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Changes in the Fibrinolytic System During Pregnancy
Normal pregnancy is accompanied by extensive changes in the hemostatic system involving, in particular, the coagulation and fibrinolytic systems. This would seem to be a physiologic adaptation which has, in the main, two interrelated functions: First, to ensure the integrity of the expanding maternal and fetal circulations at the interface of the placenta and secondly the rapid and effective control of bleeding from the placental site at the time of placental separation. The extensive changes in the coagulation system during normal pregnancy would appear to be consistent with a continuing low-grade local activation of coagulation resulting in a compensatory synthesis of fibrinogen and other clotting factors combined with a slight decrease in coagulation inhibitors. Using electron microscopy, fibrin deposition can be readily demonstrated in the uteroplacental vasculature.1 In pregnancy, the elastic lamina and smooth muscle of the expanding spiral arteries supplying the placenta are replaced by trophoblast surrounded by a matrix containing fibrin. This enables the enlargement of the lumen of these vessels to accommodate an increasing blood flow to the placenta as well as reducing the pressure in the arterial blood flowing to the intervillous space of the placenta (Figs. 1, 2). As pregnancy advances, a substantial reserve of hemostatic components, especially fibrinogen and other coagulation proteins, is present in the circulating blood in addition to an increased blood volume. These physiologic changes will provide a reserve to meet the hemostatic challenge that takes place during childbirth. The process of placental separation occurs rapidly and a maternal blood flow of approximately 700 ml/min to the From the Trinity College Department of Obstetrics and Gynaecology, St. James's Hospital, Dublin, Ireland. Reprint requests: Dr. Bonnar, Trinity College Department of Obstetrics and Gynaecology, St. James Hospital, Dublin, Ireland.
placental site has to be staunched by the combined effects of myometrial extravascular compression and thrombotic occlusion of the sheared maternal vessels. If one of these two mechanisms is defective, serious uterine hemorrhage will occur during childbirth. In normal pregnancy therefore the fibrinolytic system will have a central role in controlling the physiologic process of fibrin deposition in the uteroplacental circulation while at the same time preventing fibrin deposition in the rest of the vascular system. The physiologic changes in the fibrinolytic system during pregnancy establish a vulnerable state for disordered fibrin deposition both in the uteroplacental circulation and outside the uterus. Abnormal fibrin deposition is seen in pregnancies complicated by fetal growth retardation with and without preeclampsia. In preeclampsia, fibrin deposition is found in both the kidney and liver. In complications such as abruptio placentae and amniotic fluid embolism, massive intravascular coagulation can develop. The increased risk of venous thromboembolism in pregnancy is most likely due to the changes induced by pregnancy in the fibrinolytic system.
FIBRINOLYTIC SYSTEM IN PREGNANCY Most investigators have found that plasminogen levels increase during pregnancy and our own studies showed that the increase in plasminogen levels during pregnancy occurred pari passu with that of fibrinogen, the rise above normal levels in the third trimester being of the order of 50 to 60% with both fibrinogen and plasminogen.2 MacFarlane and Biggs3 were among the first to report that plasma proteolytic activity was reduced in healthy pregnant women. Biezenski and Moore4 subsequently reported that a gradual decrease of fibrinolysis
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FIG. 1. The spiral artery (a) of the nonpregnant uterus and (b) supplying the placenta at term. Large trophoblast cells form a pseudoendothelium in parts of the vessel wall. The elastic lamina and muscle of the artery have been removed.
occurred during pregnancy, with the lowest values present in the third trimester. Several studies on fibrinolytic activity of plasma as measured by clot lysis methods have confirmed this fibrinolytic inhibition. 1,5-10 Over the last 5 years, considerable advances have been made in our understanding of the process of fibrinolysis, but the complex relationship that exists between plasminogen activators and inhibitors during pregnancy requires further elucidation. Plasminogen activators are serine proteases that convert the inactive proenzyme plasminogen into the active protease plasmin. Two types of plasminogen activator have been identified: the tissuetype (t-PA), which is produced by endothelial cells and released into the blood; and urokinase-type (u-PA), which was isolated initially from the urine but is also produced by the kidney and other cells. The control of plasminogen activator may occur at the level of synthesis and release but also through its interaction with specific plasminogen activator inhibitors (PAIs). Present evidence indicates that there are at least three distinct PAIs: PAI-1, initially called endothelial cell PA inhibitor or fast-acting PA inhibitor; PAI-2, the placental-type inhibitor; and the protease nexin, which also inhibits plasmin. In normal circumstances, PAI-1 seems to be the most important PA-inhibitor in plasma. In pregnancy, PAI-2 of placental origin would appear to have a major role in the control of fibrinolysis during pregnancy, especially within the uteroplacental circulation.
