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Abstract
Microparticles (MP) are small membrane vesicles that are released from various cell types during cell activation and apoptosis. Their presence in vivo has been demonstrated in several studies (Combes et al., 1999, Horstman and Ahn, 1999, Mallat et al., 1999, Nieuwland et al., 2000, Nomura et al., 2000, Berckmans et al., 2001, Joop et al., 2001, Minager et al., 2001). These vesicles, resulting from an exocytic budding process, consist of cytoplasmic components, expressing negatively charged phospholipids and cell surface antigens characteristic of the cell they are derived from. Surface exposure of phosphatidylserine, and in some MP expression of active tissue factor, provide a catalytic surface that supports the assembly of clotting enzymes complexes, leading to thrombin generation (Bevers et al., 1998). Beside this procoagulant potential, MPs are able to elicit various biological responses including apoptosis (Albanese et al., 1998), platelet activation (Barry et al., 1997) or endothelial stimulation (Mesri & Altieri, 1998, 1999).
All types of cells can generate microparticles, and elevated levels may reflect increased cell activation and/or production or impairment of clearance by the reticuloendothelial system (Laude et al., 2001).
Microparticle numbers are increased in diseases involving hyper- coagulation, such as in patients with idiopathic thrombocytopenia, ischemic brain diseases and acute coronary syndromes (Mallat et al., 1999, Tate et al., 1992,Jy et al., 1992).
Furthermore, microparticles numbers are decreased in several bleeding disorders, such as Scott syndrome and Castaman’s defect (Horstman and Ahn., 1992).
Normal pregnancy is associated with extensive anatomic and functional adaptation of the cardiovascular system to accommodate the demands of pregnancy, in preeclampsia this adaptation is inadequate (VanWijk et al., 2000). Endothelial cells, leucocytes and the coagulation system all are activated in normal pregnancy, while in preeclampsia this activation seems exaggerated (Redman et al., 1999).
Preeclampsia (PE) is a vascular complication that occurs in 5 to 7% of pregnancies, the pathophysiology of which remain unclear (Khong et al., 1986). In PE, uncharacterized circulating substances originating from compromised placenta may induce pathological responses involving vascular and blood cells (Taylor et al., 1998). Biological evidence of endothelial dysfunction or damage has been shown by modified plasma levels of markers including cellular fibronectin, Von Willbrand factor, soluble thrombomodulin and adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selection (VanWijk et al., 2000, Boffa et al., 1998, Austgulen et al., 1996).
In theory, microparticles could cause the alterations that occur in preeclampsia, as they have been presented recently as a new marker of cellular activation (VanWijk et al., 2002a).
In the present study, the aim is to explore the microparticles involved in the control of hemostatic equilibrium, i.e. MP that are originating from platelets, endothelial cells and total MP defined as annexin V positive micropaticles, in normal pregnancy and in preeclampsia..
Subjects:
Fifty pregnant women who gave informed consent were recruited for the study. Twenty five women had PE were selected on the basis of the criteria as described by the International Society for the study of Hypertension in Pregnancy: diastolic blood pressure of ≥110 mmHg on any occasion or ≥90 mmHg on 2 separate occasions that were at least 4 hours apart; proteinuria of 0.3 g of protein per 24 h or more than +1 on a dip stick on two separate occasions at least four hours apart after the twenty weeks of gestation. A group of twenty five -age and gestation-matched women will be selected as healthy pregnant subjects. These healthy pregnant women were with no history of medical illness, attending the routine antenatal clinics. They must present an uneventful pregnancy at the day of inclusion with no vascular complication until the end of pregnancy. Exclusion criteria for healthy pregnant women were arterial blood pressure upper 140/90 mmHg and proteinuria > 0.3 g (Bretelle et al., 2003). Twenty five -age matched- healthy non-pregnant normotensive women were selected as a control group who were not receiving any medication (including oral contraceptives).
Methods:
1/ Urine samples were collected from all women to detect protein in urine.
2/ Blood samples were collected from them in three tubes,
a) Tube containing 0.105 M buffered sodium citrate for assessment of PT and PTT and flowcytometer analysis.
b) Tube with EDTA for complete blood picture.
c) Plain tube for assessment of Serum creatinine, liver enzymes and random blood glucose.
After preparation of PPP and isolation of MPs, the sample analyzed by flow cytometer by using FITC-labelled annexin V, specific monoclonal antibodies against endothelial and platelet antigens FITC-CD31 and PE-CD41
The results in short were:
First; total number of circulating MP in the three groups showed no significant difference.
Second; PMP (CD31+/CD41+) level were not significantly different in the three groups.
Third; EMP (CD31+/CD41-) level increased significantly in PE (compared to NP and NO). And its level in NO group not significantly different when compared to NP group.
Quantification of EMPs may be a useful objective tool in the conservative treatment of severe preeclampsia that is remote from term, if progressively increasing levels denote worsening disease in a patient who is clinically stable.
Systolic and diastolic blood pressure was significantly correlated with levels of CD31+/ CD41- EMP, which indicates that the severity of hypertension is likely indicative of progressive endothelial damage. This supports recent findings in which nonpregnant patients with severe hypertension exhibited elevated EMP values when compared with the mild hypertension and control groups (Mallat, et al., 2000).
In conclusion, the significant elevation in EMPs in this study supports the theory of endothelial injury in the pathogenesis of preeclampsia. Further studies of EMPs are needed to evaluate their diagnostic and prognostic value in preeclampsia.