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Increased circulating thrombomodulin levels in pre-eclampsia
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Clinica Chimica Acta 387 (2008) 168 – 171 www.elsevier.com/locate/clinchim
Letter to the Editor Increased circulating thrombomodulin levels in pre-eclampsia Dear Editor, Thrombomodulin (TM) is a transmembranous glycoprotein widely expressed in a variety of cells and tissues in adults and during development [1,2]. It also exists in a soluble form in plasma, generated by enzymatic cleavage of the intact protein by pro-inflammatory mediators, and is known to play a pivotal role in maintaining normal haemostatic balance. TM binds thrombin, inhibits the procoagulant functions of thrombin and acts as a cofactor for thrombin catalyzed activation of protein C, which inactivates factor (F) Va, FVIIIa, and platelet aggregation, thereby inhibiting the blood coagulation cascade [3,4]. Paradoxically however, the TM-thrombin complex accelerates the proteolytic activation of thrombin activatable fibrinolysis inhibitor, and protects the fibrin clot against lysis [5]. Thus, TM appears to have anti-coagulant and anti-fibrinolytic properties. Endothelial injury or vascular damage releases TM into the circulation. Increased plasma TM levels have been reported to occur in various disease conditions including systemic lupus erythematosus [6], disseminated intravascular coagulation [7], a variety of infections, sepsis and inflammation [8]. Interestingly, plasma TM levels were inversely correlated with the development of coronary heart disease, implying that the soluble forms of TM may be vasculoprotective [9]. Pre-eclampsia (P-EC) is a multi-system obstetric disorder, complicating 2–3% of all pregnancies (5–7% in nulliparous women). Two percent of women with P-EC will progress to eclampsia leading to convulsions and potential maternal and fetal death. It is associated with increased intravascular coagulation [10], fibrin deposition [11] and inflammatory response compared to normal pregnancy [12]. Placental thrombi, which may compromise placental perfusion and fetal development, are frequently observed in women with P-EC. Thus, pre-disposing factors to thrombosis added to the thrombotic nature of placental vasculature may cause or contributes to the development of P-EC. A few studies have determined plasma markers of endothelial disturbance, including soluble TM, in women with P-EC, but results are inconsistent [13–16]. In the present study we aimed to examine these inconsistencies and to re-assessed plasma TM levels in three groups of age matched women: healthy non-pregnant; normal pregnant; and P-EC women, at the third trimester. The validity and precision of plasma TM levels in detecting P-EC were addressed using the standard methods for sensitivity and specificity and the receiver operating characteristic (ROC) curve. 0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2007.08.015
Ethical committee approval was granted for the study by the Southampton and South West Hampshire Research Ethics Committees A and informed consent was sought from all participants. The relevant clinical details for each of the 57 subjects were recorded. These included healthy non-pregnant women (n = 22; mean 29 ± 6.0 y), normal pregnant women (n = 15; 30.4 ± 5.0 y), and women with P-EC (n = 20; 29.1 ± 5.6 y), at the third trimester. Subjects were matched for maternal age, gestational age (third trimester) and parity. Blood samples, from women with P-EC were collected at the time of hospital admission, while those from normal pregnant women were obtained during a routine outpatient prenatal visit. P-EC was defined by a diastolic blood pressure N 110 mmHg at admission, or N90 mmHg on 2 or more consecutive occasions, 4 h apart; and proteinuria (either ≥ 300 mg protein/day or an urinary protein/creatinine ratio ≥ 30 mg/mmol) occurring after 20th week of pregnancy in women who had no previous symptoms [17]. The healthy nonpregnant or normal pregnant women (control groups) had systolic/diastolic blood pressure b 120/80 mmHg and no history of hypertension or proteinuria. Exclusion criteria common for the three groups were chronic hypertension, coagulation disturbance or haemostatic abnormalities, cardiovascular diseases, cancer, diabetes, renal and hepatic diseases, connective tissue disorders, autoimmune disease, anti-coagulant or corticosteroids therapy, and smoking. None of the women had hypertension in their reproductive years or P-EC during previous pregnancies. Five ml of venous blood was collected, using a 21-gauge needle, into vacutainer tubes containing 3.8% trisodium citrate anticoagulant solution. Following centrifugation of whole blood in 1.5 ml Eppendorf tubes at 3000 rpm for 10 min at room temperature, plasma samples were immediately isolated and 100 µl aliquots were stored at −70 °C for batch-wise analysis. Samples were subsequently thawed at 37 °C and assayed. An enzymelinked immunosorbent assay (ELISA) was used to measure TM levels in diluted plasma sample (1:4) according to the manufacturer’s instructions (American Diagnostica Inc., Stamford, Connecticut, USA). In principle, the IMUBIND®Thrombomodulin ELISA assay employs a primary monoclonal antibody which recognizes the EGF1–EGF2 domains of TM and a secondary horseradish peroxidase conjugated monoclonal antibody specific for the EGF5–EGF6 domains which recognizes bound TM. The assay recognizes native and truncated functional forms of TM and TM/thrombin complexes. The manufacturer claims no significant cross-reactivity or interference with other clotting factors. The lower limit of detection for plasma samples was found to be 0.300 ng/ml. Intra-assay Coefficient of Variation = 4.0% (n = 20); Interassay Coefficient of Variation = 5.2% (n = 20).