Placental-Type Plasminogen Activator Inhibitor, PAI-2 The presence of a urokinase inhibitor in the placenta was first reported by Kawano et al,11 who purified the inhibitor from placental homogenate. In organ culture experiments, Astedt et al12 showed that placental explants released inhibitors, which inhibited the activation of plasminogen by urokinase, and plasminogen activators released by kidney and fetal vessel explants. Immunohistochemically, PAI-2 has been localized to the trophoblastic epithelium13 (Fig. 2). This is in accord with our earlier observation of the absence of fibrinolytic activity from trophoblastic cells in the spiral arteries supplying the placenta in late pregnancy.7 Apart from its presence in the placenta, PAI-2 occurs in increasing amounts in maternal plasma during pregnancy. The increase during pregnancy is almost linear and at term levels have been reported to be 100 to 300 µg/liter, with a decrease to nondetectable levels within about a week of delivery.14,15 PAI-2 has a low molecular weight form estimated to be 43 to 48 kD 16 and a high molecular weight form estimated to be about 70 kD. 17 In maternal plasma, the high molecular weight form of PAI-2 predominates. PAI-2 is also found in amniotic fluid and umbilical cord plasma; the high molecular weight form and low molecular weight form of PAI-2 were found to be about the same concentration in amniotic fluid, but
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FIG. 2. A: The spiral artery supplying the placenta: trophoblast cells are present in the wall of the vessel with deposits of fibrin at term in normal pregnancy. B: In pregnancy complicated by preeclampsia and fetal growth retardation extensive fibrin deposition and lipid-laden cells are found.
the low molecular weight form predominated in cord blood.18 The presence of two molecular weight forms of PAI-2 may relate to the control of the placental passage and distribution of PAI-2 between the maternal and fetal circulation.17
Inhibition of Fibrinolytic Activity in Pregnancy Fibrinolytic activity decreases progressively during pregnancy, even after stimulation of plasminogen activator release by venous occlusion, and remains depressed until separation of the placenta, after which it rapidly returns to nonpregnant levels. 5,19,20 In addition to the increase of PAI-2 in plasma during pregnancy, there is also an increase in the fast-acting PAI-inhibitor, PAI1. 14,21 Since both PAI-1 and PAI-2 are increased in pregnancy, the question remains as to the respective physi-
ologic role of these distinct inhibitors of plasminogen activation. Astedt et al22 showed that after removal of PAI-2 from term plasma using a PAI-2 antibody affinity column, most of the total inhibitory capacity of plasma was found to have disappeared. In addition to the increase of PAI-2 antigen, Kruithof et al14 found a marked increase of PAI-1 antigen during pregnancy as well as PAI-1 activity and a moderate increase of t-PA and u-PA antigen levels. The PAI-1 levels increase after the 20th week of pregnancy and at term were threefold higher than the nonpregnant levels. The PAI-2 levels at term showed a 25-fold increase from early pregnancy. Despite this substantial increase in PAI-1 and PAI-2, no significant change was found in plasma PA activity in diluted plasma as measured on radioactive fibrin plates. Within 1 hour of delivery, the level of t-PA antigen doubled and PAI-1 ac-
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tivity and antigen decreased and returned to control values by 3 to 5 days after delivery. PAI-2 decreased more slowly and remained elevated at 3 to 5 days after delivery. Kruithof et al14 question the conclusions that have been drawn on the decrease of fibrinolytic activity in euglobulin precipitates. Wright et al23 in a recent report confirmed a marked reduction of the fibrinolytic activity of the plasma eu globulin fraction and a parallel reduction in t-PA activity in the second and third trimester, with a rapid return to nonpregnant levels postpartum. This pattern was found in both resting and postvenous occlusion samples. PAI-2 antigen increased throughout pregnancy and remained at a high level up to 48 hours postpartum. Despite the re duction of fibrinolytic activity of the euglobulin fraction and the high level of t-PA inhibition, increased fibrin (ogen) degradation products (FDP) and D-dimer was found in the third trimester. Different results on fibri nolytic activity are found, depending on the methodol ogy and whether the assay is performed on whole blood, plasma, or euglobulins. Immunologic methods that can measure both free and complexed antigen also give rise to problems in interpretation. We cannot at this stage unravel the complex interrelationship of plasminogen ac tivation and inhibition in pregnancy. The increased levels of FDP that occur in pregnancy24 are confirmed by the recent finding of ele vated levels of x-oligomer.25 They indicate that the fi brinolytic system remains active in the general circula tion and the inhibition of fibrinolysis is most likely confined to the uteroplacental circulation both during pregnancy and for several days in the placental bed fol lowing delivery. Trophoblast, the source of PAI-2, is present in the uteroplacental vessels (see Fig. 2) and is known to persist for some days after delivery. This would have the advantage of preserving fibrin in the uteropla cental vessels following delivery and so securing nemostasis in the highly vascular placental site. t-PA exists in two forms, the native single-chain enzyme and the two-chain enzyme, which results from proteolytic cleavage by plasmin. The efficiency of the two forms of t-PA in the fibrinolytic process is appar ently the same. PAI-1 inhibits both forms of t-PA, whereas PAI-2 acts mainly against the two-chain t-PA with only a slight effect on the single-chain form. On this basis, Astedt et al26 suggested that the different pattern of t-PA inhibition that occurs with PAI-2 is of physiologic importance in pregnancy to ensure a fibrinolytic potential by leaving the single-chain t-PA essentially uninhibited in the presence of the placental inhibitor. PAI-2 will also provide an effective stop to the generation of plasmin once the necessary amount has been formed, since an excess of plasmin cleaves the single-chain t-PA to the two-chain enzyme, which will be removed by the action
of PAI-2. In the control of fibrinolysis the proteolytic cleavage of the native t-PA may be a regulatory mech anism.
