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Increased high molecular weight fibrinogen in pre-eclampsia
Thrombosis Research 111 (2003) 143 – 147
Regular Article
Increased high molecular weight fibrinogen in pre-eclampsia G.T.R. Manten *, J.M. Sikkema, A. Franx, T.M. Hameeteman, G.H.A. Visser, P.G. de Groot, H.A.M. Voorbij Department of Perinatology and Gynaecology, University Medical Center Utrecht, F05.829, PO Box 85090, 3508 AB Utrecht, The Netherlands Received 13 June 2003; received in revised form 4 August 2003; accepted 4 August 2003
Abstract Introduction: The major coagulation protein fibrinogen (Fg) is a heterogeneous protein with three main fractions: high molecular weight fibrinogen (HMW-Fg), low molecular weight fibrinogen (LMW-Fg) and low molecular weightV fibrinogen. The clottability of high molecular weight fibrinogen is highest as compared to the other fractions. Pre-eclampsia is associated with a state of hypercoagulability, and with an increase of fibrinogen concentration. The aim of the present study was to examine if the increased total fibrinogen plasma concentration in patients with pre-eclampsia is associated with a change in distribution of the main fibrinogen fractions. Material and Methods: Plasma was collected from 14 patients with pre-eclampsia and from 14 healthy pregnant matched controls. Total fibrinogen concentrations were determined according to Clauss. The percentage high molecular weight fibrinogen was assessed by SDS-electrophoresis and densitometry after isolation of fibrinogen by precipitation. The study groups were compared by the Mann – Whitney U-test. Results: The median (range) total fibrinogen concentration in the pre-eclampsia group was 5.04 (3.25 – 6.51) g/l and in the control group 4.19 (3.61 – 5.38) g/l ( p < 0.05). The median (range) percentage high molecular weight fibrinogen was 76.5 (69.6 – 84.0)% and 73.0 (69.0 – 78.9)% in the preeclampsia and control group, respectively ( p < 0.05). Conclusions: In pre-eclampsia, the concentration of total fibrinogen is increased and the percentage high molecular weight fibrinogen is also slightly higher than in normal pregnancy. These results may be a reflection of the exaggerated inflammatory response, and subsequent endothelial activation, which are currently believed to be the key pathophysiological mechanisms in pre-eclampsia. D 2003 Elsevier Ltd. All rights reserved. Keywords: Fibrinogen; High molecular weight fibrinogen; Pregnancy; Coagulation; Pre-eclampsia
1. Introduction The major coagulation protein fibrinogen (Fg) is a heterogeneous protein. It is a symmetrical glycoprotein composed of six polypeptide chains of three types: two Aa, two Bh and two g. These chains are interconnected by disulphide bridges [1]. The Aa-chains can have different lengths because segments of various lengths may lack at their carboxyl-terminal ends. When an Aa-chain has a defective carboxyl-terminal end, it becomes an AaV-chain [2,3]. On the basis of the Aa- and AaV-chain heterogeneities, three main fractions of Fg can be discerned: high molecular weight fibrinogen (HMW-Fg, mw 340,000 Da, F 70% of total Fg, both Aa-chains intact), low molecular weight fibrinogen Abbreviations: Fg, fibrinogen; HMW-Fg, high molecular weight fibrinogen; LMW-Fg, low molecular weight fibrinogen; HELLP-syndrome, hemolysis, elevated liver enzymes and low platelets syndrome. * Corresponding author. Tel.: +31-302504010. E-mail address: [email protected] (G.T.R. Manten). 0049-3848/$ - see front matter D 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2003.08.025
(LMW-Fg, mw 300,000 Da, F 26% of total Fg, one Aa- and one AaV-chain) and LMWV-Fg (mw 280,000 Da, F 4% of total Fg, two AaV-chains) [4 –6]. LMW-Fg and LMWV-Fg are cleaved from HMW-Fg by proteolysis [7]. The clottability of HMW-Fg is highest as compared to the other fractions: HMW-Fg 98%, LMW-Fg 92% and LMWV-Fg 80% clottability. Fg derivatives of lower molecular weights display higher solubility and prolonged thrombin clotting times [5]. This can be explained by the more rapid polymerization and aggregation of HMW-fibrin monomers compared to monomers formed from LMW-Fg [8,9]. Pre-eclampsia, a gestational disorder that manifests in the second half of pregnancy, is a leading cause of maternal and perinatal morbidity and mortality. Pre-eclampsia is commonly defined by hypertension and proteinuria, arising in the second half of pregnancy in a previously normotensive woman. Although the etiology of the syndrome is not fully understood yet, it has been hypothesized recently that a generalized intravascular inflammatory response, which occurs in normal pregnancy too, is more exaggerated in