Letter to the Editor
A sample size power calculation was performed. In order to detect a proportional difference of 39%, between women with P-EC and healthy non-pregnant women, with a 5% 2-sided significance level and 80% power, the sample size in each group would be 20. Data were included in a database and analyzed by Sigma Stat software system version 1.0. Data were not normally distributed, and summary statistics were expressed as medians and inter-quartile ranges (IQR). Differences between two or more groups were assessed by Mann–Whitney U-Test, Kruskal– Wallis One-Way Analysis by Ranks or Dunn’s method. A P value of b 0.05 was considered to be statistically significant. Reliability measures were calculated using the standard methods for proportion. The sensitivity and specificity were also determined by measuring the area under the curve (AUC) and the 95% confidence interval (CI) of the ROC curves, which were constructed by plotting the sensitivity (true positive values) against 1-specificity.
169
The median and IQR for the 3 groups were; healthy nonpregnant women (n = 22; median = 0.56; IQR = 0.47–0.66); normal pregnant group (n = 15; median = 0.67; IQR = 0.47–0.87); women with P-EC (n = 20; median = 0.94; IQR = 0.69–1.1). There was a significant increase in plasma TM levels in women with P-EC compared to the healthy non-pregnant women group (P = 0.004). However, no statistically significant difference was observed between women with P-EC and normal pregnant women groups or between healthy non-pregnant and normal pregnant women groups by conventional methods. The distribution of plasma TM levels in the groups studied is shown in Fig. 1A. Plasma TM levels can distinguish women with P-EC from healthy non-pregnant women, at the third trimester, with good Sensitivity (70%) and Specificity (60%). Other reliability measures include True positive = 70%; False positive = 32%; True negative = 70%; False negative = 44%. Thus, the positive and negative predictive values were 70% and 60%, respectively.
Fig. 1. A. The distribution of plasma thrombomodulin levels (ng/ml) in the groups studied. Horizontal lines represent the median value for each group. Plasma TM levels were significantly raised in women with P-EC compared to healthy non-pregnant women group (P = 0.004). B. The ROC curve for plasma thrombomodulin levels in women with pre-eclampsia vs healthy non-pregnant women. C. The ROC curve for plasma thrombomodulin levels in women with pre-eclampsia vs normal pregnant women.
