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Fibrinopeptide A levels in maternal and newborn plasma
European Journal of Obstetrics & Gynecology and Reproductive Elsevier
Biology, 30 (1989) 239-244
239
ETJO00726
Fibrinopeptide A levels in maternal and newborn plasma P.M.P. Yuen ‘,*, J.A. Yin ’ and T.T.H. Lao 2 ’ Department of Paediatrics and ’ Department of Obstetrics and Gynaecology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N. T., Hong Kong Accepted for publication 1 August 1988
Plasma fibrinopeptide A (FPA), a dynamic measure of intravascular coagulation, was determined in 70 healthy Chinese women during normal pregnancy, labour. delivery and the early puerperium and compared to a group of healthy non-pregnant adult controls. In the normal controls the plasma FPA level (mean f SD) was 1.43 f 0.46 ng/ml. During pregnancy and labour, the FPA levels were 3.05 + 0.98 ng/ml and 11.47 f 4.43 ng/ml, respectively, and it reached a peak of 32.95 k 11.66 ng/ml at parturition, then falling to 6.15 f 2.52 ng/ml in the early puerperium. All these levels were significantly higher (p < 0.001) compared to controls. Fifteen of the 21 mothers with blood sampling during parturition also had umbilical cord blood taken for determination of FPA level. There was no significant difference between the maternal (34.07 f 10.12 ng/ml) and cord (31.06 + 12.67 ng/ml) plasma FPA levels. It is concluded that the hypercoagulable state in women during pregnancy and the puerperium is associated with increased intravascular coagulation activity, and that increased intravascular coagulation activity also occurs in the fetus during parturition. This observation may account for the increased risk of thrombotic disorders observed in pregnant and parturient women as well as in the newborn. Fibrinopeptide A; Maternal; Newborn; Plasma
Introduction Thromboembolism is more common in pregnant and parturient women than non-pregnant women, and is an important cause of maternal mortality [l]. This is Correspondence: T.T.H. Lao, Department of Obstetrics and Gynaecology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N.T., Hong Kong. * Reprint request: P.M.P. Yuen.
0028-2243/89/$03.50
0 1989 Elsevier Science Publishers B.V. (Biomedical Division)
240
largely attributed to the hypercoagulable state of blood [2], with elevated levels of the procoagulants and changes in a wide range of other plasma haemostatic factors [3]. Direct indication of intravascular coagulation could be demonstrated by the level of fibrinopeptide A (FPA) which is generated when thrombin cleaves off the amino terminal of the Aa chain of fibrinogen, resulting in spontaneous polymerisation of fibrin monomers. Because FPA has a plasma half-life of 3-5 min [4], its plasma concentration therefore reflects the ongoing thrombin activity. Although many studies on the coagulation system during pregnancy have been reported, systematic information on FPA levels during and after pregnancy is scanty. Moreover, there is no information on the effect of pregnancy on FPA levels in places such as Hong Kong where the majority (> 90%) of the population is ethnic Chinese, and where the incidence of thromboembolism in the obstetric patients is negligible compared to reports from the U.K. [5]. This study was therefore conducted to measure plasma FPA levels in Chinese women during pregnancy, labour, parturition and in the early puerperium, and in the newborns of some of these mothers. The results were compared with a control group of healthy normal Chinese adults. Methods