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pre-eclampsia [10]. In pre-eclampsia, there is a poorly perfused fetoplacental unit, which releases blood-born materials, causing endothelial dysfunction, in the maternal circulation. The endothelial dysfunction explains all the pathophysiological characteristics of the maternal syndrome; i.e. vasospasms, reduced organ perfusion, activation of the coagulation cascade and increased capillary permeability. The fetal syndrome consists of intrauterine growth retardation. Pre-eclampsia is only cured by delivery [11,12]. In normal pregnancy, there is a hypercoagulable state with increased concentration of blood coagulation factors, decreased or unchanged concentration of blood coagulation inhibitors and impaired fibrinolysis. These changes increase with advancing gestation [13,14]. In pre-eclampsia, these changes are exaggerated resulting in increased activation of the coagulation cascade and a more decreased fibrinolysis, associated with placental infarction and fibrin formation [11,15– 17]. It has been established that the concentration of total Fg is increased in pre-clampsia as compared to normal pregnancy [15,17]. It is unknown however, if this increase is accompanied by an increase in percentage HMW-Fg. We therefore measured the total Fg and the percentage HMWFg in patients with pre-eclampsia and in matched controls with an uncomplicated pregnancy.
2. Materials and methods 2.1. Patients Twenty-eight pregnant women were recruited; 14 had pre-eclampsia and 14 were healthy women with uncomplicated pregnancies, matched for gestational age at the time of blood sampling. The latter served as a control group. Preeclampsia was defined as two repeated (4 h apart) diastolic blood pressure measurements of 90 mm Hg or greater and the presence of proteinuria ( z 300 mg/24 h), in the absence of infection or renal disease, occurring after the 20th week of gestation in a previously normotensive woman [18]. HELLP-syndrome was defined as a platelet count < 100 109/l, an amino aspartate transferase concentration >70 U/ l and a lactic dehydrogenase concentration >600 U/l [19]. Gestational age was determined from the best estimate according to menstrual history or ultrasound measurement in early pregnancy. The pre-eclamptic women were admitted to the Department of Obstetrics of the University Medical Center Utrecht, The Netherlands. The control women received antenatal care from midwives in private practices, and had pregnancies with a normal course and outcome. None of the patients or controls was affected by any medical condition or was taking any medication. The Institutional Review Board of the hospital had approved the study protocol prior to the start of the study,
and informed consent was obtained from all the women before enrollment. 2.2. Blood sampling and storage One blood sample from each of the patients and the controls in the study population was drawn in 10-ml sodium citrate tubes. Within 30 min, the blood was centrifuged for 15 min at 1500 g at 4 jC to separate the plasma, and stored at 70 jC until analysis. 2.3. Laboratory methods Total Fg concentrations were determined according to Clauss using the Sta-R automatic coagulation analyser with STA Fibrinogen reagent (Diagnostica Stago, France) [20]. The test is based on clot formation of citrated plasma after adding an excess of thrombin. The percentage HMW-Fg was measured using the isolation method of Fg described by Vila et al. [21]. Briefly, Fg was isolated and purified by precipitation with 4% polyethylene glycol 6000 followed by 30% saturated ammonium sulfate precipitation. Afterwards, it was dissolved in phosphate buffer 0.018 M and pH 7.8. Four-microgram Fg was put on SDS-polyacrylamide gel. Electrophoresis was performed on a Mini Protean II system (Bio-Rad laboratories, Richmond, USA) for a total of 3 h at 30 mA. The gels were stained with Coomassie Brilliant Blue and the fractions quantified by densitometric scanning (Hyrys densitometer, Sebia, Brussels, Belgium). The test was evaluated using the selective salt precipitation method for Fg according to Holm et al. [5] (data not shown). In each gel, seven samples and a standard Fg preparation were Table 1 Baseline and outcome characteristics of the study groups Characteristic N Age (years) Pre-pregnancy BMI (body mass index, kg/m2) Parity -nulliparous (n) -multiparous (n) Booking systolic blood pressure (mm Hg) Booking diastolic blood pressure (mm Hg) Highest systolic blood pressure (mm Hg) Highest diastolic blood pressure (mm Hg) Proteinuria (g/24 h) Gestational age at sampling (days) Gestational age at delivery (days) Birth weight (g)
Pre-eclampsia
Controls
14 32 (19 – 41) 22 (20 – 29)
14 30 (25 – 39) 23 (20 – 27)
9 5 125 (110 – 150)
14 0 118 (90 – 140)
80 (60 – 95)*
70 (55 – 85)
180 (150 – 210)*
118 (100 – 130)
120 (100 – 125)*
78 (60 – 95)
6.5 (1.1 – 26.2)* 218 (178 – 258)
0 218 (174 – 257)
222 (179 – 259)*
278 (250 – 294)
1280 (525 – 2890)*
3390 (2570 – 4215)
Data are expressed as medians (range). * Statistically significant difference ( p < 0.05) from the controls.
