Cross-Sectional Characterization of Albumin Glycation State in Cerebrospinal Fluid and Plasma from Alzheimer's Disease Patients.

We determined albumin post-translational modifications (PTMs) by mass spectrometry (MS) in plasma and cerebrospinal fluid (CSF) from 31 Alzheimer's disease (AD) patients (with 27 samples of paired plasma-CSF from the same patients). Results were cross-sectionally compared with healthy controls. For percentage of relative intensity of glycated isoforms, plasma albumin was globally more glycated in AD patients than in healthy controls (P<0.01). MS results in plasma were confirmed by a quantitative enzymatic assay (Lucica GA-L) for albumin early-glycation detection. In CSF there were no global glycation differences detected by MS, although a different pattern of glycated isoforms was observed. Oxidized+glycated and cysteinylated+glycated isoforms were increased in both plasma and CSF of AD patients in comparison with healthy controls (P<0.001). Furthermore, AD patients showed higher glycation in plasma than in CSF (P<0.01). Our data support the role of glycation and oxidative stress in AD.

Native plasma-derived FVIII/VWF complex has lower sensitivity to FVIII inhibitors than the combination of isolated FVIII and VWF proteins. Impact on Bethesda assay titration of FVIII inhibitors.

Sensitivity to FVIII inhibitors of the native plasma-derived (pd) FVIII/VWF complex vs. the complexes formed after exogenous FVIII infusion in the haemophilic patient has not been thoroughly studied. The role of VWF in the interaction of FVIII with inhibitors was studied in vitro using different combinations of VWF and FVIII concentrates. Normal plasma, pdFVIII/VWF and isolated FVIII (recombinant FVIII, B-domain deleted and pdFVIII) were used. Titre (BU) was kinetically determined (up to 2 h) in serial dilutions of inhibitor IgG (purified from a pool of plasmas with inhibitors) mixed with VWF and then incubated with the different FVIII. Inhibitor was also added to previously mixed VWF+FVIII. Residual FVIII:C was determined. TGA assays were performed with FVIII-deficient plasma spiked with the FVIII-VWF mixtures with/without an ESH-8 antibody. Inhibitor titres for plasma and pdFVIII/VWF were comparable at all time points. Titres for all concentrates of isolated FVIII were significantly higher than those for plasma or pdFVIII/VWF (1.4-1.9 fold) even after preincubation with VWF. At t = 0 h, titres for plasma or pdFVIII/VWF were unquantifiable, but were detectable for isolated FVIII (0.6-1.6 BU). In contrast to pdFVIII/VWF, the decrease in thrombin generation parameters by isolated FVIII in the presence of ESH-8 was significant (P < 0.01) even when previously combined with VWF. In conclusion, VWF protection against FVIII inhibitor activity might be higher with native pdFVIII/VWF complex than with the corresponding compound formed from the isolated proteins. Bethesda assay titration using different FVIII concentrates would be advisable to guide the treatment of inhibitor patients.

Kinetics of the interaction between anti-FVIII antibodies and FVIII from therapeutic concentrates, with and without von Willebrand factor, assessed by surface plasmon resonance.

