Role Of Haemostasis And Thrombosis In The Pathogenesis Of Preeclampsia

Preeclampsia is a frequent complication of pregnancies and is one of the leading causes of maternal and neonatal mortality and morbidity worldwide.Although the exact pathogenesis of preeclampsia is not fully understood,it is confirmed that the core of pathophysiological mechanisms is the dysfunction of maternal vascular endothelium. Alterations in the haemostatic system resulting in the pathological hypercoagulability and thrombophilias may contribute to the clinical spectrum of preeclampsia.However,the role of the endothelium dysfunction in hypercoagulability and thrombophilias and the importance of the various critical molecules of haemostatic system in the development of proteinuria and hypertension during preeclampsia remain to be elucidated.In this research,we first studied the alternations of haemostasis and thrombosis in normal pregnancy.Secondly,we investigate the alternations of haemostasis and thrombosis in preeclampsia and analyzed the relationship between these alternations and severity. Finally,we applied low molecular weight heparin(LMWH) to treat women with early onset preeclampsia and observed the changes of haemoststic system.We proposed to explore the mechanisms of the alternations of haemostasis and thrombosis in preeclampsia,to search more sensitive and effective haemostatic parameters for the monitoring of condition of preeclampsia,further we will investigate the role of haemostasis and thrombosis in the pathogenesis of preeclampsia.TFPI-2 is a special anticoagulant factor which has several functions including remolding of cell matrix,angiogenesis,regulating of tumor invasion and cell apoptosis.The physiological functions of TFPI-2 in pregnancy are poorly understood.In order to invetigate the role of TFPI-2 in normal pregnancy and in the pathogenesis of preeclampsia,we used time-resolved fluoroimmunoassay(TRFIA) and immunohistochemistry to evaluate maternal serum concentrations and placental,expression of TFPI-2 in nonpregnant women,normal pregnant women at different trimesters and preeclamptic women. Section 1 Maternal and Fetal alternations of haemostasis and thrombosis in normal pregnancySection 1.1 Maternal alternations of haemostasis and thrombosis in normal pregnancyObjective:To explore the maternal changes of haemostasis and thrombosis, including vascular endothelial functions,coagulant activation, anticoagulant activation and fibrinolytic activation,in normal pregnancy before and after delivery.Methods:Maternal serum concentrations of von willebrand factor(vWF), tissue factor(TF),tissue factor pathway inhibitor(TFPI), fibrinopeptide A(FPA) and D-Dimer were measured by ELISA in forty normal pregnant women and twenty healthy women without pregnancy.Results:Compared with serum of nonpregnant women,serum levels of vWF, TFPI and D- Dimer in pregnant women increased significantly before and after delivery(P＜0.01),and serum levels of TF increased significantly at delivery(P＜0.05).Serum levels of FPA were similar between pregnant and nonpregnant women(P＞0.05).A lower trend of maternal serum concentrations of vWF and TF were observed after delivery,companied with a higher trend of maternal serum concentrations of TFPI and D- Dimer(P＞0.05).Conclusion:During normal pregnancy the activation of coagulation is counterbalanced by the activation of fibrinolysis and anticoagulation, which maintains the haemostatic balance.The decreased coagulation and increased anticoagulation and fibrinolysis resulted in the fibrinolytic accentuation after delivery.Section 1.2 Fetal alternations of haemostasis and thrombosis in normal pregnancyObjective:To explore the fetal changes of haemostasis and thrombosis in normal pregnancy,including vascular endothelial functions,platelet activation,coagulant activation,anticoagulant activation and fibrinolytic activation.Methods:Fetal serum concentrations of vWF,TF,TFPI,soluble P-selectin (sP-selectin),antithrombaseⅢ(AT-Ⅲ),FPA and D-Dimer were measured by ELISA in forty normal pregnant women.Results:Fetal serum levels of TF and sP-selectin(68.97±5.95ng/L and 91.78±7.84μg/L) were significantly higher than maternal serum (56.17±5.72ng/L and 77.43±6.57μg/L)(P＜0.05,respectively),while fetal serum levels of vWF,TFPI and AT-Ⅲ(871.44±118.97U/L, 41.72±3.78μg/L and 265.13±37.31mg/L) were significantly lower than maternal serum(2435.99±155.09U/L,93.39±4.85μg/L and 710.09±183.94mg/L)(P＜0.05,respectively).Fetal serum levels of FPA and D-Dimer were higher than maternal serum(P＞0.05).Conclusion:The hypercoagulable state in fetus is even more prominent with increased coagulation and decreased anticoagulation and fibrinolysis as compared with mother.Section 2 Maternal and Fetal alternations of haemostasis and thrombosis in preeclampsiaSection 2.1 Maternal alternations of haemostasis and thrombosis in preeclampsiaObjective:To investigate the maternal changes of haemostasis and