Thrombomodulin and Antithrombin-III as Evidence for Varied Activation of the Coagulation Cascade

Oct 28, 2011

Pre-eclampsia first described more than hundred years ago, remains a leading cause of maternal and fetal morbidity and mortality. Despite extensive researches, our knowledge of the aetiology and pathophysiology is still limited (Chappel & Bewley, 1998).

INTRODUCTION :  Pre-eclampsia first described more than hundred years ago, remains a leading cause of maternal and fetal morbidity and mortality. Despite extensive researches, our knowledge of the aetiology and pathophysiology is still limited (Chappel & Bewley, 1998).

Recently, there has been interest in the role of vascular endothelial damage in the pathogenesis of this disorder. It has been proposed that endothelial activation is the primary event in the multisystem disorder of pre-eclampsia. Evidence of endothelial involvement in this condition abounds. It is now known that endothelial activation contributes to the coagulation abnormalities observed in this disease (Dekker and Sibai, 1998).

In women with severe pre-eclampsia serum thrombomodulin (TM) was found to be significantly elevated indicating its circulatory release by damaged endothelium (Hsu et al., 1995). Also a low level of Antithrombin III has been reported in pre-eclamptic women (Verduzco & Gonzmlez, 1998).

AIM OF THE WORK :  To study the changes of two natural anticoagulant factors, circulating thrombomodulin level (TM) and anti-thrombin III (AT III) activity in both maternal and fetal cord plasma of women with pre-eclampsia and its correlation with fetal morbidity and postpartum regression of pre-eclampsia.

METHODS:  This study was conducted on 60 pregnant women, attending the obstetric clinics for follow up, at Ghamra Military Hospital during the period between September 1998 and May 1999. Subjects were all primigravidae with Singleton pregnancy in the third trimester. Their ages ranged from 25 – 35 years old. All of them signed informed consent for inclusion in the study.

These cases were divided into two main groups; the first comprised a control group of 20 healthy normal pregnant women and the second comprised a study group of 40 cases having pre-eclampsia. The pre-eclampsia group was further subdivided into mild pre-eclamptics (20 patients) and severe pre-eclamptics (20 patients).The criteria of severe pre-eclampsia were:

  • Diastolic blood pressure of 110 mmHg or higher.
  • Presence of proteinuria of 2+ or more or total proteins of 4-5 gm/litre/24hours.
  • Presence of symptoms, specifically haedache, visual disturbances&upper abdominal pains.

A full obstetric sheet planned for this work was fulfilled for every case. Thorough physical examination and laboratory investigations, as full blood picture, serum urea, creatinine, uric acid, serum transaminases (ALT, AST) total bilirubin and qualitative assessment of protein in urine with reagent paper strips were carried out.
A blood sample was taken from each subject on two occasions (before delivery and within 48 hours of delivery). Before delivery venous blood samples were obtained at the same gestational age in both the pre-eclampsia and the control group (34.4 + 1.9 weeks’ gestation) In pre-eclampsia blood samples were taken at diagnosis. Cord blood samples of about 5 ml. were also obtained at the time of delivery from the fetal side. Fetal birthright and Apgar score at one and five minutes were recorded.

Plasma was prepared from all samples by collection into glass tubes containing 0.109-M trisodium citrate 9 parts blood, 1 part anticoagulant). The samples were then centrifuged for 15 min. at 3000 rpm. Aliquots of plasma were stored at 80C until the assay was performed. Plasma specimens were tabled with only the date of sampling and the patient’s identification number.

DISCUSSION:  The current study was conducted to understand the relevance of the endothelium to subsequent activation of the coagulation cascade and to determine the changes of two natural anticoagulant factors, circulating TM levels and AT-III activity, in pre-eclamptic pregnancies. Moreover, this study aims to determine whether changes in plasma levels of TM and AT-III would correlate with maternal and fetal morbidity and with the postpartum regression of pre-eclampsia.

In this study, it was found that antepartum TM levels were significantly higher in pre-eclamptic women as compared with those of their matched controls. Also, this study correlated the severity of pre-eclampsia with TM levels. It compared the maternal plasma antepartum TM in different groups. The results showed that antepartum TM levels increase with the increase in the severity of pre-eclampsia (severe pre-eclampsia 76.50 + 6.50 ng/ml. mild pre-eclampsia 54.84 + 3.11 ng/ml, and control 41.83 + 4.01 ng/ml). The statistical difference between severe pre-eclampsia and control groups, mild pre-eclampsia and control groups, severe pre-eclampsia and mild pre-eclampsia were significant (P < 0.005).

In contrast to TM, it was found that antepartum plasma AT-III activities were significantly lower in both severely and mildly pre-eclamptic pregnancies. Moreover, we found that AT-III activities were inversely correlated with TM levels in severely pre-eclamptic pregnancies, but not in mild pre-eclamptic pregnancies (r = -0.633, P = 0.003).