The presence of VWF in plasma-derived FVIII (pdFVIII/VWF) products has been pointed out as a key difference with recombinant FVIII (rFVIII) products with regard to immunogenicity. A Surface Plasmon Resonance (SPR) study was designed to characterize in detail the interaction between anti-FVIII (IgGs) from a severe haemophilia A patient, and FVIII from concentrates of different sources. Full-length rFVIII (preincubated or not with purified VWF), B domain-deleted (BDD)-rFVIII and pdFVIII/VWF were analysed. To ensure reproducible conditions for accurate determination of kinetic constants, a capture-based assay format was developed using protein G surfaces for specific and reversible coupling of endogenous anti-FVIII antibodies. Concentration ranges (nm) of FVIII products tested were 9-0.03 (rFVIII) and 6-0.024 (pdFVIII/VWF). The association with antibodies was monitored for 3-5 min, whereas dissociation of the complex was followed for 5-20-240 min. A strong interaction of rFVIII and BDD-rFVIII with patient's IgG was detected with the K (D) values in the low picomolar range (5.9 ± 3.0 and 12.7 ± 6.9 pm, respectively) and very slow dissociation rates, while pdFVIII/VWF showed only marginal binding signals. The VWF complexed rFVIII displayed reduced binding signals compared with uncomplexed rFVIII, but the K (D) was still in the picomolar range (4.1 ± 1.9 pm) indicating insufficient complex formation. rFVIII, alone or bound to exogenously added VWF, showed high affinity for anti-FVIII IgGs from a severe haemophilia A patient whereas pdFVIII/VWF did not. These results are in agreement with those studies that point towards rFVIII concentrates to be more immunogenic than pdFVIII concentrates.

Management of bleeding disorders: basic science.

Development of factor VIII (FVIII) inhibitors is the most severe and challenging complication of haemophilia A treatment and represents the highest economic burden for a chronic disease. Therefore, major research efforts are ongoing to optimize the therapeutic approaches able to minimize this complication. FVIII inhibitors have variable immuno-reactivity against different FVIII concentrates and generally have a lower reactivity against von Willebrand factor (VWF)-containing FVIII concentrates than plasma-derived FVIII (pdFVIII) or recombinant FVIII (rFVIII) that are devoid of VWF, in particular when the inhibitors are directed against the light chain of FVIII. This paper provides an overview of several in vitro and in vivo studies that compared three clinically available clinical FVIII products (Kogenate®, Bayer AG, Leverkusen, Germany; Advate®, Baxter Healthcare, Zurich, Switzerland; and Fanhdi®, Grifols S.A., Barcelona, Spain) in order to evaluate the functional actvity of the FVIII fractions in rFVIII that cannot bind VWF; explore the use of the thrombin generation assay (TGA) as a potential tool for optimizing the choice of FVIII concentrate for use in haemophilia A patients with inhibitors; compare the kinetics of the interactions between anti-FVIII antibodies and FVIII both in the presence/absence of VWF, using surface plasmon resonance.

Understanding FVIII/VWF complex--report from a symposium of XXIX WFH meeting 2010.

von Willebrand factor (VWF) has the capacity to form a complex with factor VIII (FVIII) which may modulate the immunogenicity of FVIII. It has been proposed that a significant fraction of recombinant FVIII (rFVIII) is unable to bind VWF. In an experimental model studied at the McMaster University in Canada, this VWF-unbound rFVIII fraction showed no coagulant function. Sulphation of FVIII tyrosine (Tyr) 1680 has been reported as essential for the interaction with VWF. In a study performed at the Grifols and CNS-CSIC in Spain, Tyr1680 sulphation was observed to be incomplete in rFVIII and complete in plasma-derived FVIII (pdFVIII). This could explain the incapability of some rFVIII molecules to bind VWF. Experience with immune tolerance induction (ITI) at the Bonn Haemophilia Centre indicates that only eradication of FVIII inhibitors allows safe haemostasis control and the option of prophylactic treatment. Various clinical trials were planned to evaluate the clinical role VWF-containing FVIII concentrates (FVIII/VWF). RES.I.ST (an acronym for REScue Immunotolerance STudy) is an international, prospective study aimed at assessing whether FVIII/VWF can induce ITI in high-risk haemophilia patients (RES.I.ST naïve) and whether patients who previously failed ITI with FVIII alone can be rescued with FVIII/VWF (RES.I.ST experienced). Enrolment started in November 2009. In the FAIReSt.Will (Fanhdi and Alphanate Italian Retrospective Study in Willebrand disease) study, 120 von Willebrand disease (VWD) patients treated with Fanhdi(®) or Alphanate(®) were retrospectively analysed. Efficacy was excellent and no side effects were reported. The ongoing PRO.Will study is a prospective, multicenter trial aimed at assessing the efficacy, safety and pharmacoeconomics of secondary long-term prophylaxis in patients with severe inherited VWD.