Fibrinolysis and Preeclampsia A well-known finding in women dying with ec lampsia has been widespread fibrin deposition, especially affecting the kidney and the liver. In preeclampsia asso ciated with fetal growth retardation we have found an excess of fibrin deposition in the spiral arteries supplying the placenta (Fig. 3). 6,8 This has raised the possibility that disordered fibrinolytic activity may be of importance in the pathogenesis of preeclampsia. In early studies on fibrinolysis in preeclampsia a diminished sensitivity to urokinase-induced fibrinolysis was found,27,28 but most investigators could find no difference between fibri nolytic activity and inhibitory activity between normal pregnancy and preeclampsia.29 Recently, further inves tigations in preeclampsia have been reported using new methodology. Aznar et al30 found that in severe preec lampsia the levels of the fast-acting PAI were higher than normal pregnancy and the levels of protein C antigen and activity were decreased. The high level of t-PA inhibitor and the reduced protein C in severe preeclampsia could be related to reduced fibrinolysis and persistence of microthrombi in the uteroplacental circulation. In a further report Estelles et al31 found that in severe preeclampsia the levels of PAI-1, both antigenic and functional, were increased and the levels of PAI-2, both antigenic and functional, were decreased in comparison to normal pregnancy. Despite the evidence of a greater level of fibri nolytic inhibition in preeclampsia, most studies have shown that the levels of FDP are increased 27,31,32 and also the levels of fibrinopeptide A and B-β peptides.18,33 In a recent serial study we compared the findings in 28 healthy primigravidae with 15 primigravidae with preeclampsia during pregnancy, labor, and following delivery.34 Plasminogen activator antigen was signifi cantly higher in the preeclamptic patients and the higher levels were maintained until 3 days after delivery (Fig. 4). The levels of PAI were lower in preeclampsia at 24 to 28 weeks' gestation but not at other stages (Fig. 5). In cord blood plasminogen activator antigen and PAI levels were higher in preeclampsia than in normal pregnancy (Fig. 6). The fibrinolytic system, with its highly complex system of activators and inhibitors, is designed for local action. Studies on the circulating blood will provide only a limited perception of events taking place in a specific area of the vascular system. For this reason, we have endeavored to study the process in the uterus at the time of delivery.
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FIG. 3. Inhibition (arrows) of fibrinolysis (F) at (a) the cytotrophoblast cell (T) in contrast to (b) the endothelial cell (E) in the lining of the spiral artery in pregnancy, c: Immunologic staining with PAI-2 antisera shows the inhibitor localized to the trophoblast (arrows) of a decidual spiral artery.
FIG. 4.
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Plasminogen activator levels (antigen) in 28 healthy primigravidae and 15 primigravidae with preeclampsia.
Uteroplacental Fibrinolysis in Preeclampsia and Fetal Growth Retardation These studies have been carried out in women requiring delivery by cesarean section and the findings in normal pregnancy and pregnancy complicated by preeclampsia and fetal growth retardation have been compared in peripheral blood, uterine vein blood, and cord blood taken virtually simultaneously.9 Tissue extracts from the
FIG. 5.
placenta and the placental bed of the uterus were also studied. t-PA antigen was significantly higher in pregnancies complicated by fetal growth retardation in peripheral and uterine vein blood and cord blood and the levels in uterine blood were higher than peripheral blood; in preeclampsia levels of t-PA antigen were significantly higher in peripheral blood (Fig. 7). PAI-1 was higher in pregnancies complicated by
Plasminogen activator inhibitor levels in 28 healthy primigravidae and 15 primigravidae with preeclampsia.
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preeclampsia and fetal growth retardation in both peripheral and uterine vein blood, but the cord levels were similar (Fig. 8). The placental inhibitor PAI-2 was found to be much lower in pregnancies complicated by preeclampsia and fetal growth retardation in both peripheral and uterine vein blood, and within each group the levels of PAI-2 in peripheral and uterine vein blood were similar (Fig. 9). These findings would suggest that within the uteroplacental circulation disordered fibrinolysis occurs in preeclampsia and fetal growth retardation. The low levels of PAI-2 may be the result of the impaired placental
function in preeclampsia and intrauterine fetal growth retardation or due to the greater consumption of the inhibitor associated with the excess of fibrin that is present in the uteroplacental circulation. The higher level of t-PA antigen and PAI-1 in preeclampsia and fetal growth retardation may be the result of enhanced fibrinolysis in the circulation to counteract the lowgrade systemic intravascular coagulation that occurs in preeclampsia. Fibrin is a necessary matrix to protect the integrity of the maternal and fetal circulation at the interface of the placenta. The related disorders of bleeding and thrombosis at the placental site are responsible for a substantial
UTEROPLACENTAL FIBRINOLYSIS in PET and IUGR TISSUE PLASMINOGEN ACTIVATOR ANTIGEN
FIG. 7. Tissue plasminogen activator antigen in peripheral blood, uterine vein blood and cord blood in normal pregnancy and pregnancy complicated by preeclampsia (PET) and fetal growth retardation (IUGR).