G.T.R. Manten et al. / Thrombosis Research 111 (2003) 143–147
Fig. 1. Boxplot of total Fg concentrations in the pre-eclampsia and the control group.
included for quality control assessment. Because LMWV-Fg could not accurately be discriminated by its low concentration, the percentage HMW-Fg was measured relative to the rest of the Fg (LMW-Fg and LMWV-Fg). Both the intra- and inter-run CV for HMW-Fg concentrations were less than 2.5%. 2.4. Statistical analysis Data are reported as medians (range). The pre-eclampsia and control groups were compared by the Mann – Whitney U-test for two independent samples. p-Values < 0.05 were considered to indicate statistical significance.
3. Results Table 1 summarizes the baseline and outcome characteristics of the patients and controls. By definition, highest
145
Fig. 3. Boxplot of the percentage HMW-Fg of the total Fg in the preeclampsia and the control group.
blood pressure in pregnancy and proteinuria were significantly higher in the pre-eclampsia group; gestational age at delivery and birth weight were significantly lower in this group. Furthermore, booking diastolic blood pressure was significantly higher in the pre-eclampsia group. The control group only consisted of nulliparous women, whereas 5 of 14 of the pre-eclamptic women were multiparae. In the pre-eclampsia group, there were three cases of HELLPsyndrome. In Fig. 1, the total Fg concentration in both study groups is shown. The median (range) total Fg concentration in the pre-eclampsia group was 5.04 (3.25 –6.51) g/ l and in the control group 4.19 (3.61 – 5.38) g/l ( p < 0.05). Fig. 2 shows two examples of HMW-Fg determination, in a control woman and a woman with pre-eclampsia, by densitometric graphs with calculations of the relative densities. In Fig. 3, the percentage HMW-Fg in both study groups is shown. The median (range) percentage HMW-Fg was 76.5 (69.6 –84.0)% and 73.0 (69.0 – 78.9)% in preeclampsia and control group, respectively ( p < 0.05). Figs. 1 and 3 also demonstrate that there was considerable overlap between pre-eclampsia and control patients, in both total Fg concentration and percentage HMW-Fg. Parity did not affect the results in the pre-eclampsia group, nor was there a relation with the amount of proteinuria (data not shown).
4. Discussion
Fig. 2. Two densitometric graphs of a control woman and a woman with pre-eclampsia with calculations of the relative densities.
The results of our study show that in pre-eclampsia, the concentration of total Fg and particular the percentage HMW-Fg is slightly increased as compared to normal pregnancy. The higher concentration of total Fg in preeclampsia reconfirms results from earlier studies [15,17]. The higher percentage of HMW-Fg in pre-eclampsia has not been described before.