Effect of temperature on plasma freezing under industrial conditions.

The European Pharmacopoeia monograph on Human plasma for fractionation does not define the freezing process time but does define the freezing temperature (- 30 degrees C or below). Initial freezing conditions are crucial for the quality of plasma. These conditions were intended to preserve labile proteins such as fVIIl, but they can also be considered favourable for the plasma quality in general. This study evaluates the way the industrial plasma freezing affects labile coagulation factors. We have studied the freezing of plasma in industrial-size chambers at temperatures close to - 30 degrees C, - 25 degrees C and - 20 degrees C, and the possible differences between performing the freezing process in a chamber or in a freezer, in order to elucidate whether or not these parameters affect the quality of plasma. For this study, plasma bottles were frozen in industrial chambers set at - 30 degrees C, - 25 degrees C and - 20 degrees C, and in a freezer set at - 20 degrees C. The freezing rates were followed by means of probes in plasma control bottles. From this plasma, coagulation factors (fVIII, fIX and fibrinogen) were analysed before and after freezing, and cryoprecipitate was obtained in some cases. Statistically significant differences exist in fVIII:C recovery in thawed plasma between freezing at - 30 degrees C and at - 20 degrees C (n = 11; 85.4 +/- 4.3 % versus 74.6 +/- 6.0 % (chamber) or 79.3 +/- 6.3 % (freezer)). There is no difference between - 30 degrees C and - 25 degrees C, or between freezing at - 20 degrees C in a chamber or in a freezer. No significant loss of activity in thawed plasma is observed for fIX and fibrinogen at - 25 degrees C or - 20 degrees C versus - 30 degrees C. The fVIII and vWF recovery in cryoprecipitates does not show differences (464.2 IU fVIII/ml at - 30 degrees C, 446.7 IU fVIII/ml at - 25 degrees C, and 475.8 IU fVIII/ml at - 20 degrees C). The results obtained from this study support that plasma might also be frozen at - 25 degrees C or below without any impact on its quality, and that sporadic and short term deviations, from - 30 degrees C or below up to - 25 degrees C, in the currently required freezing temperature, would not have an effect on the labile factors recovery.

PAI-1 promoter 4G/5G genotype as an additional risk factor for venous thrombosis in subjects with genetic thrombophilic defects.

Impaired fibrinolysis as a result of increased plasminogen activator inhibitor-1 (PAI-1) levels in plasma is a common finding in patients with deep vein thrombosis (DVT). A 4G/5G polymorphism in the promoter region of the PAI-1 gene has been reported to influence the levels of PAI-1. The 4G allele was found to be associated with higher plasma PAI-1 activity (act), but contradictory results on the incidence of the 4G allele in DVT patients have been reported. The aim of this study was to analyse whether the PAI-1 promoter 4G/5G genotype increases the risk of venous thrombosis in subjects with thrombophilic defects, and to determine the distribution of the PAI-1 4G/5G genotype and its relation to plasma PAI-1 levels in 190 unrelated patients with DVT in comparison with a control group of 152 healthy subjects. No differences between the 4G/5G allele distribution in the DVT group (0.43/0.57) and in the control group (0.42/0.58) were observed. However, the presence of the 4G allele significantly increased the risk of thrombosis in patients with other thrombophilic defects. Significantly higher PAI-1 levels were observed in DVT patients than in the controls. Our results also showed significant differences in the plasma levels of PAI-1 antigen (ag) and PAI-1 act among the 4G/5G genotypes in DVT patients. A multivariate analysis revealed that, in the DVT group, PAI-1 ag levels were influenced by the 4G allele dosage, triglyceride levels and body mass index (BMI). The influence of the 4G allele dosage on PAI-1 levels was independent of the triglyceride levels and BMI. In the control group, no significant correlation between PAI-1 levels and 4G allele dosage was observed. In conclusion, the PAI-1 promoter polymorphism was found to have an influence on PAI-1 levels in DVT patients and on the risk of venous thrombosis in subjects with other genetic thrombophilic defects.