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FIG. 6. Plasminogen activator antigen and plasminogen activator inhibitor levels in maternal and cord blood at time of delivery in 28 healthy primigravidae and in 15 primigravidae with preeclampsia.
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UTEROPLACENTAL FIBRINOLYSIS in PET and IUGR
FIG. 8. Plasminogen activator inhibitor (PAI-1) in peripheral blood, uterine vein blood and cord blood in normal pregnancy and pregnancy complicated by preeclampsia (PET) and fetal growth retardation (IUGR).
UTEROPLACENTAL FIBRINOLYSIS in PET and IUGR PLASMINOGEN ACTIVATOR INHIBITOR 2
FIG. 9. Placental inhibitor (PAI-2) in peripheral and uterine vein blood in normal pregnancy and pregnancy complicated by preeclampsia (PET) and fetal growth retardation (IUGR).
proportion of fetal mortality and morbidity. Further research in this area may open up new possibilities for protecting the fetus in utero from the impaired placental function that arises from disordered local coagulation and fibrinolysis in the maternal blood supply to the placenta.
REFERENCES 1. Sheppard B, J Bonnar: The ultrastructure of the arterial supply of the human placenta in early and late pregnancy. J Obstet Gynaecol Br Commonw 81:497-511, 1974. 2. Bonnar J, GP McNichol, AS Douglas: Fibrinolytic enzyme system and pregnancy. Br Med J 3:387-395, 1969.
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PLASMINOGEN ACTIVATOR INHIBITOR 1
3. MacFarlane RG, R Biggs: Observations on fibrinolysis; spontaneous activity associated with surgical operations, trauma and c. Lancet 2:862-864, 1946. 4. Biezenski JJ, HC Moore: Fibrinolysis in normal pregnancy. J Clin Pathol 11:306-310, 1958. 5. Shaper AG, DM Macintosh, CM Evans, J Kyope: Fibrinolysis and plasminogen levels in pregnancy and the puerperium. Lancet 2:706-708, 1965. 6. Sheppard BL, J Bonnar: The ultrastructure of the arterial supply of the human placenta in pregnancy complicated by fetal growth retardation. Br J Obstet Gynaecol 83:948-959, 1976. 7. Sheppard BL, J Bonnar: Fibrinolysis in decidual spiral arteries in late pregnancy. Thromb Haemost 39:751-758, 1978. 8. Sheppard BL, J Bonnar: An ultrastructural study of utero-placental spiral arteries in hypertensive and normotensive pregnancy and fetal growth retardation. Br J Obstet Gynaecol 88:695-705, 1981. 9. Sheppard BL, C Boyle, N Gleeson, M Jordan, L Daly, J Bonnar: Uteroplacental tissue plasminogen activator and inhibitor at Caesarean section in normal pregnancy, hypertensive pregnancy and intrauterine fetal growth retardation. (Submitted for publication.) 10. Stirling Y, L Woolf, WRS North, MJ Seghatchian, TW Meade: Haemostatis in normal pregnancy. Thromb Haemost 52:176-182, 1984. 11. Kawano T, K Morimoto, Y Uemura: Urokinase inhibitor in human placenta. Nature 217:253-254, 1968. 12. Astedt B, M Pandolphi, IM Nilsson: Inhibitory effect of placenta on plasminogen activation in human organ culture. Proc Soc Exp Biol Med 139:1421-1424, 1972. 13. Astedt B, I Hagerstrom, I Lecander: Cellular localisation in placenta of placental type plasminogen activator inhibitor. Thromb Haemost 56:63-65, 1986. 14. Kruithof EKO, C Tran-Thang, A Gudinchet, J Hauert, G Nicoloso, C Genton, H Welti, F Bachmann: Fibrinolysis in pregnancy—a study of plasminogen activator inhibitors. Blood 69:460-466, 1987. 15. Lecander I, B Astedt: Isolation of a new specific plasminogen activator inhibitor from pregnancy plasma. Br J Haematol 62:221228, 1986. 16. Astedt B, I Lecander, T Brodin, A Lundblad, K Low: Purification of a specific placental plasminogen activator by monoclonal antibody and its complex formation with plasminogen activator. Thromb Haemost 53:122-125, 1985. 17. Astedt B, I Lecander, T Ny: Review.The placental type plasminogen activator inhibitor, PAI 2. Fibrinolysis 1:203-208, 1987. 18. Douglas JT, M Shah, GDO Lowe, JJF Belch, CD Forbes, CRM Prentice: Plasma fibrinopeptide A and beta-thromboglobulin in pre-eclampsia and pregnancy hypertension. Thromb Haemost 47:54-55, 1982. 19. Astedt B: On fibrinolysis in pregnancy, labour, puerperium and during treatment with sex hormones. Acta Obstet Gynecol Scand 51 (Suppl 18):l-26, 1972. 20. Bonnar J, GP McNicol, AS Douglas: Coagulation and fibrinolytic