The study at hand speculates that this may reflect a different degree of vascular endothelial damage and subsequent activation of the coagulation cascade between women with severe and mild pre-eclampsia. Consequently, there is an increase in the shedding of TM antigen into the blood of pre-eclamptic women and decreased availability of binding sites for thrombin and subsequently decreases circulating AT-III levels. Thus, increased unbound thrombin in pre-eclampsia subsequently enhances the consumption of AT-III and increases thrombin-AT-III complex levels (Reinthaller et al., 1990). In addition, injured endothelial cells could also lead to decreased secretion of heparin sulfate proteoglycan and impede the activity of AT-III on thrombin (Galstian et al., 1992). Thus, decreased AT-III levels may in part reflect the binding of AT-III to denuded areas of injured endothelium and subsequently shorten the activity elimination half-life (Weiner et al., 1990). 

These results are in agreement with the results of Minakami et al., (1993) and Bontis et al., (1995). However Hsu et al., (1993) and Hsu et al., (1995) found that the severely, but not mildly, pre-eclamptic women had significantly higher circulating TM levels, than those in their matched controls did. They concluded that this might indicate a different mechanism of disease entity in severely versus mildly pre-eclamptic pregnancies. In women with mild pre-eclampsia they speculated that perturbation of the endothelium might not be severe enough to increase the circulating TM levels and decrease the secretion of heparin sulfate proteoglycan. However, a functional change of TM conformation might decrease the activation of the TM-protein C anticoagulant pathway, which eventually increases unbound thrombin and subsequently decreases circulating AT-III levels.

This study compared the cord plasma TM and plasma AT III in different pre-eclamptic groups. It was found that the cord plasma TM levels were significantly higher in severely (P = 0.03), but not mildly (P = 0.13), pre-eclamptic women as compared with those of their matched controls. In contrast to TM, we found that cord plasma At-III levels were significantly lower in severely (P = 0.00), but not mildly (P = 0.43) pre-eclamptic women as compared with those of their matched controls.

The explanation of this can possibly be that, the high cord plasma TM and the low cord plasma AT-III may be originated from the maternal side and cross the placental barrier. In this study, there was a significant positive correlation between antepartum plasma TM level and cord plasma TM in the severely (r=0.678), but not mildly (r 0.109) pre-eclamptic groups. However, there was no significant correlation between antepartum AT-III level and cord plasma AT-III in the control (r=0.020) as well as in the pre-eclamptic groups. Further researches on the placental role in transferring TM and AT-III may be of benefit.

Another explanation of this could be that it might come from the fetal side affected by the severity of pre-eclampsia. One of the main complications of pre-eclampsia is fetal hypoxia. In this study, there was a significant negative correlation between cord plasma TM and Apgar score at l-min in mildly (r = 0.847) and severely (r =0.822) pre-eclamptic groups. However, there was a significant positive correlation between cord plasma AT-III and Apgar score at l-min. in mildly (r=0.818) and severely (r =0.961) pre-eclamptic groups.

Nako et al., (1997) concluded that hypoxia would injury vascular endothelial cells and consequently raised plasma TM levels in asphyxiated infants. They studied the plasma TM concentration at birth in 11 asphyxiated infants was significantly elevated compared with that in 48 infants without asphyxia (38.1 Vs 27.0 ug/l, P <0.0001).

Thus, the present study demonstrated the role of antepartum plasma TM and cord plasma TM in the prediction of fetal outcome in pre-eclamptic pregnancies and in reflecting the state of uteroplacental insufficiency, resulting in antepartum or intrapartum apoxia and also intrauterine fetal growth restriction. SO, the elevated levels of antepartum plasma TM and cord plasma TM, either done by cordocentesis or after delivery of the fetus should alert the clinician to the possibility of fetal jeopardy and the need for immediate interference.

Should elevated levels of antepartum plasma TM be used as an indicator for impairment of uteroplacental circulation and consequently poor fetal outcome necessitating termination of pregnancy is still subjected to further studies.

Table (1): Clinical Data in Severely and Mildly Pre-Eclamptic Groups

PARAMETER Control Mild Pre-eclampsia Severe Pre-eclampsia
MATERNAL AGE (yrs.)28.5 + 2.529.1+ 3.129.5 y + 2.7
Range26 – 31 y26 – 31 y25 – 35 y
Diastolic76 .4 + 10.796.4 + 13.1*103.5 + 14.3*
Range60 – 8590 – 10590 - 120
Systolic119.5 + 11.2138.8 + 14.1163.3 + 16.4*
Range105 – 135120 – 160145 - 190
GESTATIONAL Age (wk)    
At Sampling34.9 + 2.134.2 + 1.934.1 + 2.5
At Delivery39.1 + 1.738.2 + 2.136.4 + 1.9 *
Range37 – 4236 – 3934 - 37

Values are mean +/- SD * P < 0.05

 

