This report defines the nature of the molecules responsible for the increased plasma plasminogen activator inhibitor (PAI) activity in preeclamptic patients and the relationship of these inhibitors to the severity of placental damage in preeclampsia. Clinical groups consisting of pregnant women with either severe preeclampsia or chronic hypertension with superimposed severe preeclampsia, as well as normal pregnant and nonpregnant women, were analyzed in a panel of functional and immunologic assays for PAI-1 and PAI-2. Pure severe preeclamptic patients in their third trimester showed a significant increase in both antigenic (136 ng/mL) and functional (5.76 U/mL) type 1 PAI (PAI-1) as compared with normal third-trimester pregnant women (34.8 ng/mL and 2.57 U/mL, respectively). In contrast, antigenic (186 ng/mL) and functional (5.76 U/mL) levels of type 2 PAI (PAI-2) were significantly lower in the pure severe preeclampsia group as compared with the values of the normal pregnant group (269 ng/mL and 9.58 U/mL, respectively). The patients with chronic hypertension and superimposed severe preeclampsia exhibited PAI-2 levels comparable to those of the pure preeclamptic group, whereas their antigenic and functional PAI-1 levels were intermediate (94 ng/mL and 3.25 U/mL, respectively) between the normal pregnant and the pure preeclamptic groups. During early puerperium of both normal pregnant women and patients, plasma PAI-1 antigen and activity decreased within one day to approximately the levels detected in normal nonpregnant women, while PAI-2 levels remained elevated for over 11 days. Similar results were obtained in plasma samples obtained from citrated blood and blood collected with an anticoagulant/antiplatelet mixture, suggesting that increased PAI-1 levels in preeclamptic patients were not due to platelet activation in vitro. In preeclamptic patients, a positive correlation between birth weight and PAI-2 values was observed (r = .64, P less than .05), whereas birth weight was inversely correlated with both PAI-1 levels and total PAI activity (r = -.6, P less than .005 and r = -.76, P less than .005 respectively). Preeclamptic patients with extensive placental infarction exhibited higher plasma PAI activity (24.1 U/mL v 11.6 U/mL) and PAI-1 values (305 ng/mL v 80.9 ng/mL) than preeclamptic patients without extensive placental infarction. In contrast, PAI-2 levels were reduced in preeclamptic patients with infarction in comparison with those of patients without infarction (141 ng/mL v 212.9 ng/mL). Our data indicate that increases in the level of PAI-1 accounts for the high plasma PAI activity in severe preeclampsia as measured using single-chain t-PA.

The mechanism by which activated protein C stimulates fibrinolysis was studied in a simple radiolabeled clot lysis assay system containing purified tissue-type plasminogen activator, bovine endothelial plasminogen activator inhibitor (PAI), plasminogen, 125I-fibrinogen and thrombin. Fibrinolysis was greatly enhanced by the addition of purified bovine activated protein C; however, in the absence of PAI, activated protein C did not stimulate clot lysis, thus implicating this inhibitor in the mechanism. In clot lysis assay systems containing washed human platelets as a source of PAI, bovine-activated protein C-dependent fibrinolysis was associated with a marked decrease in PAI activity as detected using reverse fibrin autography. Bovine-activated protein C also decreased PAI activity of whole blood and of serum. In contrast to the bovine molecule, human-activated protein C was much less profibrinolytic in these clot lysis assay systems and much less potent in causing the neutralization of PAI. This species specificity of activated protein C in clot lysis assays reflect the known in vivo profibrinolytic species specificity. When purified bovine-activated protein C was mixed with purified PAI, complex formation was demonstrated using immunoblotting techniques after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. These observations suggest that a major mechanism for bovine protein C- dependent fibrinolysis in in vitro clot lysis assays involves a direct neutralization of PAI by activated protein C.

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.

The extracellular matrix (ECM) of cultured bovine aortic endothelial cells (BAEs) was analyzed by immunoblotting and reverse fibrin autography and shown to contain type 1 plasminogen activator inhibitor (PAI-1). Most PAI-1 in the ECM formed complexes with exogenously added tissue-type plasminogen activator (tPA), demonstrating that this PAI-1 was functionally active. The resulting tPA/PAI-1 complexes were recovered in the reaction solution, indicating that the PAI-1 in such complexes no longer bound to ECM. The PAI-1 could not be removed by incubating ECM in high salt (2 mol/L NaCl), sugars (1 mol/L galactose, 1 mol/L mannose), glycosaminoglycans (10 mmol/L heparin, 10 mmol/L dermatan sulfate), or epsilon-aminocaproic acid (0.1 mol/L). However, PAI-1 could be extracted from ECM by treatment with either arginine (0.5 mol/L) or potassium thiocyanate (2 mol/L), or by incubation under acidic conditions (pH 2.5). ECM depleted of PAI-1 by acid extraction was able to bind both the active and latent forms of PAI-1. In this instance, most of the bound PAI-1 did not form complexes with tPA, indicating that the latent form was not activated as a consequence of binding to ECM. Although the PAI-1 activity in conditioned medium decayed with a half-life (t 1/2) of less than 3 hours, the t 1/2 of ECM- associated PAI-1 was greater than 24 hours. These data suggest that PAI- 1 is produced by cultured BAEs in an active form and is then either released into the medium where it is rapidly inactivated or into the subendothelium where it binds to ECM. The specific binding of PAI-1 to ECM protects it from this inactivation.

Vitronectin (Vn) regulates proteolytic enzyme systems, as well as cell migration and tissue remodelling. These processes have been implicated in the pathogenesis of atherosclerosis. In this study, the distribution of Vn antigen in apparently normal and atherosclerotic human blood vessels was evaluated. Normal and diseased vessels showed Vn immunostaining in the lamina elastica interna and externa, and in strand-like structures in the adventitia. In most of these instances, the Vn antigen appeared to be located in the proximity of elastin. In pulmonary arteries, Vn staining was additionally detected in the media. The intima was devoid of Vn antigen in all vessels studied. In general, there was increased deposition of Vn antigen in the atherosclerotic arteries. In particular, strong Vn staining was apparent in amorphous material adjacent to cholesterol clefts and in acellular fibrous tissue, in plaques present in the carotic artery and aorta. Collagen layers and fresh fibrin depositions were devoid of Vn antigen. In spite of the abundance of Vn immunostaining throughout the normal and diseased vessel wall, the Vn transcript was not detectably by in situ hybridization. These results indicate that Vn is a constituent of the normal vessel wall and raise the possibility that increased local deposition of Vn may be related to the development of atherosclerotic vascular disease.