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mechanisms during and after normal childbirth. Br Med J 2:200203, 1970. Wiman B, Csemiczky, L Marsk, H Robbe: The fast inhibitor of tissue plasminogen activator in plasma during pregnancy. Thromb Haemost 52:124-126, 1984. Astedt B, I Lecander, IM Nilsson: Depression of PA activity during pregnancy by the placenta inhibitor. Fibrinolysis. Proc. Eighth Intern Congr Fibrinolysis, 1986, p 185. Wright JG, P Cooper, B Astedt, I Lecander, JT Wilde, FE Preston, M Greaves: Fibrinolysis during normal human pregnancy: Complex inter-relationships between plasma levels of tissue plasminogen activator and inhibitors and the euglobulin clot lysis time. Br J Haematol 69:253-258, 1988. Fletcher AP, NK Alkjaersig, R Burstein: The influence of pregnancy upon blood coagulation and plasma fibrinolytic enzyme function. Am J Obstet Gynecol 134:743-751, 1979. Gaffney PJ, LJ Creighton, M Callus, R Thorpe: Monoclonal antibodies to crosslinked fibrin degradation products (XL-FDP): II. Evaluation in a variety of clinical conditions. Br J Haematol 68:9196, 1988. Astedt B, B Bladh, U Christensen, I Lecander: Different inhibition of one and two chain tissue plasminogen activator by a placental inhibitor studied with two tripeptide-p-nitroanilide substrates. Scand J Clin Lab Invest 45:429-435, 1985. Bonnar J, GP McNicol, AS Douglas: Coagulation and fibrinolytic systems in pre-eclampsia and eclampsia. Br Med J 2:12— 16, 1971. Howie PW, CRM Prentice, GP McNicol: Coagulation, fibrinolysis and platental function in pre-eclampsia, essential hypertension, and placental insufficiency. J Obstet Gynaecol Br Commow 78:992-1003, 1971. Gow L, DM Campbell, D Ogston: The fibrinolytic system in preeclampsia. J Clin Pathol 37:56-58, 1984. Aznar J, J Gilabert, A Estelles, F Espana: Fibrinolytic activity and protein C in pre-eclampsia. Thromb Haemost 55:314-317, 1986. Estelles A, J Gilabert, J Aznar, RR Scheef: Plasminogen activator inhibitors type 1 and type 2 (PAI-1 and PAI-2) in normal pregnancy and in patients with severe pre-eclampsia. Fibrinolysis 2:162, 1988. Giles C: Intravascular coagulation in gestational hypertension and pre-eclampsia: The value of haematological screening tests. Clin Lab Haematol 4:351-358, 1982. Borok Z, J Weitz, J Owen, M Auerbach, HL Nossel: Fibrinogen proteolysis and platelet alpha-granule release in pre-eclampsia/ eclampsia. Blood 63:525-531, 1984. Daly L, J Bonnar: Fibrinolytic activation and inhibition in normal pregnancy and in pre-eclampsia. (Submitted for publication.) Brakman P: The fibrinolytic system in human blood during pregnancy. Am J Obstet Gynecol 94:14-20, 1966. Gibson B, D Hunter, PB Neame, JG Kelton: Thrombocytopenia in pre-eclampsia and eclampsia serum. Thromb Haemost 8:234-247, 1982.
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FIBRINOLYTIC CHANGES DURING PREGNANCY—BONNAR, DALY, SHEPPARD
Changes in the Fibrinolytic System During Pregnancy
Normal pregnancy is accompanied by extensive changes in the hemostatic system involving, in particular, the coagulation and fibrinolytic systems. This would seem to be a physiologic adaptation which has, in the main, two interrelated functions: First, to ensure the integrity of the expanding maternal and fetal circulations at the interface of the placenta and secondly the rapid and effective control of bleeding from the placental site at the time of placental separation. The extensive changes in the coagulation system during normal pregnancy would appear to be consistent with a continuing low-grade local activation of coagulation resulting in a compensatory synthesis of fibrinogen and other clotting factors combined with a slight decrease in coagulation inhibitors. Using electron microscopy, fibrin deposition can be readily demonstrated in the uteroplacental vasculature.1 In pregnancy, the elastic lamina and smooth muscle of the expanding spiral arteries supplying the placenta are replaced by trophoblast surrounded by a matrix containing fibrin. This enables the enlargement of the lumen of these vessels to accommodate an increasing blood flow to the placenta as well as reducing the pressure in the arterial blood flowing to the intervillous space of the placenta (Figs. 1, 2). As pregnancy advances, a substantial reserve of hemostatic components, especially fibrinogen and other coagulation proteins, is present in the circulating blood in addition to an increased blood volume. These physiologic changes will provide a reserve to meet the hemostatic challenge that takes place during childbirth. The process of placental separation occurs rapidly and a maternal blood flow of approximately 700 ml/min to the From the Trinity College Department of Obstetrics and Gynaecology, St. James's Hospital, Dublin, Ireland. Reprint requests: Dr. Bonnar, Trinity College Department of Obstetrics and Gynaecology, St. James Hospital, Dublin, Ireland.