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In normal pregnancy, there are hemostatic changes in the direction of hypercoagulability which progressively increase towards term. There is an increase in blood coagulation factors as Fg, a decreased number of circulating platelets and increased platelet adhesiveness, increased amounts of soluble fibrin and inhibition of fibrinolysis [13 – 15]. Despite these hemostatic changes, the micro-circulation in the placenta seems to be unaltered during normal pregnancy [13,15]. In pre-eclampsia, this hypercoagulability is more outspoken. There is further inhibition of fibrinolysis without simultaneous inhibition of fibrin formation, probably contributing to impaired uteroplacental circulation and placental infarctions, which may lead to intrauterine growth retardation [15 –17]. In normal pregnancy, the Fg concentration is raised but the percentage HMW-Fg and the percentage LMW-Fg do not change [22]. The rise in total Fg concentration is most probably a hormonal (estrogen) effect, as observed in women taking oral contraceptives and hormonal replacement therapy [14,23]. After delivery, a rise in C-reactive protein and a further increase in the Fg concentration take place, suggestive of an acute phase response. At this stage, the rise in total Fg is associated with an increase in the HMW fraction rather than the LMW fraction [22], as in other acute phase conditions [4,8]. We speculate that the higher total Fg and percentage HMW-Fg we found in preeclampsia as compared to normal pregnancy are an expression of the more exaggerated inflammatory response that is currently considered to occur in pre-eclamptic pregnancies [10]. Human neutrophil elastase seems to be responsible for the proteolysis of HMW-Fg into LMW-Fg [22,24]. It has been suggested that the elastase is activated in pregnancy and probably responsible for maintaining a stable ratio of HMW-Fg and LMW-Fg despite of the raised total Fg concentration [22]. We hypothesize that in acute phase conditions, like pre-eclampsia, delivery, extensive myocardial infarction or major surgery, the proteolytic capacity of neutrophil elastase may be insufficient to keep up with the raised number of Fg molecules, resulting in a rise in the percentage HMW-Fg. The clottability of HMW-Fg is highest as compared to the other fractions [4,8,9]. Therefore, we speculate that the increased concentrations of total Fg and in particular the percentage HMW-Fg may contribute to the increased thrombus formation and the hampered fibrinolysis in pre-eclampsia. It is unlikely that the small and not significant difference in age in both groups contributed to the higher percentage HMW-Fg in the pre-eclampsia group. Although total Fg increases with age, the percentage HMW-Fg is rather lower in elderly [4]. We cannot exclude that the significant higher diastolic booking blood pressure in the pre-eclampsia group is a confounding factor. It is known that patients who develop pre-eclampsia more often have or develop chronic hypertension [25]. There is very little data about the relationship between chronic hypertension and Fg, and no
data about chronic hypertension and HMW-Fg. A recent study showed that patients with chronic hypertension have higher concentrations of total Fg. These patients also had higher concentrations of von Willebrand factor, a known marker for endothelial damage. The authors speculated that there is an association between the higher concentrations of Fg and endothelial dysfunction in patients with chronic hypertension [26]. Unfortunately, they did not determine the percentage HMW-Fg. Therefore, we cannot exclude that the higher percentage HMW-Fg in our pre-eclampsia group is associated with chronic hypertension, rather than with pre-eclampsia itself. We are presently considering a study which will address this problem. In conclusion, we found slightly higher concentrations of total Fg and percentage HMW-Fg in pre-eclamptic patients as compared to controls with uncomplicated pregnancies. These phenomena may be part of the exaggerated inflammatory response, and subsequent endothelial activation, which are currently believed to be the key pathophysiological mechanisms in pre-eclampsia.
Acknowledgements We thank Dr. M. de Maat and W. Nieuwenhuizen for their support and critical discussions.
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G.T.R. Manten et al. / Thrombosis Research 111 (2003) 143–147 [11] Roberts JM, Redman CWG. Pre-eclampsia: more than pregnancyinduced hypertension. Lancet 1993;341:1447 – 51. [12] Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK. Preeclampsia: an endothelial cell disorder. Am J Obstet Gynecol 1989;161:1200 – 4. [13] Kjellberg U, Andersson NE, Rose´n S, Tengborn L, Hellgren M. APC resistance and other haemostatic variables during pregnancy and puerperium. Thromb Haemost 1999;81:527 – 31. [14] Stirling Y, Woolf L, North WRS, Seghatchian MJ, Meade TW. Haemostasis in normal pregnancy. Thromb Haemost 1984;52:176 – 82. [15] McKay DG. Chronic intravascular coagulation in normal pregnancy and preeclampsia. Contrib Nephrol 1981;25:108 – 19. [16] Kanfer A, Bruch JF, Nguyen G, He CJ, Delarue F, Flahault A, et al. Increased placental antifibrinolytic potential and fibrin deposits in pregnancy-induced hypertension and preeclampsia. Lab Invest 1996; 74:253 – 8. [17] Maki M. Coagulation, fibrinolysis, platelet and kinin forming systems during toxemia of pregnancy. Biol Res Pregnancy 1983;4: 152 – 4. [18] Davey DA, MacGillivray I. The classification and definition of the hypertensive disorders of pregnancy. Am J Obstet Gynecol 1988; 158:892 – 8. [19] Sibai BM, Taslimi MM, El-Nazer A, Amon E, Mabie BC, Ryan GM. Maternal – perinatal outcome associated with the syndrome of hemolysis, elevated liver enzymes, and low platelets in severe preeclampsia – eclampsia. Am J Obstet Gynecol 1986;155:501 – 9.