thrombosis in preeclampsia,including vascular endothelial functions, platelet activation,coagulant activation,anticoagulant activation and fibrinolytic activation.Methods:Maternal serum concentrations of vWF,TF,TFPI,sP-selectin, AT-Ⅲ,FPA and D-Dimer were measured by ELISA in forty-six women with preeclampsia and forty normal pregnant women before and after delivery.Results:Compared with maternal serum in normal pregnancy,higher concentrations of maternal serum TF,vWF and sP-selectin as well as lower concentrations of maternal serum TFPI and D-Dimer in preeclampsia were observed before and after delivery.Maternal serum levels of AT-Ⅲdecreased at delivery and increased after delivery.A lower trend of maternal serum concentrations of vWF,TF and sP-selectin were found after delivery,companied with a higher trend of maternal serum concentrations of TFPI,AT-Ⅲand D- Dimer.Maternal serum concentrations of FPA were similar between two groups before and after delivery.Conclusions:Pathological hypercoagulability and thrombophilias resulted from changes in the haemostatic system which included impaired endothelial functions,increased coagulation and platelet activation and decreased anticoagulation and fibrinolysis are the important pathophysiological alternations in preeclampsia.Section 2.2 Fetal alternations of haemostasis and thrombosis in preeclampsiaObjective:To investigate the fetal changes of haemostasis and thrombosis in preeclampsia,including vascular endothelial functions,platelet activation,coagulant activation,anticoagulant activation and fibrinolytic activation.Methods:Fetal serum concentrations of vWF,TF,TFPI,sP-selectin,AT-Ⅲ, FPA and D-Dimer were measured by ELISA in forty-six women with preeclampsia and forty normal pregnant women.Results:Compared with maternal serum,fetal serum concentrations of vWF, TFPI and AT-Ⅲwere decreased significantly in preeclampsia,while TF concentrations were increased significantly.Fetal serum concentrations of sP-selectin and D-Dimer were lower than maternal serum although the difference had no significance.Serum levels of FPA were similar between mothers and fetus.Compared with fetal serum in normal pregnancy,fetal serum concentrations of TF were markedly increased in preeclampsia, accompanied with a higher level of vWF and a lower level of sP-selectin but without statistical significance.Fatal serum levels of other haemostatic parameters were similar between normal pregnancy and preeclampsia.Conclusion:The hypercoagulable state and thrombophilias of fetus in preeclampsia is even more prominent than its mothers and fetus in normal pregnancy which resulted in the higher incidence of complications such as FGR,PAS and abortion. Section 3 Relationship between alternations of haemostasis and thrombosis and severity of preeclampsiaObjective:To explore the relationship between alternations of haemostasis and thrombosis and severity of preeclampsia and search sensitive and effective haemostatic parameters to monitor the condition of preeclampsia.Methods:Based on above research,we used bivariate correlation and t test to analyze the relationship between maternal and fetal changes of haemostasis and thrombosis and severity of preeclampsia which were judged by the clinical parameters including blood pressure,proteinuria,liver and renal functions,blood routine examination,birth weeks,birth weight, Apgar scores and maternal and fetal complications.Results:1.There were significant positive relationships between SBP, DBP and TF(r=0.218 and r=0.234;p＜0.05,respectively).And also, significant positive relationships between proteinuria and TF, sP-selectinwere observed(r=0.225 and r=0.229;p＜0.05,respectively). No marked relationships were observed between other clinical parameters and the hemostatic parameters.2.According to SBP as 160mmHg,we further divided these pregnant women into two groups.Maternal Serum concentrations of TF were significantly increased in hypertensive group than in normotensive group before and after delivery.On the other hand, a higher trend of vWF,FPA,sP-selectin,TFPI and a lower trend of AT-Ⅲwere observed in hypertensive groups before and after delivery, especially the differences of vWF at delivery and FPA on day 5 after postpartum had significance.Maternal serum levels of D-Dimer and routine haemostatic examinations were similar between two groups.3.Similarly, all participants were divided into abnormal group(proteinuria≥++) and normal group(proteinuria＜++).Maternal serum concentrations of sP-selectin were significantly increased before and after delivery,well as maternal serum concentrations of TF were increased significantly at delivery and on day 5 after postpartum in abnormal group than in normal group.In addition,maternal serum levels of other parameters and routine haemostatic examinations were similar between two groups.4.Acording to maternal complications,all pregnant women were divided into two groups. Fatal serum