and their relevance for cardiovascular diseases. Journal of Leukocyte Biology, v. 85, n. 2, p. 195-204, Feb. 2009. van IERSSEL, S. H.; van CRAENENBROECK, E. M.; CONRAADS, V. M.; et al. Flow cytometric detection of endothelial microparticles (EMP): effects of centrifugation and storage alter with the phenotype studied. Thrombosis Research. v. 125, p. 332339, 2010. VANTROYEN, B.; VANSTRAELEN, D. Management of essential thrombocytemia during pregnancy with aspirn, interferon alpha-2a ando no treatment. A comparative analysis of the literature. Acta Haematology. v. 107, n. 3, p. 158-169, 2002. van WIJK, M. J.; BOER, K.; BERKMANS, R. J.; MEIJERS, C. M.; van der POST, J. A. M.; STURK, A.; van BRAVEL, E.; NIEUWLAND, R. Enhanced coagulation activation in preeclampsia: the role of APC resistance, microparticles and other plasma constituents. Thrombosis and Haemostasis. v. 88, p. 415-420, 2002. VZQUEZ-RODRIGUEZ, J. G.; RICO-TREJO, E. I. Role of uric acid in preeclampsia-eclampsia. Ginecologa y Obstetricia de Mxico. v. 79, n. 5, p. 292-297, May 2011. VIEIRA, L. M.; DUSSE, L. M. S; FERNANDES, A. P.; MARTINS-FILHO, O. M.; BASTOS, M.; FERREIRA, M. F. R.; COOPER, A. J.; LWALEED, B. A.; CARVALHO, M. G. Monocytes and plasma tissue factor levels in normal individuals and patients with deep venous thrombosis of the lower limbs: Potential diagnostic tools? Thrombosis Research. v. 119, n. 2, p. 157-165, 2007.

von DADELSZEN, P.; WATSON, R. W.; NOORWALE, F.; MARSHALL, J. C.; PARODO, J.; FARINE, D.; LYE, S. J.; RITCHIE, J. W.; ROTSTEIN, O. D. Maternal neutrophil apoptosis in normal pregnancy, preeclampsia, and normotensive intrauterine growth restriction. American Journal of Obstetrics and Gynecology. v. 181, n. 2, p. 408-414, Aug, 1999. WALSH, M. T.; RYAN, M.; HILLMANN, A.; CONDREN, R.; KENNY, D.; DINAN, T.; THAKORE, J. H. Elevated expression of integrin alpha(IIb) beta(IIIa) in drugnave, firstepisode schizophrenic patients. Biological Psychiatry. v. 52, p. 874879, 2002. WANG, Y.; GU, Y.; LUCAS, M. J. Expression of thrombin receptors in endothelial cells and neutrophils from normal preeclamptic pregnancies. The Journal of Clinical Endocrinology & Metabolism. v. 87, n. 8, p. 3728-3734, 2002. WANGA, Y.; GU, Y.; PHILIBERTA, L.; LUCAS, M. J. Neutrophil activation induced by placental factors in normal and pre-eclamptic pregnancies in vitro. Placenta. v. 22, n. 6, p. 560-565, Jul. 2001. WAUGH, J. J.; CLARK, T. J.; KHAN, K. S.; KILBY, M. D. Accuracy of urinalysis dipstick techniques in predicting significant proteinuria in pregnancy. Obstetrics & Gynecology. v. 103, n. 4, p. 769-777, Apr. 2004. WILSON, B. J.; WATSON, M. S.; PRESCOTT, G. J, et al. British Medical Journal. v.326, p. 1-7, 2003. WOHNER, N. Role of cellular elements in thrombus formation and dissolution. Cardiovascular and Hematological Agents in Medicinal Chemistry. v. 6, n.3, p. 224-228, 2008. WORLD HEALTH ORGANIZATION. World health report: Make every mother and child count. Genebra: World Health Organization, 2005. YANG, H.; LANG, S.; ZHAI, Z.; LI, L.; KAHR, W. H.; CHEN, P.; BRKI, J.; SPRING, C. M.; FLICK, M. J.; DEGEN, J. L.; FREEDMAM, J.; NI, H. Fibrinogen is required for maintenance of platelet intracellular and cell-surface P-selectin expression. Bood, v. 114, n. 2, Jul. 2009. YONEYAMA, Y.; SUZUKI, S.; SAWA, R.; KIYOKAWA, Y.; POWER, G. G.; ARAKI, T. Plasma adenosine levels and P-selectin expression on platelets in preeclampsia. Obstetrics and Gynecology. v. 97, n. 3, p. 366-370, Mar, 2001. YOUNG, B. C.; LEVINE, R. J.; KARUMANCHI, S. Ananth. Pathogenesis of preeclampsia. Annual Review of Pathology. v. 5, p. 173-192, 2010. ZHONG, Y.; TUULI, M.; ODIBO, A. O. First-trimester assessment of placenta function and the prediction of preeclampsia and intrauterine growth restriction. Prenatal Diagnosis. v. 30, p. 293-308, Feb. 2010.