Only women with uncomplicated pregnancy, delivery and puerperium were included in the study. They were divided into four groups according to the timing of the blood sample collection. Group A (antenatal) consisted of 24 women attending the antenatal clinic over the three trimesters. Group B (predelivery) consisted of 9 women in the first and second stages of labour. Group C (parturition) consisted of 21 women in the third stage of labour with the cord clamped and cut. In 15 women, paired cord blood was also obtained for comparison. Group D (puerperium) consisted of 16 women in the puerperium with blood sampling at 6 hours following delivery of the placenta to 5 days afterwards. Normal healthy adults were used as controls. Informed consent was obtained prior to blood sampling. Blood samples were obtained by the double-syringe technique, following a clean venepuncture, and placed into anticoagulant mixtures in the ratio of 9 : 1 in plastic tubes containing 0.11 M trisodium citrate, 1000 IU/ml heparin and 1 TIU/ml approtinin or the ready-for-use anticoagulant mixture provided in the commercial FUA kit manufactured by Mallinckrodt Inc. (St. Louis, MO, U.S.A.} containing EDTA, aprotinin and a thrombin inhibitor. The samples were placed in crushed ice immediately after collection and centrifuged at 4000 rpm for 2 min at 4 o C in a Hermle ZK 364 (BHG) refrigerated centrifuge. Plasma collected was aliquoted for immediate storage at -70°C until assay. Time of storage was under 2 months. All plasma specimens were pretreated with bentonite before assay to remove antigenitally cross-reacting fibrinogen and fibrin split products. Plasma FPA level was measured by radioimmunoassay method [6] using a commercial kit available from Mallinckrodt Inc. This is based on competitive binding between radiolabelled FPA and unlabelled (test) FPA for a limited number of binding sites on exogenously added FPA antiserum. FPA in the five groups was compared using Mann-Whitney U test and the unpaired Student’s t test, using BIOSTATISTS III.
241
Results
The results of maternal FPA levels are shown in Fig. 1 and the mean values of FPA in the control and study groups are shown in Table I. In the antenatal period, there was a 2-fold elevation of FPA above that in the normal controls, a difference that was statistically significant (p < 0.001). During labour, there was a further 4-fold increase in plasma FPA level reaching a peak that equals a further lo-fold
TABLE I Fibrinopeptide
FPA levels
f
A levels in normal pregnant women and non-pregnant
controls. Results are in means + SD
Non pregnant controls (n = 7)
Antenatal
Predelivery
Parturition
Puerperium
group A (n = 24)
group B (n = 9)
group c (n = 21)
group D (n = 16)
1.43 f 0.46
3.01 f 0.96
11.47It4.43
32.01 f 12.23
6.15 + 2.52
p < 0.001
p < 0.001
p < 0.001
p < 0.001
Puerperium
Controls
(ng/mU test with controls
. .
i .. . . .
.
Antenatal
Predelivery
Parturition
Fig. 1. Plasma FPA levels in normal women during various periods in pregnancy and controls.
242
.
. .
l* . .
.
: l
. .
.
.
20 Cord
Fig. 2. Correlation
Plasma
40 FPA
60
(ng/ml)
between maternal and umbilical cord plasma FPA levels.
increase during parturition. Following delivery of the placenta, there was a decline in FPA level with time. However, FPA level still remained significantly elevated (p < 0.001) even on postpartum day 5 when compared to the controls. In the 15 paired maternal and cord blood samples (Fig. 2), there was no significant difference between the maternal FPA level of 34.07 ( f 10.12) ng/ml and the cord blood FPA level of 31.06 ( f 12.67) ng/ml. Despite a correlation coefficient (r) of 0.46 between maternal and cord levels of FPA, it was not statistically significant.