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[20] Clauss A. Gerinnungsphysiologische schnellmethode zur bestimmung des fibrinogens. Acta Haematol 1957;17:237 – 46. [21] Vila V, Reganon E, Llopis F, Aznar J. A rapid method for isolation of fibrinogen from human plasma by precipitation with polyethylene glycol 6000. Thromb Res 1985;39:651 – 6. [22] Adler G, Duchinski T, Jasinska A, Piotrowska U. Fibrinogen fractions in the third trimester of pregnancy and in puerperium. Thromb Res 2000;97:405 – 10. [23] Meade TW, Brozovic M, Chakrabarti R, Howarth DJ, North WRS, Stirling Y. An epidemiological study of the haemostatic and other effects of oral contraceptives. Br J Haematol 1976;34:353 – 64. [24] Weitz JI, Landman SL, Crowley KA, Birken S, Morgan FJ. Development of an assay for in vivo human neutrophil elastase activity. Increased elastase activity in patients with alpha 1-proteinase inhibitor deficiency. J Clin Invest 1986;78:155 – 62. [25] He S, Silveira A, Hamsten A, Blomba¨ck M, Bremme K. Haemostatic, endothelial and lipoprotein parameters and blood pressure levels in women with a history of preeclampsia. Thromb Haemost 1999; 81:538 – 42. [26] Lip GYH, Edmunds E, Hee FLLS, Blann AD, Beevers DG. A crosssectional, diurnal, and follow up study of platelet activation and endothelial dysfunction in malignant phase hypertension. Am J Hypertens 2001;14:823 – 8.
Regular Article
Increased high molecular weight fibrinogen in pre-eclampsia G.T.R. Manten *, J.M. Sikkema, A. Franx, T.M. Hameeteman, G.H.A. Visser, P.G. de Groot, H.A.M. Voorbij Department of Perinatology and Gynaecology, University Medical Center Utrecht, F05.829, PO Box 85090, 3508 AB Utrecht, The Netherlands Received 13 June 2003; received in revised form 4 August 2003; accepted 4 August 2003
Abstract Introduction: The major coagulation protein fibrinogen (Fg) is a heterogeneous protein with three main fractions: high molecular weight fibrinogen (HMW-Fg), low molecular weight fibrinogen (LMW-Fg) and low molecular weightV fibrinogen. The clottability of high molecular weight fibrinogen is highest as compared to the other fractions. Pre-eclampsia is associated with a state of hypercoagulability, and with an increase of fibrinogen concentration. The aim of the present study was to examine if the increased total fibrinogen plasma concentration in patients with pre-eclampsia is associated with a change in distribution of the main fibrinogen fractions. Material and Methods: Plasma was collected from 14 patients with pre-eclampsia and from 14 healthy pregnant matched controls. Total fibrinogen concentrations were determined according to Clauss. The percentage high molecular weight fibrinogen was assessed by SDS-electrophoresis and densitometry after isolation of fibrinogen by precipitation. The study groups were compared by the Mann – Whitney U-test. Results: The median (range) total fibrinogen concentration in the pre-eclampsia group was 5.04 (3.25 – 6.51) g/l and in the control group 4.19 (3.61 – 5.38) g/l ( p < 0.05). The median (range) percentage high molecular weight fibrinogen was 76.5 (69.6 – 84.0)% and 73.0 (69.0 – 78.9)% in the preeclampsia and control group, respectively ( p < 0.05). Conclusions: In pre-eclampsia, the concentration of total fibrinogen is increased and the percentage high molecular weight fibrinogen is also slightly higher than in normal pregnancy. These results may be a reflection of the exaggerated inflammatory response, and subsequent endothelial activation, which are currently believed to be the key pathophysiological mechanisms in pre-eclampsia. D 2003 Elsevier Ltd. All rights reserved. Keywords: Fibrinogen; High molecular weight fibrinogen; Pregnancy; Coagulation; Pre-eclampsia