Plasminogen activator inhibitor-1 (PAI-1) promoter 4G/5G genotype and increased PAI-1 circulating levels in postmenopausal women with coronary artery disease.

Increased circulating levels of type 1 plasminogen activator inhibitor (PAI-1) have been associated with coronary artery disease (CAD). However, genetic and environmental determinants of PAI-1 expression are only partially understood. The levels of PAI-1 have been found to relate to 4/5 guanosine (4G/5G) polymorphism in the promoter region of the PAI-1 gene. The 4G allele in this polymorphism has been associated with higher levels of plasma PAI-1 activity, but despite the strong correlation between PAI-1 activity and antigen, no association has been found between PAI-1 antigen levels and the PAI-1 promoter 4G/5G genotype. The aim of the present study was to analyze the influence of the PAI-1 promoter 4G/5G genotype on PAI-1 levels in post-menopause women with coronary disease in comparison with healthy women in pre and postmenopausal status, and the influence of this genotype on variations in PAI-1 levels after hormone replacement therapy (HRT). No differences between 4G/5G allele distribution in the groups studied were observed. The group of postmenopausal women with CAD showed significantly increased PAI-1 antigen and activity levels in comparison with the control groups, and the levels of PAI-1 correlated with the 4G/5G genotype. A multivariate analysis revealed that in the CAD group there was a high correlation between 4G allele dosage and PAI-1 antigen levels, which were also influenced by the triglyceride levels but not by estrogen or glucose levels. After hormone replacement therapy the decrease in PAI-1 levels was correlated with the 4G allele dosage. We conclude that in the group of postmenopausal women with CAD the influence of the PAI-1 promoter 4G/5G genotype on PAI-1 levels is more evident than in the control groups, and that the decrease in PAI-1 levels after HRT in CAD women correlates with the 4G allele dosage.

Lipoprotein(a) levels and isoforms and fibrinolytic activity in postmenopause--influence of hormone replacement therapy.

Epidemiological studies suggest that hormone replacement therapy (HRT) decreases the risk of cardiovascular disease in postmenopausal women via several mechanisms, including modifications in the fibrinolytic system and lipoprotein(a) [Lp(a)] levels. The aim of this study was to examine the influence of the levels and isoforms of Lp(a) on fibrinolytic activity in 91 postmenopausal women in comparison with premenopause and analyze the effect of HRT on those parameters. In postmenopause, an increase in plasma Lp(a) and plasminogen activator inhibitor-1 (PAI-1) levels was found. A significant inverse correlation was observed between Lp(a) or PAI-1 levels and plasmin generation. Plasma samples with low molecular weight (MW) apo(a) isoforms showed higher plasmin inhibition than plasmas with high MW apo(a) isoforms and similar levels of total Lp(a) and PAI-1. HRT induced a significant decrease in Lp(a) and PAI-1 levels and an increase in estradiol levels, as well as an increase in fibrinolytic activity. A significant correlation was found between the percentages of variation in Lp(a) levels and in plasmin generation and between the percentages of variation in PAI-1 levels and in the euglobulin lysis time under HRT. In conclusion, the increase in fibrinolytic activity observed in women under HRT could be explained by two independent mechanisms: (a) the decrease in PAI-1 and (b) the decrease in the inhibition of plasmin generation due to the decrease in Lp(a) levels.

Factor V Leiden and antibodies against phospholipids and protein S in a young woman with recurrent thromboses and abortion.

We describe the case of a 39-year-old woman who suffered two iliofemoral venous thromboses, a cerebral ischemic infarct and recurrent fetal loss. Initial studies showed high levels of antiphospholipid antibodies (APAs) and a moderate thrombocytopenia. After her second miscarriage, laboratory diagnosis revealed that the woman was heterozygous for the factor V Leiden mutation and had a functional protein S deficiency as well as anti-protein S and anti-beta 2-glycoprotein I antibodies. The impairment of the protein C pathway at various points could well explain the recurrent thromboses in the patient and supports the role of a disturbed protein C system in the pathophysiology of thrombosis in patients with APAs.

