A molecular variant of plasminogen activator inhibitor of rat decidual tissue.

Artificially induced rat decidual tissue expresses plasminogen activator inhibitor (PAI). This PAI, isolated and purified employing chromatographic techniques, is a low molecular weight protein unlike the known PAIs. The final purified preparation resolves into a single band following SDS-PAGE and has an approximate molecular weight of 29 kDa. The properties studied include specificity for urokinase-type (uPA) and tissue-type (tPA) plasminogen activators, binding to conA and heparin, inhibition of thrombin, plasmin and trypsin. Decidual PAI is immunogenic in rabbit and a monospecific antiserum raised against the decidual inhibitor cross reacts with an extract of human placenta.

Inhibition of tissue plasminogen activators and urokinase by human saliva.

The fibrinolytic activity found in body fluids is mediated through the generation of plasmin from plasminogen by plasminogen activator. Mixed fresh human saliva was found to have plasminogen activator inhibitory activity that has not yet been characterized. In the present study, mixed fresh human saliva was found to contain an inhibitor for tissue plasminogen activator and for urokinase. The plasminogen activator inhibitory activity was mainly found in the mixed fresh human saliva cellular pellet containing buccal epithelial cells and not in the supernatant salivary fluid. Saliva collected by Stenson's duct cannulation does not have any PA-inhibitor activity, although saliva collected from submandibular glands contains small amounts of such activity. The salivary plasminogen activator inhibitor is unstable, and its activity is lost during storage.

Plasminogen activator inhibitor-1 binds to fibrin and inhibits tissue-type plasminogen activator-mediated fibrin dissolution.

Plasminogen activator inhibitor-1 (PAI-1) accumulates within thrombi and forming whole blood clots. To explore this phenomenon at the molecular level, PAI-1 binding to fibrin was examined. The experiments were performed by adding 125I-PAI-1, which retains its complete tissue-type plasminogen (t-PA) inhibitory activity, to fibrin matrices formed in 2-cm2 tissue culture wells. Guanidine HCl-activated PAI-1 binding was reversible and was inhibited in the presence of excess, unlabeled PAI-1. Activated 125I-PAI-1 recognized 2 sites on fibrin: a very small number of high affinity sites (Kd less than 1 nM) and principally a large number of low affinity sites with an approximate Kd of 3.8 microM. Latent PAI-1 bound to fibrin at a site indistinguishable from the lower affinity site recognized by activated PAI-1. Fibrin, pretreated with activated PAI-1, was protected from t-PA-mediated plasmin degradation in a PAI-1 dose-responsive manner (IC50 = 12.3 nM). Clot protection correlated with partial occupancy of the low affinity PAI-1 binding site on fibrin and was due to the formation of sodium dodecyl sulfate-stable, PAI-1.t-PA complexes. Latent PAI-1 (27 nM) did not protect the fibrin from dissolution. The localization of PAI-1 to a thrombus by virtue of its fibrin binding potential could result in significant protection of the thrombus from the degradative effects of the fibrinolytic system.

Regulation of gene expression by SPARC during angiogenesis in vitro. Changes in fibronectin, thrombospondin-1, and plasminogen activator inhibitor-1.

Angiogenesis in vitro, the formation of capillary-like structures by cultured endothelial cells, is associated with changes in the expression of several extracellular matrix proteins. The expression of SPARC, a secreted collagen-binding glycoprotein, has been shown to increase significantly during this process. We now show that addition of purified SPARC protein, or an N-terminal synthetic peptide (SPARC4-23), to strains of bovine aortic endothelial cells undergoing angiogenesis in vitro resulted in a dose-dependent decrease in the synthesis of fibronectin and thrombospondin-1 and an increase in the synthesis of type 1-plasminogen activator inhibitor. SPARC decreased fibronectin mRNA by 75% over 48 h, an effect that was inhibited by anti-SPARC immunoglobulins. Levels of thrombospondin-1 mRNA were diminished by 80%. Over a similar time course, both mRNA and protein levels of type 1-plasminogen activator inhibitor (PAI-1) were enhanced by SPARC and the SPARC4-23 peptide. The effects were dose-dependent with concentrations of SPARC between 1 and 30 micrograms/ml. In contrast, no changes were observed in the levels of either type I collagen mRNA or secreted gelatinases. Half-maximal induction of PAI-1 mRNA or inhibition of fibronectin and thrombospondin mRNAs occurred with 2-5 micrograms/ml SPARC and approximately 0.05 mM SPARC4-23. Strains of endothelial cells that did not form cords and tubes in vitro had reduced or undetectable responses to SPARC under identical conditions. These results demonstrate that SPARC modulates the synthesis of a subset of secreted proteins and identify an N-terminal acidic sequence as a region of the protein that provides an active site. SPARC might therefore function, in part, to achieve an optimal ratio among different components of the extracellular matrix. This activity would be consistent with known effects of SPARC on cellular morphology and proliferation that might contribute to the regulation of angiogenesis in vivo.

A hybrid protein of urokinase growth-factor domain and plasminogen-activator inhibitor type 2 inhibits urokinase activity and binds to the urokinase receptor.