1. Introduction The major coagulation protein fibrinogen (Fg) is a heterogeneous protein. It is a symmetrical glycoprotein composed of six polypeptide chains of three types: two Aa, two Bh and two g. These chains are interconnected by disulphide bridges [1]. The Aa-chains can have different lengths because segments of various lengths may lack at their carboxyl-terminal ends. When an Aa-chain has a defective carboxyl-terminal end, it becomes an AaV-chain [2,3]. On the basis of the Aa- and AaV-chain heterogeneities, three main fractions of Fg can be discerned: high molecular weight fibrinogen (HMW-Fg, mw 340,000 Da, F 70% of total Fg, both Aa-chains intact), low molecular weight fibrinogen Abbreviations: Fg, fibrinogen; HMW-Fg, high molecular weight fibrinogen; LMW-Fg, low molecular weight fibrinogen; HELLP-syndrome, hemolysis, elevated liver enzymes and low platelets syndrome. * Corresponding author. Tel.: +31-302504010. E-mail address: [email protected] (G.T.R. Manten). 0049-3848/$ - see front matter D 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2003.08.025
(LMW-Fg, mw 300,000 Da, F 26% of total Fg, one Aa- and one AaV-chain) and LMWV-Fg (mw 280,000 Da, F 4% of total Fg, two AaV-chains) [4 –6]. LMW-Fg and LMWV-Fg are cleaved from HMW-Fg by proteolysis [7]. The clottability of HMW-Fg is highest as compared to the other fractions: HMW-Fg 98%, LMW-Fg 92% and LMWV-Fg 80% clottability. Fg derivatives of lower molecular weights display higher solubility and prolonged thrombin clotting times [5]. This can be explained by the more rapid polymerization and aggregation of HMW-fibrin monomers compared to monomers formed from LMW-Fg [8,9]. Pre-eclampsia, a gestational disorder that manifests in the second half of pregnancy, is a leading cause of maternal and perinatal morbidity and mortality. Pre-eclampsia is commonly defined by hypertension and proteinuria, arising in the second half of pregnancy in a previously normotensive woman. Although the etiology of the syndrome is not fully understood yet, it has been hypothesized recently that a generalized intravascular inflammatory response, which occurs in normal pregnancy too, is more exaggerated in
144
G.T.R. Manten et al. / Thrombosis Research 111 (2003) 143–147
pre-eclampsia [10]. In pre-eclampsia, there is a poorly perfused fetoplacental unit, which releases blood-born materials, causing endothelial dysfunction, in the maternal circulation. The endothelial dysfunction explains all the pathophysiological characteristics of the maternal syndrome; i.e. vasospasms, reduced organ perfusion, activation of the coagulation cascade and increased capillary permeability. The fetal syndrome consists of intrauterine growth retardation. Pre-eclampsia is only cured by delivery [11,12]. In normal pregnancy, there is a hypercoagulable state with increased concentration of blood coagulation factors, decreased or unchanged concentration of blood coagulation inhibitors and impaired fibrinolysis. These changes increase with advancing gestation [13,14]. In pre-eclampsia, these changes are exaggerated resulting in increased activation of the coagulation cascade and a more decreased fibrinolysis, associated with placental infarction and fibrin formation [11,15– 17]. It has been established that the concentration of total Fg is increased in pre-clampsia as compared to normal pregnancy [15,17]. It is unknown however, if this increase is accompanied by an increase in percentage HMW-Fg. We therefore measured the total Fg and the percentage HMWFg in patients with pre-eclampsia and in matched controls with an uncomplicated pregnancy.