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Letter to the Editor
The AUC and the 95% CI of the ROC curve for P-EC women against healthy non-pregnant women groups were (0.79; 95% CI: 0.7–0.94%; P b 0.01; Fig. 1B). However, when the P-EC group was assessed against normal pregnant women the AUC and the 95% CI were (0.66; 95% CI: 0.5–0.84%; P N 0.05; Fig. 1C). Vascular damage is increasingly recognized as a prominent factor in the development of atherosclerosis and vascular complications known to occur in diabetics and in women with P-EC [18]. In fact there are striking similarities between P-EC and atherosclerosis [19]. In P-EC thrombin formation is uncontrolled, as evidenced by the presence of fibrin deposits occluding the spiral arteries and in the capillaries of renal glomeruli [20,21]. In addition, many placental villi from women with P-EC have been shown to contain fibrin deposits on TM-negative endothelium [22]. Circulating TM levels are associated with various disease conditions complicated by vascular endothelial cell damage and could be useful early indices for P-EC [23]. They may also represent essential clinical markers for differentiating between P-EC and other causes of pregnancy induced hypertension [15]. Patients with chronic renal failure have increased plasma TM levels and reduced urinary TM levels [24]. Therefore, increased plasma TM levels in women with P-EC may reflect a decrease in renal clearance rather than vascular endothelial cell damage [13]. However, this view is not supported by Hsu et al who showed that urinary TM levels in women with P-EC were not significantly reduced and that urinary TM-creatinine ratio was significantly higher in P-EC women compared to controls [25]. Two recent studies by independent groups showed that serum creatinine levels did not correlate significantly with serum TM levels in a group of women with pregnancy complications including P-EC, gestational hypertension, or chronic hypertension [15,26]. Serum TM levels were also significantly raised prior to the onset of P-EC but not in gestational hypertension, or chronic hypertension [15]. Collectively, these results lend unequivocal support to the fact that elevated plasma TM levels in women with P-EC are not secondary to renal impairment. It is more likely that plasma TM is derived from placental or vascular endothelial cells subsequent to activation or damage, and that vascular endothelial cells damage is the primary underlying cause of P-EC. In the present study we report a significant increase in circulating TM levels in women with P-EC compared to healthy non-pregnant women (P = 0.004; Fig. 1) but not normal pregnant women. We observed no statistical difference between healthy non-pregnant and normal pregnant women groups. The lack of statistical difference between these groups could potentially be due to sample size of the normal pregnant women group. However, our finding confirms the increased in plasma TM levels seen in cross sectional studies of P-EC and eclamptic women during 32–38 week of gestation [13–15,22,23,26]. These results support the concept that a direct relationship between plasma TM levels and endothelial cell damage in women with P-EC does exist. The pathogenic mechanism involved in increased plasma TM levels in women with P-EC is not fully understood. TM fragments detected in the blood are not secreted under normal physiological conditions [24,27], and
increased plasma TM levels in various clinical conditions appear secondary to vascular endothelial cells damage [28]. Such damage is thought to be involved in the development of P-EC [16]. It is also possible that neutrophil activation and superoxide generation, occurring during the last weeks of gestation [29], trigger endothelial TM proteolysis and increased plasma TM levels [30,31]. Plasma TM levels measured at week 32 may predict the development of gestational hypertension or P-EC [23]. Indeed, we found that circulating TM antigen levels can distinguish women with P-EC from healthy non-pregnant women with considerable sensitivity, specificity, positive and negative predictive values. However there was no statistically significant difference in plasma TM levels in P-EC compared to normal pregnant women in our study although there was an upward trend in the P-EC group. These results may reflect variations in TM levels occurring during the third trimester and/or a requirement for stricter criteria in the determination of the PEC group. Clearly TM levels are raised in pregnancy and P-EC. Further work is required to monitor TM levels against progression of gestation (in weeks) during the third trimester to determine if measures of TM have clinical usefulness in the early recognition and management of P-EC. In summary, taken together, previous data and our current finding suggest that understanding endothelial pathophysiology in P-EC, including its functional disturbance, could provide important insights into the clinical management of P-EC. Previous studies indicated that TM levels may potentially be a significant clinical marker for differentiating P-EC from other causes of pregnancy induced hypertension. Here, we reported considerable sensitivity, specificity, positive and negative predictive values for circulating TM levels in detecting P-EC against healthy non-pregnant women. Whilst we acknowledge that this result has no direct clinical value of itself, it strongly supports the need for further work to examine the relationship between plasma TM levels, progression of gestation and early detection of P-EC. Considering the current shortcomings in the diagnosis of P-EC (in both clinical as well as laboratory settings) it is important that efforts to understand the pathophysiological mechanisms occurring in P-EC and to develop a screening test for susceptibility/early stages of P-EC continue. Acknowledgements We thank Mr Rajnikant L. Mehta, Research and Development Unit, School of Medicine, University of Southampton, for his help with the statistical analysis, and the staff and the community midwifes at Princess Anne Hospital, Southampton, UK, for their professional skills and assistance with subject recruitments and sample collections. The study was support in part by CAPES, Brazil (Grant number: BEX 2694.05.0). The work was presented in the 47th annual scientific meeting of the British Society for Haematology, the 12th congress of the European Haematology Association and the XXIst Congress of the International Society on Thrombosis and Haemostasis.