Abnormal expression of type 1 plasminogen activator inhibitor and tissue factor in severe preeclampsia.

Preeclampsia is a multisystemic obstetric disease of unknown etiology that is commonly associated with fibrin deposition, occlusive lesions in placental vasculature, and intrauterine fetal growth retardation. We previously reported that type 1 plasminogen activator inhibitor (PAI-1) levels are significantly increased in plasma and placenta from pregnant women with preeclampsia compared to normal pregnant women. In the present report we localize the expression of placental PAI-1 in greater detail and compare it with that of tissue factor (TF), a procoagulant molecule, and vitronectin (Vn), a PAI-1 cofactor. We also examine the expression of two cytokines, tumor necrosis factor alpha (TNFalpha) and interleukin-1 (IL-1), in order to begin to define the underlying mechanisms responsible for the elevated levels of PAI-1 and fibrin deposits observed in placenta from preeclampsia. We demonstrate a significant increase in PAI-1, TF and TNFalpha antigen and PAI-1 and TF mRNA in placentas from preeclamptic patients. PAI-1 mRNA was increased not only in syncytiotrophoblast and infarction areas, but also in fibroblasts and in some endothelial cells of fetal vessels in placentas from preeclamptic patients. However, there was no colocalization between PAI-1, TF, Vn and TNFalpha in placental villi. The elevated TNFalpha in the placenta may induce PAI-1 and TF, and thus promote the thrombotic alterations associated with preeclampsia.

Activated protein C resistance phenotype in patients with antiphospholipid antibodies.

The effect of antiphospholipid antibodies (aPL) on the action of activated protein C (APC) was examined in 32 patients: 19 with lupus anticoagulant (LA), 6 with anticardiolipin antibodies (aCL), and 7 with LA and aCL. Eighteen patients had a ratio of activated partial thromboplastin time (APTT) with APC to APTT without APC (APTT ratio) <2.06 (cut-off level) and no factor V Leiden mutation; these patients showed APC-resistance (APC-R) phenotype. The mean prolongation of APTT after addition of APC in a control group was 45.3 seconds, with a lower limit of 31.4 seconds. Only 3 of the 18 patients with low APTT ratio had a prolongation of <31.4 seconds; they were classified as true APC-R phenotype, whereas the other 15 patients were classified as spurious APC-R. Of the 3 patients with true APC-R, 2 had deep venous thrombosis, 1 with pulmonary embolism, and the third had recurrent abortion. Of the other 15 patients, 2 had had ischemic stroke, 1 had recurrent abortion, and 12 were asymptomatic. Circulating APC level was measured in 14 of the 18 aPL patients with a low APTT ratio; it was lower than the normal lower limit in 4 patients and within the lower limit in 2. Three of the 4 patients with reduced APC levels had a history of thrombosis. We conclude that patients with aPL who show APC-R phenotype due to a low APTT ratio without the factor V Leiden mutation can be classified into two groups: true and spurious APC-R phenotype. Since those with true APC-R phenotype could have greater thrombotic risk, adequate classification of these patients is important. Moreover, aPL can sometimes interfere with the activation of protein C, thus reducing the circulating levels of APC, and this could constitute another thrombotic risk factor.

Decreased expression of PAI-2 mRNA and protein in pregnancies complicated with intrauterine fetal growth retardation.