Table (2): Lab Data in Severely and Mildly Pre-Eclamptic Groups

PARAMETER  CONTROLMILD Pre-ECLAMPSIASEVERE Pre-ECLAMPSIA
Proteinuria01 (+)3 (+)
S. Urea (mg/dl) 20.85 + 2.35 30.95 + 5.17 *37.65 + 7.25 *
S. Creatinine (mg/dl)0.86 + 0.161.26 + 0.27*1.52 + 0.34 *
S.Urate (mg/dl) 3.95 + 0.594.99 + 0.85 * 5.62 + 1.13 *
S.AST (U/ml)21.6 + 0.9131.7 + 0.75 *35.35 + 7.58*
S.ALT (U/ml)23.1 + 3.0034.7 + 7.66 * 38.25 + 8.25*
S. Bilirubin (mg/dl)0.8 + 0.11 + 0.091.09 + 0.09*
Antepartum TM (nglml) 41.83 + 4.0154.85 + 3.11 *76.51 + 6.51 *
Antepartum ATIII (U/ml) 308.78 + 8.40227.255 + 2.63*163.325 + 8.631*
TM/cr Ratio47.09 + 10.2745.26 + 9.0752.63 + 11.44*
AT III/cr ratio370.368 + 75.109187.731+ 37.635*113.388 + 21.473
Postpartum TM (ng/dl)32.39 + 3.5035.50 + 3.90*55.88 + 5.02*
Postpartum ATIII (U/ml)321.88 + 8.93284.22 + 5.47*269 .735+ 3.316*
Cord TM (ng/ml)29.45 + 7.3134.64 + 13.1343.745 + 26.551*
Cord ATIII (U/ml)289.63 + 5.01284.40 + 28.64259.165 + 50.464

Values are mean +/- SD * P < 0.05

 


   
  ????? ??????? ??????????????? ????? ???? ????????? 3 ????? ??? ????? ???? ?????? ??????? ?? ????? ????? ???????? ??????
( ???? ????? )

?0 ???? ???? ????
????? ????? ?????? ???????? ???????
???? ???? ????? ??????


??? ????? ?? ??? ??? ???? ????? ?? ) ????? ???????? ?????? ) ??? ???? ?????? ? ???? ?? ???? ?? ??????? ???????? ?????? ??????? ??????? 0

??? ???? ??? ????? ?????? ?????? ??????????? ????? ????? ??????????????? ????? ????????? 3 ?? ????? ???? ???? ???? ???? ?????? ?????? ?? ???? ??? ?????? ???? ???? ????? ????? 0

??? ?????? ????? ????? ???? ????? ??????????????? 3 ?? ??? ??????? ??????? ???? ???? ??? ??? ??? ?? ??? ???????? ?????? ( ???? ????? ) ????? ?? ?????? ????? ??? ?????? ?????? ???? ????????? ????????? ???? ?????? ????? 0 ????? ?????? ?????? ???? ?????? ???? ?? ?????? ????? ????????? ???????? ???? ???????? ???? ?????? 0

References:

REFERENCES:

Bontis, J., Vavilis, D., Agorastos, T. et al., (1995): Maternal plasma level of thrombomodulin is increased in mild pre-eclampsia. Eur. J. Obstet. Gynaecol. 60: 139: 141.

Chappell, L. and Bewley, S. (1998): Pre-eclamptic toxaemia: the role of uterine artery Doppler. Br. J. Obstet. Gynaecol. 105: 379-382.

Dekker, G.A., Sibai, B.M. (1998): Etiology and pathogenesis of pre-eclampsia: Current concepts. Am. J. Obstet. Gynaecol. 179: 1359 – 1375.

Hsu, C.D.; Iriye, B.; Johnson, T.R.B. et al., (1993): Elevated circulating thrombomodulin levels in severe pre-eclampsia. Am. J. Obstet. Gynaecol. 169:148-149.

Hsu, C.D.; Johnson, T.R.B., Hong, S.F. et al., (1995): Altered circulating thrombomodulin and antithrombin III activities as evidence for varied activation of the coagulation cascade in severe versus mild pre-eclampsia. J. Matern. Fetal Invest. 5: 140-143.

Galstian, A., Beer, A.E.; Roberts, J. M. et al., (1992): Immunology of pre-eclampsia. In: Coulam, C.B., Faulk, W.P. McIntyre, J.A. edit. Immunological Obstetrics. New York: W.W. Norton and Company Inc. 502 – 516.

Minakami, H.; Takahashi, T.; Izumi, A. et al., (1993): Increased levels of plasma thrombomodulin in pre-eclampsia. Gynaecol. Obstet. Invest. 36: 208-210.

Nako, Y.; Tomomasa, T.; Morikawa, A. (1997): Plasma thrombomodulin level in newborn infants with and without perinatal asphyxia. Acta Paediatr. 86:91-95.

Reinthaller, A.; Mursch-Edlmayr, G.; Tatra, G. (1990): Thrombin-antithrombin III complex levels in normal pregnancy and hypertensive disorders and after delivery. Br. J. Obstet. Gynaecol. 97:506-510.

Verduzco, R.L.; Gonzmlez, P.E. (1998): Antithrombin III in pre-eclampsia-eclampsia. Gynaecol. Obstet. Mex. 66: 343-346.

Weiner, C.P.; Herrig, J.E., Peizer, G.D. et al., (1990): Elimination of antithrombin III concentrate in healthy pregnant and pre-eclamptic women with an acquired antithrombin III deficiency. Thromb. Res. 58:395-401.