placental site has to be staunched by the combined effects of myometrial extravascular compression and thrombotic occlusion of the sheared maternal vessels. If one of these two mechanisms is defective, serious uterine hemorrhage will occur during childbirth. In normal pregnancy therefore the fibrinolytic system will have a central role in controlling the physiologic process of fibrin deposition in the uteroplacental circulation while at the same time preventing fibrin deposition in the rest of the vascular system. The physiologic changes in the fibrinolytic system during pregnancy establish a vulnerable state for disordered fibrin deposition both in the uteroplacental circulation and outside the uterus. Abnormal fibrin deposition is seen in pregnancies complicated by fetal growth retardation with and without preeclampsia. In preeclampsia, fibrin deposition is found in both the kidney and liver. In complications such as abruptio placentae and amniotic fluid embolism, massive intravascular coagulation can develop. The increased risk of venous thromboembolism in pregnancy is most likely due to the changes induced by pregnancy in the fibrinolytic system.
FIBRINOLYTIC SYSTEM IN PREGNANCY Most investigators have found that plasminogen levels increase during pregnancy and our own studies showed that the increase in plasminogen levels during pregnancy occurred pari passu with that of fibrinogen, the rise above normal levels in the third trimester being of the order of 50 to 60% with both fibrinogen and plasminogen.2 MacFarlane and Biggs3 were among the first to report that plasma proteolytic activity was reduced in healthy pregnant women. Biezenski and Moore4 subsequently reported that a gradual decrease of fibrinolysis
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JOHN BONNAR, M.D., F.R.C.O.G., LEISHA DALY, Ph.D., and BRIAN L SHEPPARD, D.Phil. MRC.Path.
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FIG. 1. The spiral artery (a) of the nonpregnant uterus and (b) supplying the placenta at term. Large trophoblast cells form a pseudoendothelium in parts of the vessel wall. The elastic lamina and muscle of the artery have been removed.
occurred during pregnancy, with the lowest values present in the third trimester. Several studies on fibrinolytic activity of plasma as measured by clot lysis methods have confirmed this fibrinolytic inhibition. 1,5-10 Over the last 5 years, considerable advances have been made in our understanding of the process of fibrinolysis, but the complex relationship that exists between plasminogen activators and inhibitors during pregnancy requires further elucidation. Plasminogen activators are serine proteases that convert the inactive proenzyme plasminogen into the active protease plasmin. Two types of plasminogen activator have been identified: the tissuetype (t-PA), which is produced by endothelial cells and released into the blood; and urokinase-type (u-PA), which was isolated initially from the urine but is also produced by the kidney and other cells. The control of plasminogen activator may occur at the level of synthesis and release but also through its interaction with specific plasminogen activator inhibitors (PAIs). Present evidence indicates that there are at least three distinct PAIs: PAI-1, initially called endothelial cell PA inhibitor or fast-acting PA inhibitor; PAI-2, the placental-type inhibitor; and the protease nexin, which also inhibits plasmin. In normal circumstances, PAI-1 seems to be the most important PA-inhibitor in plasma. In pregnancy, PAI-2 of placental origin would appear to have a major role in the control of fibrinolysis during pregnancy, especially within the uteroplacental circulation.
Placental-Type Plasminogen Activator Inhibitor, PAI-2 The presence of a urokinase inhibitor in the placenta was first reported by Kawano et al,11 who purified the inhibitor from placental homogenate. In organ culture experiments, Astedt et al12 showed that placental explants released inhibitors, which inhibited the activation of plasminogen by urokinase, and plasminogen activators released by kidney and fetal vessel explants. Immunohistochemically, PAI-2 has been localized to the trophoblastic epithelium13 (Fig. 2). This is in accord with our earlier observation of the absence of fibrinolytic activity from trophoblastic cells in the spiral arteries supplying the placenta in late pregnancy.7 Apart from its presence in the placenta, PAI-2 occurs in increasing amounts in maternal plasma during pregnancy. The increase during pregnancy is almost linear and at term levels have been reported to be 100 to 300 µg/liter, with a decrease to nondetectable levels within about a week of delivery.14,15 PAI-2 has a low molecular weight form estimated to be 43 to 48 kD 16 and a high molecular weight form estimated to be about 70 kD. 17 In maternal plasma, the high molecular weight form of PAI-2 predominates. PAI-2 is also found in amniotic fluid and umbilical cord plasma; the high molecular weight form and low molecular weight form of PAI-2 were found to be about the same concentration in amniotic fluid, but
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FIG. 2. A: The spiral artery supplying the placenta: trophoblast cells are present in the wall of the vessel with deposits of fibrin at term in normal pregnancy. B: In pregnancy complicated by preeclampsia and fetal growth retardation extensive fibrin deposition and lipid-laden cells are found.