Information on FPA levels in pregnancy is scanty. Van Royen and Ten Cate [7] have reported an elevated FPA level in late pregnancy but they did not include control subjects in their study. Suarez et al. [8] and Suarez et al. [9] have also reported their findings of activation of the clotting process at delivery, using FPA as a marker. Elevated FPA level has been reported in normal and pre-eclamptic pregnant women before and during labour [lO,ll]. But in one study [lo], two of the nine normal pregnant women had undergone Caesarean section before the onset of labour. However, the changes in FPA levels in relation to pregnancy, parturition and the puerperium remain uncertain, because in the forementioned reports [7-111, only one point in time was chosen for each study. Our approach was to study coagulation as a continuum in women from pregnancy through labour and into the early puerperium. Our results suggest that there is increased activity of the coagulation system during pregnancy as is shoti by the elevated plasma FPA levels in group A. This hypercoagulable state during pregnancy, which is associated with increased coagulation activity, is believed to result from increasing uteroplacental coagulation [12] owing to a low-grade chronic intravascular coagulation within the placental bed. The marked increase in FPA level during labour and parturition indicates an acceleration of the clotting process, and the rapid decline in FPA level after delivery suggests that adequate haemostasis was achieved at the placental site. As the FPA
243
level remained significantly elevated in the early puerperium compared to non-pregnant controls, this may mean that, while contraction of the myometrium is probably the major factor in preventing postpartum haemorrhage from the placental site, it is the activated coagulation system that plays an important role in placental-site haemostasis after the first 24 hours. The umbilical cord plasma FPA level, which is similar to the maternal level at parturition, suggests that the fetal coagulation system is also activated. It is likely that the fetal coagulation system is involved to a similar extent as the maternal one during parturition, and this may account for the increased incidence of fetal thrombotic complications following complicated labour and delivery [ 131. The lack of statistical significance in the correlation between maternal and cord plasma FPA levels could be due to the small number of sample size in the study. Our results confirm the findings of previous workers that there is an elevated FPA level during pregnancy and in the puerperium. This may reflect a progressive enhancement of coagulation and the observed increased in vivo tendency for thromboembolism. Other factors, such as the efficiency of the anticoagulant system in plasma and of fibrinolysis, may account for the lower incidence of thromboembolism in the Chinese obstetric patients. Further studies in this direction are now in progress. Acknowledgements
We thank Professor D.P. Davies for his enthusiastic help and advice in preparing the manuscript, Dr. D. Leung for advice on statistics, and Mrs. Angela Chu for typing. References 1 Department of Health and Social Security (D.H.S.S.) Report on confidential enquiries into maternal deaths in England and Wales 1976-1978. Her Majesty’s Stationary Office, London 1982. 2 Pecht L, Alexander B. Increased clotting factors in pregnancy. N EngJ J Med 1961;265:1093-1097. 3 Ratnoff OD, Holland TR. Coagulation components in normal and abnormal pregnancies. Ann NY Acad Sci 1959;75:626-633. 4 Nossel HL, Yudelman RE, Canfield RE, et al. Measurement of fibrinopeptide A in human blood. J Clin Invest 1974;54:45-53. 5 Lao TT, De Swiet M, Smith V, Walters BNJ. Prophylaxis of thromboembolism in pregnancy: an alternative. Br J Obstet Gynaecol 1985;92:202-206. 6 Nossel HL, Younger LR, Wilner GD, Procupez T, Canfield RE, Butler VP Jr. Radioimmunoassay of human fibrinopeptide A. Proc Nat1 Acad Sci USA 1971;68:2350-2353. 7 Van Royen EA, Ten Cate JW. Generation of a thrombin-like activity in late pregnancy. Thromb Res 1976;8:487-491. 8 Weiner G, Keller S. Pre-eclampsia is not associated with excess fetal clotting. Obstet Gynecol 1986;68:871-872. 9 Suarez CR, Walenga J, Mangogna LC, Fareed J. Neonatal & maternal fibrinolysis: activation at time of birth. Am J Hematol 1985;19:365-372. 10 Suarez CR, Menendez CE, Walenga JM, Fareed J. Neonatal and maternal hemostasis: value of molecular markers in the assessment of hemostatic status. Semin Thromb Hemost 1984;10:282-284. 11 Wallmo L, Karlsson K, Teger-Nilsson AC. Fibrinopeptide A and intravascular coagulation in
244 normotensive and hypertensive pregnancy and parturition. Acta Obstet Gynecol Stand 1984;63:637640. 12 Sheppard BL, Bonnar J. The ultrastructure of the human placenta in early and late pregnancy. J Obstet Gynecol Br Commonw 1974;81:497-511. 13 Mahasandana C, Hathaway WE. Circulating anticoagulants in the newborn: relation to hypercoagulability and the idiopathic respiratory distress syndrome. Pediat Res 1973;7:670-673.