Letter to the Editor
References [1] Boffa M-C, Burke B, Haudenschild C. Preservation of thrombomodulin antigen on vascular and extravascular surfaces. J Histochem Cytochem 1987;35:1267–79. [2] Imada M, Imada S, Iwasaki H, Kume A, Yamaguchi H, Moore E. : Marker surface protein of fetal development which is modulatable by cyclicAMP. Dev Biol 1987;122:483–91. [3] Esmon C. The roles of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem 1989;264:4743–6. [4] Esmon C. Thrombomodulin as a model of molecular mechanisms that modulate protease specificity and function at the vessel surface. FASEB J 1995;9:946–55. [5] Bajzar L, Morser J, Nesheim M. TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombin– thrombomodulin complex. J Biol Chem 1996;271:16603–8. [6] Takaya M, Ichikawa Y, Kobayashi N, et al. Serum thrombomodulin and anticardiolipin antibodies in patients with systemic lupus erythematosus. Clin Exp Rheumatol 1991;9:495–9. [7] Asakura H, Jokaji H, Saito M, et al. Plasma levels of soluble thrombomodulin increase in cases of disseminated intravascular coagulation with organ failure. Am J Hematol 1991;38:281–7. [8] Lohi O, Urban S, Freeman M. Diverse substrate recognition mechanisms for rhomboids: thrombomodulin is cleaved by mammalian rhomboids. Curr Biol 2004;14:236–41. [9] Wu KK. Soluble thrombomodulin and coronary heart disease. Curr Opin Lipidol 2003;14:373–5. [10] Brown MA. The physiology of preeclampsia. Clin Exp Pharmacol Physiol 1995;22:781–91. [11] Bonnar J, McNicol GP, Douglas AS. Coagulation and fibrinolytic systems in pre-eclampsia and eclampsia. Br Med J 1973;2:12–6. [12] Redmann CWG, Sacks GP, Sargent IL. Preeclampsia: an excessive maternal inflammatory response to pregnancy. Am J Obstet Gynecol 1999;180:499–506. [13] Minakami H, Takahashi T, Izumi A, Tamada T. Increased levels of plasma thrombomodulin in preeclampsia. Gynecol Obstet Invest 1993;36:208–10. [14] Hsu CD, Iriye B, Johnson TR, Witter FR, Hong SF, Chan DW. Elevated circulating thrombomodulin in severe preeclampsia. Am J Obstet Gynecol 1993;169:148–9. [15] Hsu CD, Copel JA, Hong SF, Chan DW. Thrombomodulin levels in preeclampsia, gestational hypertension, and chronic hypertension. Obstet Gynecol 1995;86:897–9. [16] Hayashi M, Inoue T, Hoshimoto K, Negishi H, Ohkura T, Inaba N. Characterization of five marker levels of the hemostatic system and endothelial status in normotensive pregnancy and pre-eclampsia. Eur J Haematol 2002;69:297–302. [17] National High Blood Pressure Education Program Working Group Report on High Pressure in Pregnancy. Am J Obstet Gynecol 1990;163:1691–712. [18] van Hinsbergh VWM. The endothelium: vascular control of haemostasis. Eur J Obstet Gynecol Reprod Biol 2001;95:198–201. [19] Roberts JM. Endothelial dysfunction in preeclampsia. Semin Reprod Endocrinol 1998;16:5–15. [20] Perry Jr KG, Martin Jr JN. Abnormal hemostasis and coagulopathy in preeclampsia and eclampsia. Clin Obstet Gynecol 1992;35:338–50. [21] Hustin J, Foidart JM, Lambotte R. Maternal vascular lesions in preeclampsia and intrauterine growth retardation: light microscopy and immunofluorescence. Placenta 1983;4:489–98.