An increase in plasma plasminogen activator inhibitors (PAIs), fundamentally PAI type 2 (PAI-2), has been described in normal pregnancy probably because the placenta is the main source of the high plasma levels of this protein. Although we have previously described plasmatic alterations of these inhibitors in pregnancies complicated with intrauterine fetal growth retardation (IUGR), no reports have been published about placental PAI-2 mRNA expression. In the present study, the placental PAI-2 expression determined in pregnancies complicated with IUGR and in severe preeclamptic patients was compared with that of normal pregnancies in order to identify the placental cell types expressing PAI-2 and to determine whether the production of PAI-2 is altered in placentas from IUGR. In situ hybridization analyses show that the syncytiotrophoblasts are the cells with the greatest PAI-2 expression in placenta. We report that the significant decrease in plasma and placental PAI-2 levels in IUGR groups is fundamentally due to a diminished expression of PAI-2 mRNA in placenta.

Abnormal expression of plasminogen activator inhibitors in patients with gestational trophoblastic disease.

We previously reported significantly elevated levels of plasminogen activator inhibitor type 1 (PAI-1) in plasma and placenta from pregnant women with severe pre-eclampsia, and pre-eclampsia is a frequent problem in molar pregnancies. As increases in PAI-1 may contribute to the placental alterations that occur in pre-eclampsia, we have begun to investigate changes in PAI-1 as well as PAI-2 and several other components of the fibrinolytic system in patients with trophoblastic disease. Significant increases in plasma PAI-1 and decreases in plasma PAI-2 levels were observed in molar pregnancies when compared with the levels in normal pregnant women of similar gestational age. PAI-1 antigen levels also were increased, and PAI-2 levels were decreased in placenta from women with molar pregnancies compared with placenta obtained by spontaneous abortion. Immunohistochemical analysis revealed strong positive and specific staining of PAI-1 in trophoblastic epithelium in molar pregnancies and relatively weak staining of PAI-2. No association between the distribution of PAI-1 and vitronectin was found, and no specific signal for tissue type PA, urokinase type PA, tumor necrosis factor-alpha, or interleukin-1 was detected. In situ hybridization revealed an increase in PAI-1 but not PAI-2 mRNAs in placenta from molar pregnancies in comparison with placenta from abortions. These results demonstrate increased PAI-1 protein and mRNA in trophoblastic disease and suggest that localized elevated levels of PAI-1 may contribute to the hemostatic problems associated with this disorder.

The effect of estrogen replacement therapy with or without progestogen on the fibrinolytic system and coagulation inhibitors in postmenopausal status.

OBJECTIVE: The aim of this study was to analyze several fibrinolytic components and coagulation inhibitors in postmenopausal women and had to evaluate the effect of hormone replacement therapy. STUDY DESIGN: Several hemostatic parameters were evaluated in 75 postmenopausal women before and after 3 to 4 and 12 months of hormone therapy. RESULTS: An increase in plasma fibrinolytic activity primarily related to a significant increase in tissue-type plasminogen activator and a decrease in plasminogen activator inhibitor type 1 was observed in women receiving hormone replacement therapy. A significant decrease in protein S and lipoprotein(a) was detected under therapy. No modifications in tissue-type plasminogen activator/plasminogen activator inhibitor-1 and activated protein C/alpha 1-antitrypsin complexes, urokinase activity, plasminogen, and antithrombin III were detected. CONCLUSIONS: The increase in fibrinolytic activity and the decrease in lipoprotein(a) levels observed in women receiving hormone replacement therapy could help decrease the risk of coronary disease associated with the postmenopausal state.

Fibrinolytic system and reproductive process with special reference to fibrinolytic failure in pre-eclampsia.

Here we summarize the recent progress in research on the role of the fibrinolytic system in reproduction, with a special emphasis on the role of the plasminogen activator inhibitors in fetal development. Trophoblasts produce fibrinolytic proteins that can promote normal implantation and regulate blood flow to the fetus and placenta throughout pregnancy. Normal pregnancy is associated with a hypofibrinolytic state that is fundamentally caused by an increase in plasminogen activator inhibitors types 1 and 2. In pre-eclampsia, a fibrinolytic failure, resulting from an increase in plasma and placental concentrations of plasminogen activator inhibitor-1, was observed. The localized elevated concentrations of placenta plasminogen activator inhibitor-1 protein and mRNA observed in pre-eclamptic patients would be expected to foster the deposition of fibrin and thus play a role in the complications associated with this disease. The decreased plasminogen activator inhibitor-2 concentrations in placenta and plasma from intrauterine fetal growth retardation pregnancies and the positive correlation between plasma/placenta plasminogen activator inhibitor-2 concentration and birthweight suggest that this inhibitor could be considered an adequate marker of placental function.