Biology, 30 (1989) 239-244
239
ETJO00726
Fibrinopeptide A levels in maternal and newborn plasma P.M.P. Yuen ‘,*, J.A. Yin ’ and T.T.H. Lao 2 ’ Department of Paediatrics and ’ Department of Obstetrics and Gynaecology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N. T., Hong Kong Accepted for publication 1 August 1988
Plasma fibrinopeptide A (FPA), a dynamic measure of intravascular coagulation, was determined in 70 healthy Chinese women during normal pregnancy, labour. delivery and the early puerperium and compared to a group of healthy non-pregnant adult controls. In the normal controls the plasma FPA level (mean f SD) was 1.43 f 0.46 ng/ml. During pregnancy and labour, the FPA levels were 3.05 + 0.98 ng/ml and 11.47 f 4.43 ng/ml, respectively, and it reached a peak of 32.95 k 11.66 ng/ml at parturition, then falling to 6.15 f 2.52 ng/ml in the early puerperium. All these levels were significantly higher (p < 0.001) compared to controls. Fifteen of the 21 mothers with blood sampling during parturition also had umbilical cord blood taken for determination of FPA level. There was no significant difference between the maternal (34.07 f 10.12 ng/ml) and cord (31.06 + 12.67 ng/ml) plasma FPA levels. It is concluded that the hypercoagulable state in women during pregnancy and the puerperium is associated with increased intravascular coagulation activity, and that increased intravascular coagulation activity also occurs in the fetus during parturition. This observation may account for the increased risk of thrombotic disorders observed in pregnant and parturient women as well as in the newborn. Fibrinopeptide A; Maternal; Newborn; Plasma
Introduction Thromboembolism is more common in pregnant and parturient women than non-pregnant women, and is an important cause of maternal mortality [l]. This is Correspondence: T.T.H. Lao, Department of Obstetrics and Gynaecology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N.T., Hong Kong. * Reprint request: P.M.P. Yuen.
0028-2243/89/$03.50
0 1989 Elsevier Science Publishers B.V. (Biomedical Division)
240
largely attributed to the hypercoagulable state of blood [2], with elevated levels of the procoagulants and changes in a wide range of other plasma haemostatic factors [3]. Direct indication of intravascular coagulation could be demonstrated by the level of fibrinopeptide A (FPA) which is generated when thrombin cleaves off the amino terminal of the Aa chain of fibrinogen, resulting in spontaneous polymerisation of fibrin monomers. Because FPA has a plasma half-life of 3-5 min [4], its plasma concentration therefore reflects the ongoing thrombin activity. Although many studies on the coagulation system during pregnancy have been reported, systematic information on FPA levels during and after pregnancy is scanty. Moreover, there is no information on the effect of pregnancy on FPA levels in places such as Hong Kong where the majority (> 90%) of the population is ethnic Chinese, and where the incidence of thromboembolism in the obstetric patients is negligible compared to reports from the U.K. [5]. This study was therefore conducted to measure plasma FPA levels in Chinese women during pregnancy, labour, parturition and in the early puerperium, and in the newborns of some of these mothers. The results were compared with a control group of healthy normal Chinese adults. Methods