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[22] Nadar SK, Yemeni EA, Blann AD, Lip GYH. Thrombomodulin, von Willebrand factor and E-selectin as plasma markers of endothelial damage/ dysfunction and activation in pregnancy induced hypertension. Thromb Res 2004;113:123–8. [23] Boffa MC, Valsecchi L, Fausto A, et al. Predictive value of plasma thrombomodulin in preeclampsia and gestational hypertension. Thromb Haemost 1998;79:1092–5. [24] Ishii H, Nakano M, Tsubouchi J, et al. Establishment of enzyme immunoassay of human thrombomodulin in plasma and urine using monoclonal antibodies. Thromb Haemost 1990;63:157–62. [25] Hsu CH, Lucas RB, Jonson TR, Hong SF, Chan DW. Elevated urine thombomodulin/creatinine ratio in severely preeclamptic pregnancies. Am J Obstet Gynecol 1994;171:854–6. [26] Bontis J, Vavilis D, Agorastos T, Zournatzi V, Konstantinidis T, Tagou K. Maternal plasma level of thrombomodulin is increased in mild preeclampsia. Eur J Obstet Gynecol Reprod Biol 1995;60:139–41. [27] Takano S, Kimura S, Ohdama S, Aoki N. Plasma thrombomodulin in health and diseases. Blood 1990;76:2024–9. [28] Asakura H, Jokaji H, Saito M, et al. Plasma levels of soluble thrombomodulin increase in cases of disseminated intravascular coagulation with organ failure. Am J Hematol 1991;38:281–7. [29] Tsukimori K, Maeda H, Ishida K, Nagata H, Koyanagi T, Nakano H. The superoxide generation of neutrophils in normal and preeclamptic pregnancies. Obstet Gynecol 1993;81:536–40. [30] Abe H, Okajima K, Okabe H, Takatsuki K, Binder BR. Granulocyte proteases and hydrogen peroxide synergistically inactivate thrombomodulin of endothelial cells in vitro. J Lab Clin Med 1994;123:874–81. [31] Boehme MW, Deng Y, Raeth U, et al. Release of thrombomodulin from endothelial cells by concerted action of TNF-alpha and neutrophils: in vivo and in vitro studies. Immunology 1996;87:134–40.
Luci M. Dusse* Maria G. Carvalho Faculty of Pharmacy, Federal University of Minas Gerais, Brazil E-mail address: [email protected]. ⁎ Corresponding author. Kathryn Getliffe David Voegeli Bashir A. Lwaleed School of Nursing and Midwifery, University of Southampton, Southampton, UK Alan J. Cooper Department of Biomedical Sciences, Portsmouth University, Portsmouth, UK Bashir A. Lwaleed Department of Urology, Southampton University Hospitals NHS Trust, Southampton, UK 27 June 2007
Clinica Chimica Acta 387 (2008) 168 – 171 www.elsevier.com/locate/clinchim
Letter to the Editor Increased circulating thrombomodulin levels in pre-eclampsia Dear Editor, Thrombomodulin (TM) is a transmembranous glycoprotein widely expressed in a variety of cells and tissues in adults and during development [1,2]. It also exists in a soluble form in plasma, generated by enzymatic cleavage of the intact protein by pro-inflammatory mediators, and is known to play a pivotal role in maintaining normal haemostatic balance. TM binds thrombin, inhibits the procoagulant functions of thrombin and acts as a cofactor for thrombin catalyzed activation of protein C, which inactivates factor (F) Va, FVIIIa, and platelet aggregation, thereby inhibiting the blood coagulation cascade [3,4]. Paradoxically however, the TM-thrombin complex accelerates the proteolytic activation of thrombin activatable fibrinolysis inhibitor, and protects the fibrin clot against lysis [5]. Thus, TM appears to have anti-coagulant and anti-fibrinolytic properties. Endothelial injury or vascular damage releases TM into the circulation. Increased plasma TM levels have been reported to occur in various disease conditions including systemic lupus erythematosus [6], disseminated intravascular coagulation [7], a variety of infections, sepsis and inflammation [8]. Interestingly, plasma TM levels were inversely correlated with the development of coronary heart disease, implying that the soluble forms of TM may be vasculoprotective [9]. Pre-eclampsia (P-EC) is a multi-system obstetric disorder, complicating 2–3% of all pregnancies (5–7% in nulliparous women). Two percent