the low molecular weight form predominated in cord blood.18 The presence of two molecular weight forms of PAI-2 may relate to the control of the placental passage and distribution of PAI-2 between the maternal and fetal circulation.17
Inhibition of Fibrinolytic Activity in Pregnancy Fibrinolytic activity decreases progressively during pregnancy, even after stimulation of plasminogen activator release by venous occlusion, and remains depressed until separation of the placenta, after which it rapidly returns to nonpregnant levels. 5,19,20 In addition to the increase of PAI-2 in plasma during pregnancy, there is also an increase in the fast-acting PAI-inhibitor, PAI1. 14,21 Since both PAI-1 and PAI-2 are increased in pregnancy, the question remains as to the respective physi-
ologic role of these distinct inhibitors of plasminogen activation. Astedt et al22 showed that after removal of PAI-2 from term plasma using a PAI-2 antibody affinity column, most of the total inhibitory capacity of plasma was found to have disappeared. In addition to the increase of PAI-2 antigen, Kruithof et al14 found a marked increase of PAI-1 antigen during pregnancy as well as PAI-1 activity and a moderate increase of t-PA and u-PA antigen levels. The PAI-1 levels increase after the 20th week of pregnancy and at term were threefold higher than the nonpregnant levels. The PAI-2 levels at term showed a 25-fold increase from early pregnancy. Despite this substantial increase in PAI-1 and PAI-2, no significant change was found in plasma PA activity in diluted plasma as measured on radioactive fibrin plates. Within 1 hour of delivery, the level of t-PA antigen doubled and PAI-1 ac-
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tivity and antigen decreased and returned to control values by 3 to 5 days after delivery. PAI-2 decreased more slowly and remained elevated at 3 to 5 days after delivery. Kruithof et al14 question the conclusions that have been drawn on the decrease of fibrinolytic activity in euglobulin precipitates. Wright et al23 in a recent report confirmed a marked reduction of the fibrinolytic activity of the plasma eu globulin fraction and a parallel reduction in t-PA activity in the second and third trimester, with a rapid return to nonpregnant levels postpartum. This pattern was found in both resting and postvenous occlusion samples. PAI-2 antigen increased throughout pregnancy and remained at a high level up to 48 hours postpartum. Despite the re duction of fibrinolytic activity of the euglobulin fraction and the high level of t-PA inhibition, increased fibrin (ogen) degradation products (FDP) and D-dimer was found in the third trimester. Different results on fibri nolytic activity are found, depending on the methodol ogy and whether the assay is performed on whole blood, plasma, or euglobulins. Immunologic methods that can measure both free and complexed antigen also give rise to problems in interpretation. We cannot at this stage unravel the complex interrelationship of plasminogen ac tivation and inhibition in pregnancy. The increased levels of FDP that occur in pregnancy24 are confirmed by the recent finding of ele vated levels of x-oligomer.25 They indicate that the fi brinolytic system remains active in the general circula tion and the inhibition of fibrinolysis is most likely confined to the uteroplacental circulation both during pregnancy and for several days in the placental bed fol lowing delivery. Trophoblast, the source of PAI-2, is present in the uteroplacental vessels (see Fig. 2) and is known to persist for some days after delivery. This would have the advantage of preserving fibrin in the uteropla cental vessels following delivery and so securing nemostasis in the highly vascular placental site. t-PA exists in two forms, the native single-chain enzyme and the two-chain enzyme, which results from proteolytic cleavage by plasmin. The efficiency of the two forms of t-PA in the fibrinolytic process is appar ently the same. PAI-1 inhibits both forms of t-PA, whereas PAI-2 acts mainly against the two-chain t-PA with only a slight effect on the single-chain form. On this basis, Astedt et al26 suggested that the different pattern of t-PA inhibition that occurs with PAI-2 is of physiologic importance in pregnancy to ensure a fibrinolytic potential by leaving the single-chain t-PA essentially uninhibited in the presence of the placental inhibitor. PAI-2 will also provide an effective stop to the generation of plasmin once the necessary amount has been formed, since an excess of plasmin cleaves the single-chain t-PA to the two-chain enzyme, which will be removed by the action
of PAI-2. In the control of fibrinolysis the proteolytic cleavage of the native t-PA may be a regulatory mech anism.