Only women with uncomplicated pregnancy, delivery and puerperium were included in the study. They were divided into four groups according to the timing of the blood sample collection. Group A (antenatal) consisted of 24 women attending the antenatal clinic over the three trimesters. Group B (predelivery) consisted of 9 women in the first and second stages of labour. Group C (parturition) consisted of 21 women in the third stage of labour with the cord clamped and cut. In 15 women, paired cord blood was also obtained for comparison. Group D (puerperium) consisted of 16 women in the puerperium with blood sampling at 6 hours following delivery of the placenta to 5 days afterwards. Normal healthy adults were used as controls. Informed consent was obtained prior to blood sampling. Blood samples were obtained by the double-syringe technique, following a clean venepuncture, and placed into anticoagulant mixtures in the ratio of 9 : 1 in plastic tubes containing 0.11 M trisodium citrate, 1000 IU/ml heparin and 1 TIU/ml approtinin or the ready-for-use anticoagulant mixture provided in the commercial FUA kit manufactured by Mallinckrodt Inc. (St. Louis, MO, U.S.A.} containing EDTA, aprotinin and a thrombin inhibitor. The samples were placed in crushed ice immediately after collection and centrifuged at 4000 rpm for 2 min at 4 o C in a Hermle ZK 364 (BHG) refrigerated centrifuge. Plasma collected was aliquoted for immediate storage at -70°C until assay. Time of storage was under 2 months. All plasma specimens were pretreated with bentonite before assay to remove antigenitally cross-reacting fibrinogen and fibrin split products. Plasma FPA level was measured by radioimmunoassay method [6] using a commercial kit available from Mallinckrodt Inc. This is based on competitive binding between radiolabelled FPA and unlabelled (test) FPA for a limited number of binding sites on exogenously added FPA antiserum. FPA in the five groups was compared using Mann-Whitney U test and the unpaired Student’s t test, using BIOSTATISTS III.
241
Results
The results of maternal FPA levels are shown in Fig. 1 and the mean values of FPA in the control and study groups are shown in Table I. In the antenatal period, there was a 2-fold elevation of FPA above that in the normal controls, a difference that was statistically significant (p < 0.001). During labour, there was a further 4-fold increase in plasma FPA level reaching a peak that equals a further lo-fold
TABLE I Fibrinopeptide
FPA levels
f
A levels in normal pregnant women and non-pregnant
controls. Results are in means + SD
Non pregnant controls (n = 7)
Antenatal
Predelivery
Parturition
Puerperium
group A (n = 24)
group B (n = 9)
group c (n = 21)
group D (n = 16)
1.43 f 0.46
3.01 f 0.96
11.47It4.43
32.01 f 12.23
6.15 + 2.52
p < 0.001
p < 0.001
p < 0.001
p < 0.001
Puerperium
Controls
(ng/mU test with controls
. .
i .. . . .
.
Antenatal
Predelivery
Parturition
Fig. 1. Plasma FPA levels in normal women during various periods in pregnancy and controls.
242
.
. .
l* . .
.
: l
. .
.
.
20 Cord
Fig. 2. Correlation
Plasma
40 FPA
60
(ng/ml)
between maternal and umbilical cord plasma FPA levels.
increase during parturition. Following delivery of the placenta, there was a decline in FPA level with time. However, FPA level still remained significantly elevated (p < 0.001) even on postpartum day 5 when compared to the controls. In the 15 paired maternal and cord blood samples (Fig. 2), there was no significant difference between the maternal FPA level of 34.07 ( f 10.12) ng/ml and the cord blood FPA level of 31.06 ( f 12.67) ng/ml. Despite a correlation coefficient (r) of 0.46 between maternal and cord levels of FPA, it was not statistically significant.