levels of TF,TFPI and vWF were increased significantly in group with maternal complications compared with group without maternal complications.Maternal serum levels of TF and vWF were highter in group with maternal complications than in group without maternal complications (74.33±22.32ng/L and 2629.21±226.40U/L vs 49.17±3.97ng/L and 2106.79±159.64U/L;P=0.06,P=0.07,respectively).Maternal and fetal serum levels of other parameters were similar between two groups.5. Acording to fetal complications,all pregnant women were divided into two groups.Maternal and fetal serum concentrations of TF increased significantly in group with fetal complications compared with group without fetal complications(86.21±19.65ng/L and 161.63±74.39ng/L vs 51.62±4.50ng/L and 72.38±5.17ng/L;P＜0.05,respectively).Maternal and fetal serum levels of other parameters were similar between two groups.Conclusion:The degree of bypercoagulable state is positively related to the severity of preeclampsia,especially the serum levels of TF is the most sensitive changes which can be used to monitor the severity of preeclampsia.Section 4 Expectant treatment of early onset preeclampsiaObjective:It is found that the degree of hypercoagulable state is positively related to the severity of preeclampsia through above studies. Therefore we investigated the feasibility of expectant treatment which used LMWH to correct the hypercogulable state of women with early onset preeclampsia.Methods:Maternal serum concentrations of vWF,TF,TFPI and FPA were measured by ELISA in sixteen women with early onset preeclampsia who received 5000IU/d LMWH about seven days and twenty-seven women with early onset preeclampsia who received routine treatmen.Results:SBP and DBP were decreased significantly and gestational weeks were prolonged significantly in study group compared with the control group.Maternal serum levels of IF decreased significantly in study group than in the control group before and after delivery,well as maternal serum levels of vWF,FPA and sP-selectin decreased significantly in study group at delivery.Conclusion:Our study revealed that LMWH can effectively improve the hypercoagulable state of women with early onset preeclampsia,smoothing the clinical symptoms and decrease the incidence of maternal and fetal complications.The pathological changes of haemostasis and thrombosis may play an important role in the pathogenesis of preeclampsia and result to aggravate the illness.The abundant release of TF resulted from impairment of vascular endothelial cells,which triggered the extrinsic pathway of coagulation,may play a key role in the pathogenesis of preeclampsia.Section 5 Role of TFPI-2 in the pathogenesis of preeclampsiaObjective:TFPI-2 is a special anticoagulant factor which had several functions including remolding of cell matrix,angiogenesis,regulating of tumor invasion and cell apoptosis.In order to explore the physiological functions of TFPI-2 in normal pregnancy and the role in the pathogenesis of preeclampsia,we evaluated maternal serum concentration and placental expression of TFPI-2 in normal pregnancy and preeclampsia.Methods:Maternal serum and placental concentrations of TFPI-2 were measured by time resolved fluoroimmunoassay(TRFIA) and Immunohistochemistry in twenty women without pregnancy,five hundred and eighteen normal pregnant women at different trimesters,forty women in normal pregnancy and forty-six women in preeclampsia before and after delivery.Results:1.Maternal serum levels of TFPI-2 in normal pregnant women at 13 weeks of gestation increased 9.3 times as compared with healthy women without pregnancy(149.3±17.1 vs 16.0±3.6ng/ml).This was followed by a gradual increase towards term,reached a maximum mean value of 282.6±17.1ng/ml at 39 weeks of gestation and then gradual decreased to 259.3±49.8ng/ml at 42 weeks of gestation.2.Maternal serum concentrations of TFPI-2 were significantly higher in normal pregnancy than inpreeclampsia(263.4±8.7 vs 193.8±18.8ng/ml),but there were no significant differences in this value which dramatically decreased to non-pregnant levels after delivery between two groups.3.TFPI-2 was detected in the cytoplasm of syncytiotrophoblasts,but not in any other type of cell such as cytotrophoblasts,decidual cells,stromal cells,or chorionic vascular endothelial cells.Placental expression of TFPI-2 was low during the first trimester,increased gradually in the second trimester and reached a maximum level in the third trimester.Placental expression of TFPI-2 decreased significantly in preeclampsia compared to normal pregnancy,well as it decreased significantly in severe preeclampsia than in mild preeclampsia.Conclusion:TFPI-2 is secreted from the cytotrophoblasts in placenta and may play an important role in maintaining the normal pregnancy.TFPI-2 may also play a role in the pathogenesis of preeclampsia through affecting invasion,apoptosis,differentiation and proliferation of trophoblasts.