2. Materials and methods 2.1. Patients Twenty-eight pregnant women were recruited; 14 had pre-eclampsia and 14 were healthy women with uncomplicated pregnancies, matched for gestational age at the time of blood sampling. The latter served as a control group. Preeclampsia was defined as two repeated (4 h apart) diastolic blood pressure measurements of 90 mm Hg or greater and the presence of proteinuria ( z 300 mg/24 h), in the absence of infection or renal disease, occurring after the 20th week of gestation in a previously normotensive woman [18]. HELLP-syndrome was defined as a platelet count < 100 109/l, an amino aspartate transferase concentration >70 U/ l and a lactic dehydrogenase concentration >600 U/l [19]. Gestational age was determined from the best estimate according to menstrual history or ultrasound measurement in early pregnancy. The pre-eclamptic women were admitted to the Department of Obstetrics of the University Medical Center Utrecht, The Netherlands. The control women received antenatal care from midwives in private practices, and had pregnancies with a normal course and outcome. None of the patients or controls was affected by any medical condition or was taking any medication. The Institutional Review Board of the hospital had approved the study protocol prior to the start of the study,
and informed consent was obtained from all the women before enrollment. 2.2. Blood sampling and storage One blood sample from each of the patients and the controls in the study population was drawn in 10-ml sodium citrate tubes. Within 30 min, the blood was centrifuged for 15 min at 1500 g at 4 jC to separate the plasma, and stored at 70 jC until analysis. 2.3. Laboratory methods Total Fg concentrations were determined according to Clauss using the Sta-R automatic coagulation analyser with STA Fibrinogen reagent (Diagnostica Stago, France) [20]. The test is based on clot formation of citrated plasma after adding an excess of thrombin. The percentage HMW-Fg was measured using the isolation method of Fg described by Vila et al. [21]. Briefly, Fg was isolated and purified by precipitation with 4% polyethylene glycol 6000 followed by 30% saturated ammonium sulfate precipitation. Afterwards, it was dissolved in phosphate buffer 0.018 M and pH 7.8. Four-microgram Fg was put on SDS-polyacrylamide gel. Electrophoresis was performed on a Mini Protean II system (Bio-Rad laboratories, Richmond, USA) for a total of 3 h at 30 mA. The gels were stained with Coomassie Brilliant Blue and the fractions quantified by densitometric scanning (Hyrys densitometer, Sebia, Brussels, Belgium). The test was evaluated using the selective salt precipitation method for Fg according to Holm et al. [5] (data not shown). In each gel, seven samples and a standard Fg preparation were Table 1 Baseline and outcome characteristics of the study groups Characteristic N Age (years) Pre-pregnancy BMI (body mass index, kg/m2) Parity -nulliparous (n) -multiparous (n) Booking systolic blood pressure (mm Hg) Booking diastolic blood pressure (mm Hg) Highest systolic blood pressure (mm Hg) Highest diastolic blood pressure (mm Hg) Proteinuria (g/24 h) Gestational age at sampling (days) Gestational age at delivery (days) Birth weight (g)
Pre-eclampsia
Controls
14 32 (19 – 41) 22 (20 – 29)
14 30 (25 – 39) 23 (20 – 27)
9 5 125 (110 – 150)
14 0 118 (90 – 140)
80 (60 – 95)*
70 (55 – 85)
180 (150 – 210)*
118 (100 – 130)
120 (100 – 125)*
78 (60 – 95)
6.5 (1.1 – 26.2)* 218 (178 – 258)
0 218 (174 – 257)
222 (179 – 259)*
278 (250 – 294)
1280 (525 – 2890)*
3390 (2570 – 4215)
Data are expressed as medians (range). * Statistically significant difference ( p < 0.05) from the controls.
G.T.R. Manten et al. / Thrombosis Research 111 (2003) 143–147
Fig. 1. Boxplot of total Fg concentrations in the pre-eclampsia and the control group.
included for quality control assessment. Because LMWV-Fg could not accurately be discriminated by its low concentration, the percentage HMW-Fg was measured relative to the rest of the Fg (LMW-Fg and LMWV-Fg). Both the intra- and inter-run CV for HMW-Fg concentrations were less than 2.5%. 2.4. Statistical analysis Data are reported as medians (range). The pre-eclampsia and control groups were compared by the Mann – Whitney U-test for two independent samples. p-Values < 0.05 were considered to indicate statistical significance.
3. Results Table 1 summarizes the baseline and outcome characteristics of the patients and controls. By definition, highest
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Fig. 3. Boxplot of the percentage HMW-Fg of the total Fg in the preeclampsia and the control group.
blood pressure in pregnancy and proteinuria were significantly higher in the pre-eclampsia group; gestational age at delivery and birth weight were significantly lower in this group. Furthermore, booking diastolic blood pressure was significantly higher in the pre-eclampsia group. The control group only consisted of nulliparous women, whereas 5 of 14 of the pre-eclamptic women were multiparae. In the pre-eclampsia group, there were three cases of HELLPsyndrome. In Fig. 1, the total Fg concentration in both study groups is shown. The median (range) total Fg concentration in the pre-eclampsia group was 5.04 (3.25 –6.51) g/ l and in the control group 4.19 (3.61 – 5.38) g/l ( p < 0.05). Fig. 2 shows two examples of HMW-Fg determination, in a control woman and a woman with pre-eclampsia, by densitometric graphs with calculations of the relative densities. In Fig. 3, the percentage HMW-Fg in both study groups is shown. The median (range) percentage HMW-Fg was 76.5 (69.6 –84.0)% and 73.0 (69.0 – 78.9)% in preeclampsia and control group, respectively ( p < 0.05). Figs. 1 and 3 also demonstrate that there was considerable overlap between pre-eclampsia and control patients, in both total Fg concentration and percentage HMW-Fg. Parity did not affect the results in the pre-eclampsia group, nor was there a relation with the amount of proteinuria (data not shown).