Information on FPA levels in pregnancy is scanty. Van Royen and Ten Cate [7] have reported an elevated FPA level in late pregnancy but they did not include control subjects in their study. Suarez et al. [8] and Suarez et al. [9] have also reported their findings of activation of the clotting process at delivery, using FPA as a marker. Elevated FPA level has been reported in normal and pre-eclamptic pregnant women before and during labour [lO,ll]. But in one study [lo], two of the nine normal pregnant women had undergone Caesarean section before the onset of labour. However, the changes in FPA levels in relation to pregnancy, parturition and the puerperium remain uncertain, because in the forementioned reports [7-111, only one point in time was chosen for each study. Our approach was to study coagulation as a continuum in women from pregnancy through labour and into the early puerperium. Our results suggest that there is increased activity of the coagulation system during pregnancy as is shoti by the elevated plasma FPA levels in group A. This hypercoagulable state during pregnancy, which is associated with increased coagulation activity, is believed to result from increasing uteroplacental coagulation [12] owing to a low-grade chronic intravascular coagulation within the placental bed. The marked increase in FPA level during labour and parturition indicates an acceleration of the clotting process, and the rapid decline in FPA level after delivery suggests that adequate haemostasis was achieved at the placental site. As the FPA
243
level remained significantly elevated in the early puerperium compared to non-pregnant controls, this may mean that, while contraction of the myometrium is probably the major factor in preventing postpartum haemorrhage from the placental site, it is the activated coagulation system that plays an important role in placental-site haemostasis after the first 24 hours. The umbilical cord plasma FPA level, which is similar to the maternal level at parturition, suggests that the fetal coagulation system is also activated. It is likely that the fetal coagulation system is involved to a similar extent as the maternal one during parturition, and this may account for the increased incidence of fetal thrombotic complications following complicated labour and delivery [ 131. The lack of statistical significance in the correlation between maternal and cord plasma FPA levels could be due to the small number of sample size in the study. Our results confirm the findings of previous workers that there is an elevated FPA level during pregnancy and in the puerperium. This may reflect a progressive enhancement of coagulation and the observed increased in vivo tendency for thromboembolism. Other factors, such as the efficiency of the anticoagulant system in plasma and of fibrinolysis, may account for the lower incidence of thromboembolism in the Chinese obstetric patients. Further studies in this direction are now in progress. Acknowledgements
We thank Professor D.P. Davies for his enthusiastic help and advice in preparing the manuscript, Dr. D. Leung for advice on statistics, and Mrs. Angela Chu for typing. References 1 Department of Health and Social Security (D.H.S.S.) Report on confidential enquiries into maternal deaths in England and Wales 1976-1978. Her Majesty’s Stationary Office, London 1982. 2 Pecht L, Alexander B. Increased clotting factors in pregnancy. N EngJ J Med 1961;265:1093-1097. 3 Ratnoff OD, Holland TR. Coagulation components in normal and abnormal pregnancies. Ann NY Acad Sci 1959;75:626-633. 4 Nossel HL, Yudelman RE, Canfield RE, et al. Measurement of fibrinopeptide A in human blood. J Clin Invest 1974;54:45-53. 5 Lao TT, De Swiet M, Smith V, Walters BNJ. Prophylaxis of thromboembolism in pregnancy: an alternative. Br J Obstet Gynaecol 1985;92:202-206. 6 Nossel HL, Younger LR, Wilner GD, Procupez T, Canfield RE, Butler VP Jr. Radioimmunoassay of human fibrinopeptide A. Proc Nat1 Acad Sci USA 1971;68:2350-2353. 7 Van Royen EA, Ten Cate JW. Generation of a thrombin-like activity in late pregnancy. Thromb Res 1976;8:487-491. 8 Weiner G, Keller S. Pre-eclampsia is not associated with excess fetal clotting. Obstet Gynecol 1986;68:871-872. 9 Suarez CR, Walenga J, Mangogna LC, Fareed J. Neonatal & maternal fibrinolysis: activation at time of birth. Am J Hematol 1985;19:365-372. 10 Suarez CR, Menendez CE, Walenga JM, Fareed J. Neonatal and maternal hemostasis: value of molecular markers in the assessment of hemostatic status. Semin Thromb Hemost 1984;10:282-284. 11 Wallmo L, Karlsson K, Teger-Nilsson AC. Fibrinopeptide A and intravascular coagulation in
244 normotensive and hypertensive pregnancy and parturition. Acta Obstet Gynecol Stand 1984;63:637640. 12 Sheppard BL, Bonnar J. The ultrastructure of the human placenta in early and late pregnancy. J Obstet Gynecol Br Commonw 1974;81:497-511. 13 Mahasandana C, Hathaway WE. Circulating anticoagulants in the newborn: relation to hypercoagulability and the idiopathic respiratory distress syndrome. Pediat Res 1973;7:670-673.