Altered expression of plasminogen activator inhibitor type 1 in placentas from pregnant women with preeclampsia and/or intrauterine fetal growth retardation.

Elevated plasma levels of type 1 plasminogen activator inhibitor (PAI-1) have been implicated in mediating the fibrin deposition and occlusive lesions that occur within the placental vasculature in preeclampsia (PE) and intrauterine growth retardation (IUGR). In this report we identify the cells within the normal-appearing villous tissue that are responsible for the local production of PAI-1 in women with PE and IUGR. Levels for another fibrinolytic inhibitor (ie, type 2 plasminogen activator inhibitor [PAI-2]) were determined for comparative purposes. Elevated levels of PAI-1 were detected in placenta extracts from PE/IUGR patients (121 +/- 38 ng/mg, n = 8) when compared with the levels in placenta extracts from normal women (43 +/- 17 ng/mg, n = 10) or women with IUGR but not PE (51 +/- 22 ng/mg, n = 11). Immunohistochemical analysis of paraffin sections showed an increased immunoreactivity for PAI-1 in the placental villous syncytiotrophoblasts from PE/IUGR women compared with the immunostaining of placental samples from the normal or IUGR group. In contrast, antigen levels and immunostaining for PAI-2 were reduced in the placentas harvested from not only the PE/IUGR women (209 +/- 144 ng/mg) but also the IUGR group (169 +/- 106 ng/mg) in comparison with the PAI-2 levels in normal placentas (535 +/- 98 ng/mg). To document that the increased immunoreactivity for PAI-1 in PE/IUGR syncytiotrophoblasts was mediated by an increased production of PAI-1 within these cells, in situ hybridization analysis was performed. A strong positive signal for PAI-1 mRNA in villous syncytiotrophoblasts from PE patients (n = 5) was obtained after 2 weeks of exposure to the NTB2 emulsion in comparison with the weak signal for PAI-1 mRNA that required a 10-week exposure of the normal placenta sections (n = 10). Northern blotting for PAI-1 mRNA showed that both transcripts (ie, 3.2 and 2.3 kb) were elevated in samples of two PE patients in comparison with the PAI-1 mRNA transcripts present in a normal placenta and an IUGR placental sample. These results show increased PAI-1 and mRNA levels in placentas from PE patients and raise the possibility that localized elevated levels of PAI-1 may play a role in the initiation of placental damage, as well as in the thrombotic complications associated with this disease.

Evaluation of plasminogen activators and plasminogen activator inhibitors in plasma and amniotic fluid in pregnancies complicated with intrauterine fetal growth retardation.

Several fibrinolytic parameters were determined in plasma and amniotic fluid from normotensive pregnancies complicated by intrauterine fetal growth retardation (IUGR) and severe preeclamptic (PE) patients with IUGR and compared with data from normal pregnancies. A significant decrease in plasminogen activator type 2 (PAI-2) and urokinase levels in plasma and amniotic fluid was observed in IUGR groups in comparison with normal pregnancy. No significant differences were observed between the control and IUGR groups in relation to the other fibrinolytic parameters, except for plasma PAI type 1 and tissue-type plasminogen activator levels, which were significantly increased in the PE group. A significant positive correlation was observed between birth weight and PAI-2 levels in both plasma and amniotic fluid, but the plasma PAI-2 levels showed a higher correlation. In conclusion, these results suggest that the PAI-2 level measured in plasma is a more adequate marker of placental function than the PAI-2 level measured in amniotic fluid.