Fibrinolysis and Preeclampsia A well-known finding in women dying with ec lampsia has been widespread fibrin deposition, especially affecting the kidney and the liver. In preeclampsia asso ciated with fetal growth retardation we have found an excess of fibrin deposition in the spiral arteries supplying the placenta (Fig. 3). 6,8 This has raised the possibility that disordered fibrinolytic activity may be of importance in the pathogenesis of preeclampsia. In early studies on fibrinolysis in preeclampsia a diminished sensitivity to urokinase-induced fibrinolysis was found,27,28 but most investigators could find no difference between fibri nolytic activity and inhibitory activity between normal pregnancy and preeclampsia.29 Recently, further inves tigations in preeclampsia have been reported using new methodology. Aznar et al30 found that in severe preec lampsia the levels of the fast-acting PAI were higher than normal pregnancy and the levels of protein C antigen and activity were decreased. The high level of t-PA inhibitor and the reduced protein C in severe preeclampsia could be related to reduced fibrinolysis and persistence of microthrombi in the uteroplacental circulation. In a further report Estelles et al31 found that in severe preeclampsia the levels of PAI-1, both antigenic and functional, were increased and the levels of PAI-2, both antigenic and functional, were decreased in comparison to normal pregnancy. Despite the evidence of a greater level of fibri nolytic inhibition in preeclampsia, most studies have shown that the levels of FDP are increased 27,31,32 and also the levels of fibrinopeptide A and B-β peptides.18,33 In a recent serial study we compared the findings in 28 healthy primigravidae with 15 primigravidae with preeclampsia during pregnancy, labor, and following delivery.34 Plasminogen activator antigen was signifi cantly higher in the preeclamptic patients and the higher levels were maintained until 3 days after delivery (Fig. 4). The levels of PAI were lower in preeclampsia at 24 to 28 weeks' gestation but not at other stages (Fig. 5). In cord blood plasminogen activator antigen and PAI levels were higher in preeclampsia than in normal pregnancy (Fig. 6). The fibrinolytic system, with its highly complex system of activators and inhibitors, is designed for local action. Studies on the circulating blood will provide only a limited perception of events taking place in a specific area of the vascular system. For this reason, we have endeavored to study the process in the uterus at the time of delivery.
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FIG. 3. Inhibition (arrows) of fibrinolysis (F) at (a) the cytotrophoblast cell (T) in contrast to (b) the endothelial cell (E) in the lining of the spiral artery in pregnancy, c: Immunologic staining with PAI-2 antisera shows the inhibitor localized to the trophoblast (arrows) of a decidual spiral artery.
FIG. 4.
SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 16, NO. 3, 1990
Plasminogen activator levels (antigen) in 28 healthy primigravidae and 15 primigravidae with preeclampsia.
Uteroplacental Fibrinolysis in Preeclampsia and Fetal Growth Retardation These studies have been carried out in women requiring delivery by cesarean section and the findings in normal pregnancy and pregnancy complicated by preeclampsia and fetal growth retardation have been compared in peripheral blood, uterine vein blood, and cord blood taken virtually simultaneously.9 Tissue extracts from the
FIG. 5.
placenta and the placental bed of the uterus were also studied. t-PA antigen was significantly higher in pregnancies complicated by fetal growth retardation in peripheral and uterine vein blood and cord blood and the levels in uterine blood were higher than peripheral blood; in preeclampsia levels of t-PA antigen were significantly higher in peripheral blood (Fig. 7). PAI-1 was higher in pregnancies complicated by
Plasminogen activator inhibitor levels in 28 healthy primigravidae and 15 primigravidae with preeclampsia.
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preeclampsia and fetal growth retardation in both peripheral and uterine vein blood, but the cord levels were similar (Fig. 8). The placental inhibitor PAI-2 was found to be much lower in pregnancies complicated by preeclampsia and fetal growth retardation in both peripheral and uterine vein blood, and within each group the levels of PAI-2 in peripheral and uterine vein blood were similar (Fig. 9). These findings would suggest that within the uteroplacental circulation disordered fibrinolysis occurs in preeclampsia and fetal growth retardation. The low levels of PAI-2 may be the result of the impaired placental
function in preeclampsia and intrauterine fetal growth retardation or due to the greater consumption of the inhibitor associated with the excess of fibrin that is present in the uteroplacental circulation. The higher level of t-PA antigen and PAI-1 in preeclampsia and fetal growth retardation may be the result of enhanced fibrinolysis in the circulation to counteract the lowgrade systemic intravascular coagulation that occurs in preeclampsia. Fibrin is a necessary matrix to protect the integrity of the maternal and fetal circulation at the interface of the placenta. The related disorders of bleeding and thrombosis at the placental site are responsible for a substantial
UTEROPLACENTAL FIBRINOLYSIS in PET and IUGR TISSUE PLASMINOGEN ACTIVATOR ANTIGEN
FIG. 7. Tissue plasminogen activator antigen in peripheral blood, uterine vein blood and cord blood in normal pregnancy and pregnancy complicated by preeclampsia (PET) and fetal growth retardation (IUGR).
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FIG. 6. Plasminogen activator antigen and plasminogen activator inhibitor levels in maternal and cord blood at time of delivery in 28 healthy primigravidae and in 15 primigravidae with preeclampsia.
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UTEROPLACENTAL FIBRINOLYSIS in PET and IUGR
FIG. 8. Plasminogen activator inhibitor (PAI-1) in peripheral blood, uterine vein blood and cord blood in normal pregnancy and pregnancy complicated by preeclampsia (PET) and fetal growth retardation (IUGR).
UTEROPLACENTAL FIBRINOLYSIS in PET and IUGR PLASMINOGEN ACTIVATOR INHIBITOR 2
FIG. 9. Placental inhibitor (PAI-2) in peripheral and uterine vein blood in normal pregnancy and pregnancy complicated by preeclampsia (PET) and fetal growth retardation (IUGR).
proportion of fetal mortality and morbidity. Further research in this area may open up new possibilities for protecting the fetus in utero from the impaired placental function that arises from disordered local coagulation and fibrinolysis in the maternal blood supply to the placenta.
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