of women with P-EC will progress to eclampsia leading to convulsions and potential maternal and fetal death. It is associated with increased intravascular coagulation [10], fibrin deposition [11] and inflammatory response compared to normal pregnancy [12]. Placental thrombi, which may compromise placental perfusion and fetal development, are frequently observed in women with P-EC. Thus, pre-disposing factors to thrombosis added to the thrombotic nature of placental vasculature may cause or contributes to the development of P-EC. A few studies have determined plasma markers of endothelial disturbance, including soluble TM, in women with P-EC, but results are inconsistent [13–16]. In the present study we aimed to examine these inconsistencies and to re-assessed plasma TM levels in three groups of age matched women: healthy non-pregnant; normal pregnant; and P-EC women, at the third trimester. The validity and precision of plasma TM levels in detecting P-EC were addressed using the standard methods for sensitivity and specificity and the receiver operating characteristic (ROC) curve. 0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2007.08.015
Ethical committee approval was granted for the study by the Southampton and South West Hampshire Research Ethics Committees A and informed consent was sought from all participants. The relevant clinical details for each of the 57 subjects were recorded. These included healthy non-pregnant women (n = 22; mean 29 ± 6.0 y), normal pregnant women (n = 15; 30.4 ± 5.0 y), and women with P-EC (n = 20; 29.1 ± 5.6 y), at the third trimester. Subjects were matched for maternal age, gestational age (third trimester) and parity. Blood samples, from women with P-EC were collected at the time of hospital admission, while those from normal pregnant women were obtained during a routine outpatient prenatal visit. P-EC was defined by a diastolic blood pressure N 110 mmHg at admission, or N90 mmHg on 2 or more consecutive occasions, 4 h apart; and proteinuria (either ≥ 300 mg protein/day or an urinary protein/creatinine ratio ≥ 30 mg/mmol) occurring after 20th week of pregnancy in women who had no previous symptoms [17]. The healthy nonpregnant or normal pregnant women (control groups) had systolic/diastolic blood pressure b 120/80 mmHg and no history of hypertension or proteinuria. Exclusion criteria common for the three groups were chronic hypertension, coagulation disturbance or haemostatic abnormalities, cardiovascular diseases, cancer, diabetes, renal and hepatic diseases, connective tissue disorders, autoimmune disease, anti-coagulant or corticosteroids therapy, and smoking. None of the women had hypertension in their reproductive years or P-EC during previous pregnancies. Five ml of venous blood was collected, using a 21-gauge needle, into vacutainer tubes containing 3.8% trisodium citrate anticoagulant solution. Following centrifugation of whole blood in 1.5 ml Eppendorf tubes at 3000 rpm for 10 min at room temperature, plasma samples were immediately isolated and 100 µl aliquots were stored at −70 °C for batch-wise analysis. Samples were subsequently thawed at 37 °C and assayed. An enzymelinked immunosorbent assay (ELISA) was used to measure TM levels in diluted plasma sample (1:4) according to the manufacturer’s instructions (American Diagnostica Inc., Stamford, Connecticut, USA). In principle, the IMUBIND®Thrombomodulin ELISA assay employs a primary monoclonal antibody which recognizes the EGF1–EGF2 domains of TM and a secondary horseradish peroxidase conjugated monoclonal antibody specific for the EGF5–EGF6 domains which recognizes bound TM. The assay recognizes native and truncated functional forms of TM and TM/thrombin complexes. The manufacturer claims no significant cross-reactivity or interference with other clotting factors. The lower limit of detection for plasma samples was found to be 0.300 ng/ml. Intra-assay Coefficient of Variation = 4.0% (n = 20); Interassay Coefficient of Variation = 5.2% (n = 20).