4. Discussion
Fig. 2. Two densitometric graphs of a control woman and a woman with pre-eclampsia with calculations of the relative densities.
The results of our study show that in pre-eclampsia, the concentration of total Fg and particular the percentage HMW-Fg is slightly increased as compared to normal pregnancy. The higher concentration of total Fg in preeclampsia reconfirms results from earlier studies [15,17]. The higher percentage of HMW-Fg in pre-eclampsia has not been described before.
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In normal pregnancy, there are hemostatic changes in the direction of hypercoagulability which progressively increase towards term. There is an increase in blood coagulation factors as Fg, a decreased number of circulating platelets and increased platelet adhesiveness, increased amounts of soluble fibrin and inhibition of fibrinolysis [13 – 15]. Despite these hemostatic changes, the micro-circulation in the placenta seems to be unaltered during normal pregnancy [13,15]. In pre-eclampsia, this hypercoagulability is more outspoken. There is further inhibition of fibrinolysis without simultaneous inhibition of fibrin formation, probably contributing to impaired uteroplacental circulation and placental infarctions, which may lead to intrauterine growth retardation [15 –17]. In normal pregnancy, the Fg concentration is raised but the percentage HMW-Fg and the percentage LMW-Fg do not change [22]. The rise in total Fg concentration is most probably a hormonal (estrogen) effect, as observed in women taking oral contraceptives and hormonal replacement therapy [14,23]. After delivery, a rise in C-reactive protein and a further increase in the Fg concentration take place, suggestive of an acute phase response. At this stage, the rise in total Fg is associated with an increase in the HMW fraction rather than the LMW fraction [22], as in other acute phase conditions [4,8]. We speculate that the higher total Fg and percentage HMW-Fg we found in preeclampsia as compared to normal pregnancy are an expression of the more exaggerated inflammatory response that is currently considered to occur in pre-eclamptic pregnancies [10]. Human neutrophil elastase seems to be responsible for the proteolysis of HMW-Fg into LMW-Fg [22,24]. It has been suggested that the elastase is activated in pregnancy and probably responsible for maintaining a stable ratio of HMW-Fg and LMW-Fg despite of the raised total Fg concentration [22]. We hypothesize that in acute phase conditions, like pre-eclampsia, delivery, extensive myocardial infarction or major surgery, the proteolytic capacity of neutrophil elastase may be insufficient to keep up with the raised number of Fg molecules, resulting in a rise in the percentage HMW-Fg. The clottability of HMW-Fg is highest as compared to the other fractions [4,8,9]. Therefore, we speculate that the increased concentrations of total Fg and in particular the percentage HMW-Fg may contribute to the increased thrombus formation and the hampered fibrinolysis in pre-eclampsia. It is unlikely that the small and not significant difference in age in both groups contributed to the higher percentage HMW-Fg in the pre-eclampsia group. Although total Fg increases with age, the percentage HMW-Fg is rather lower in elderly [4]. We cannot exclude that the significant higher diastolic booking blood pressure in the pre-eclampsia group is a confounding factor. It is known that patients who develop pre-eclampsia more often have or develop chronic hypertension [25]. There is very little data about the relationship between chronic hypertension and Fg, and no
data about chronic hypertension and HMW-Fg. A recent study showed that patients with chronic hypertension have higher concentrations of total Fg. These patients also had higher concentrations of von Willebrand factor, a known marker for endothelial damage. The authors speculated that there is an association between the higher concentrations of Fg and endothelial dysfunction in patients with chronic hypertension [26]. Unfortunately, they did not determine the percentage HMW-Fg. Therefore, we cannot exclude that the higher percentage HMW-Fg in our pre-eclampsia group is associated with chronic hypertension, rather than with pre-eclampsia itself. We are presently considering a study which will address this problem. In conclusion, we found slightly higher concentrations of total Fg and percentage HMW-Fg in pre-eclamptic patients as compared to controls with uncomplicated pregnancies. These phenomena may be part of the exaggerated inflammatory response, and subsequent endothelial activation, which are currently believed to be the key pathophysiological mechanisms in pre-eclampsia.
Acknowledgements We thank Dr. M. de Maat and W. Nieuwenhuizen for their support and critical discussions.
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