Letter to the Editor
A sample size power calculation was performed. In order to detect a proportional difference of 39%, between women with P-EC and healthy non-pregnant women, with a 5% 2-sided significance level and 80% power, the sample size in each group would be 20. Data were included in a database and analyzed by Sigma Stat software system version 1.0. Data were not normally distributed, and summary statistics were expressed as medians and inter-quartile ranges (IQR). Differences between two or more groups were assessed by Mann–Whitney U-Test, Kruskal– Wallis One-Way Analysis by Ranks or Dunn’s method. A P value of b 0.05 was considered to be statistically significant. Reliability measures were calculated using the standard methods for proportion. The sensitivity and specificity were also determined by measuring the area under the curve (AUC) and the 95% confidence interval (CI) of the ROC curves, which were constructed by plotting the sensitivity (true positive values) against 1-specificity.
169
The median and IQR for the 3 groups were; healthy nonpregnant women (n = 22; median = 0.56; IQR = 0.47–0.66); normal pregnant group (n = 15; median = 0.67; IQR = 0.47–0.87); women with P-EC (n = 20; median = 0.94; IQR = 0.69–1.1). There was a significant increase in plasma TM levels in women with P-EC compared to the healthy non-pregnant women group (P = 0.004). However, no statistically significant difference was observed between women with P-EC and normal pregnant women groups or between healthy non-pregnant and normal pregnant women groups by conventional methods. The distribution of plasma TM levels in the groups studied is shown in Fig. 1A. Plasma TM levels can distinguish women with P-EC from healthy non-pregnant women, at the third trimester, with good Sensitivity (70%) and Specificity (60%). Other reliability measures include True positive = 70%; False positive = 32%; True negative = 70%; False negative = 44%. Thus, the positive and negative predictive values were 70% and 60%, respectively.
Fig. 1. A. The distribution of plasma thrombomodulin levels (ng/ml) in the groups studied. Horizontal lines represent the median value for each group. Plasma TM levels were significantly raised in women with P-EC compared to healthy non-pregnant women group (P = 0.004). B. The ROC curve for plasma thrombomodulin levels in women with pre-eclampsia vs healthy non-pregnant women. C. The ROC curve for plasma thrombomodulin levels in women with pre-eclampsia vs normal pregnant women.
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Letter to the Editor
The AUC and the 95% CI of the ROC curve for P-EC women against healthy non-pregnant women groups were (0.79; 95% CI: 0.7–0.94%; P b 0.01; Fig. 1B). However, when the P-EC group was assessed against normal pregnant women the AUC and the 95% CI were (0.66; 95% CI: 0.5–0.84%; P N 0.05; Fig. 1C). Vascular damage is increasingly recognized as a prominent factor in the development of atherosclerosis and vascular complications known to occur in diabetics and in women with P-EC [18]. In fact there are striking similarities between P-EC and atherosclerosis [19]. In P-EC thrombin formation is uncontrolled, as evidenced by the presence of fibrin deposits occluding the spiral arteries and in the capillaries of renal glomeruli [20,21]. In addition, many placental villi from women with P-EC have been shown to contain fibrin deposits on TM-negative endothelium [22]. Circulating TM levels are associated with various disease conditions complicated by vascular endothelial cell damage and could be useful early indices for P-EC [23]. They may also represent essential clinical markers for differentiating between P-EC and other causes of pregnancy induced hypertension [15]. Patients with chronic renal failure have increased plasma TM levels and reduced urinary TM levels [24]. Therefore, increased plasma TM levels in women with P-EC may reflect a decrease in renal clearance rather than vascular endothelial cell damage [13]. However, this view is not supported by Hsu et al who showed that urinary TM levels in women with P-EC were not significantly reduced and that urinary TM-creatinine ratio was significantly higher in P-EC women compared to controls [25]. Two recent studies by independent groups showed that serum creatinine levels did not correlate significantly with serum TM levels in a group of women with pregnancy complications including P-EC, gestational hypertension, or chronic hypertension [15,26]. Serum TM levels were also significantly raised prior to the onset of P-EC but not in gestational hypertension, or chronic hypertension [15]. Collectively, these results lend unequivocal support to the fact that elevated plasma TM levels in women with P-EC are not secondary to renal impairment. It is more likely that plasma TM is derived from placental or vascular endothelial cells subsequent to activation or damage, and that vascular endothelial cells damage is the primary underlying cause of P-EC. In the present study we report a significant increase in circulating TM levels in women with P-EC compared to healthy non-pregnant women (P = 0.004; Fig. 1) but not normal pregnant women. We observed no statistical difference between healthy non-pregnant and normal pregnant women groups. The lack of statistical difference between these groups could potentially be due to sample size of the normal pregnant women group. However, our finding confirms the increased in plasma TM levels seen in cross sectional studies of P-EC and eclamptic women during 32–38 week of gestation [13–15,22,23,26]. These results support the concept that a direct relationship between plasma TM levels and endothelial cell damage in women with P-EC does exist. The pathogenic mechanism involved in increased plasma TM levels in women with P-EC is not fully understood. TM fragments detected in the blood are not secreted under normal physiological conditions [24,27], and
increased plasma TM levels in various clinical conditions appear secondary to vascular endothelial cells damage [28]. Such damage is thought to be involved in the development of P-EC [16]. It is also possible that neutrophil activation and superoxide generation, occurring during the last weeks of gestation [29], trigger endothelial TM proteolysis and increased plasma TM levels [30,31]. Plasma TM levels measured at week 32 may predict the development of gestational hypertension or P-EC [23]. Indeed, we found that circulating TM antigen levels can distinguish women with P-EC from healthy non-pregnant women with considerable sensitivity, specificity, positive and negative predictive values. However there was no statistically significant difference in plasma TM levels in P-EC compared to normal pregnant women in our study although there was an upward trend in the P-EC group. These results may reflect variations in TM levels occurring during the third trimester and/or a requirement for stricter criteria in the determination of the PEC group. Clearly TM levels are raised in pregnancy and P-EC. Further work is required to monitor TM levels against progression of gestation (in weeks) during the third trimester to determine if measures of TM have clinical usefulness in the early recognition and management of P-EC. In summary, taken together, previous data and our current finding suggest that understanding endothelial pathophysiology in P-EC, including its functional disturbance, could provide important insights into the clinical management of P-EC. Previous studies indicated that TM levels may potentially be a significant clinical marker for differentiating P-EC from other causes of pregnancy induced hypertension. Here, we reported considerable sensitivity, specificity, positive and negative predictive values for circulating TM levels in detecting P-EC against healthy non-pregnant women. Whilst we acknowledge that this result has no direct clinical value of itself, it strongly supports the need for further work to examine the relationship between plasma TM levels, progression of gestation and early detection of P-EC. Considering the current shortcomings in the diagnosis of P-EC (in both clinical as well as laboratory settings) it is important that efforts to understand the pathophysiological mechanisms occurring in P-EC and to develop a screening test for susceptibility/early stages of P-EC continue. Acknowledgements We thank Mr Rajnikant L. Mehta, Research and Development Unit, School of Medicine, University of Southampton, for his help with the statistical analysis, and the staff and the community midwifes at Princess Anne Hospital, Southampton, UK, for their professional skills and assistance with subject recruitments and sample collections. The study was support in part by CAPES, Brazil (Grant number: BEX 2694.05.0). The work was presented in the 47th annual scientific meeting of the British Society for Haematology, the 12th congress of the European Haematology Association and the XXIst Congress of the International Society on Thrombosis and Haemostasis.
Letter to the Editor
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Luci M. Dusse* Maria G. Carvalho Faculty of Pharmacy, Federal University of Minas Gerais, Brazil E-mail address: [email protected]. ⁎ Corresponding author. Kathryn Getliffe David Voegeli Bashir A. Lwaleed School of Nursing and Midwifery, University of Southampton, Southampton, UK Alan J. Cooper Department of Biomedical Sciences, Portsmouth University, Portsmouth, UK Bashir A. Lwaleed Department of Urology, Southampton University Hospitals NHS Trust, Southampton, UK 27 June 2007