The binding of urokinase-type plasminogen activator (uPA) to its specific cell-surface receptor (uPAR) localises the proteolytic cascade initiated by uPA to the pericellular environment. Inhibition of uPA activity or prevention of uPA binding to uPAR might have a beneficial effect on disease states wherein this activity is deregulated, e.g. cancer and some inflammatory diseases. To this end, a bifunctional hybrid molecule consisting of the uPAR-binding growth-factor domain of uPA (amino acids 1-47; GFuPA) at the N-terminus of plasminogen-activator inhibitor type 2 (PAI-2) was produced in Saccharomyces cerevisiae. The purified protein inhibited uPA with kinetics similar to placental or recombinant PAI-2 and was also found to bind to U937 cells and to FL amnion cells. GFuPA-PAI-2 competed with uPA, the N-terminal fragment of uPA and a proteolytic fragment of uPA (amino acids 4-43) in cell binding experiments, indicating that the molecule bound to the cells via uPAR. Hence, both the uPA-inhibitory and uPAR-binding domains of the hybrid molecule were functional, demonstrating the feasibility of the novel concept of introducing an unrelated, functional domain onto a member of the serine-protease-inhibitor superfamily.

Identification of a conformationally distinct form of plasminogen activator inhibitor-1, acting as a noninhibitory substrate for tissue-type plasminogen activator.

Plasminogen activator inhibitor-1 (PAI-1), the primary physiological inhibitor of tissue-type plasminogen activator (t-PA) in plasma, is a serine proteinase inhibitor (serpin) that forms a 1:1 stoichiometric complex with its target proteinase leading to the formation of a stable inactive complex. The active, inhibitory form of PAI-1 spontaneously converts to a latent form that can be reactivated by protein denaturants. In the present study we have isolated another molecular form of intact PAI-1 that, in contrast with active PAI-1, does not form stable complexes with t-PA but is cleaved at the P1-P1' bond (Arg346-Met347). Other serine proteinases, e.g. urokinase-type plasminogen activator and thrombin, also cleaved this "substrate" form of PAI-1. Fluorescence spectroscopy revealed conformational differences between the latent, active, and substrate forms of PAI-1. This observation confirms our hypothesis that the three functionally different forms of PAI-1 are the consequence of conformational transitions. Thus PAI-1 may occur in three interconvertible conformations: latent, inhibitor, and substrate PAI-1. The identification of two distinct conformations of PAI-1 which interact with their target protease either as an inhibitor or as a substrate is a previously unrecognized phenomenon among the serpins. Conversion of substrate PAI-1 to its inactive degradation product may constitute a pathway for the physiological regulation of PAI-1 activity.

Identification of the 52 kDa cytoskeletal-like protein of cytochalasin D-stimulated normal rat kidney (NRK/CD) cells as substrate-associated glycoprotein p52 [plasminogen-activator inhibitor type-1 (PAI-1)]. Expression of p52 (PAI-1) in NRK/CD cells is regulated at the level of mRNA abundance.

Cell shape profoundly affects cellular metabolic activity, protein and nucleic acid synthesis, and cytoskeletal organization. To examine the influence of cell shape on protein expression, normal rat kidney (NRK) cells were exposed to the microfilament-disrupting drug cytochalasin D (CD), labelled with [35S]methionine, and newly synthesized cellular and cytoskeletal proteins examined by two-dimensional gel electrophoresis. CD produced dramatic changes in cell shape (from a flat to round phenotype) with concomitant 3-7-fold increases in the cellular content and cytoskeletal deposition of the microfilament-associated proteins actin, alpha-actinin, and tropomyosin isoform 1. Augmented actin protein content in NRK/CD cells was paralleled by a corresponding increase in actin mRNA abundance and was inhibited by prior addition of actinomycin D. A detergent-insoluble protein of 52 kDa was also detected at high levels in the cytoskeletal fraction of NRK/CD cells. Two-dimensional electrophoretic mapping of total cellular and cytoskeletal proteins revealed this 52 kDa protein to be the previously described glycoprotein p52 [Higgins & Ryan (1989) Biochem. J. 257, 173-182]. By using electrophoretic and immunochemical criteria, p52 was identified as plasminogen-activator inhibitor type-1 (PAI-1). Like actin, CD-induced p52(PAI-1) synthesis, cellular content, and partitioning to the detergent-insoluble cytoskeletal compartment reflected a corresponding increase in p52(PAI-1) mRNA. Such induction was similarly inhibited by actinomycin D. p52(PAI-1) expression in the NRK-cell system is thus responsive to CD-mediated shape changes and requires ongoing RNA synthesis for its induction. Differential extraction of detached cell bodies and the substrate-adherent 'remnant' fraction of NRK/CD cultures, furthermore, indicated that p52(PAI-1) was not an intrinsic internal cytoskeletal element but, rather, selectively localized to the extracellular residue. p52(PAI-1) retained its detergent-insoluble characteristics even in this isolated 'remnant' fraction, where it was also the predominant protein species resolved.

Fibrinolysis relating substances in marine creatures.

1. Extracts with physiological saline solution were obtained from about 20 species of invertebrates and seaweed. Tosyl-L-Arg-MeOH hydrolysing and fibrin plate lytic activity were detected in the invertebrates Stichopus japonicus, Crassost gigas, Tapes japonica, and Kintai-gai as well as the seaweed Codiales codium. 2. These activities were all labile against heat (at 65 degrees C for 1 hr). Except for the extract from Stichopus japonicus, lytic activities against fibrin plates with and without plasminogen were similar. 3. The extract from S. japonicus showed plasminogen activating potency as well as the existence of urokinase (UK) activity enhancing factor. 4. On the other hand, the extract of the seaweed Hizikia fusiformis showed a strong UK inhibiting activity. 5. A fraction of fibrinolytic enzyme was obtained from the extract of S. japonicus by absorption to the celite affinity chromatography. It was orally administered to rabbits at a dosage of 40 mg/kg/day. 6. Fibrinolytic activity was determined periodically on the eugloblin fraction of plasma samples collected from these animals. 7. As compared with the pretreatment value, the activity increased about 2 times (P less than 0.01) and 3 times (P less than 0.005) after 4 and 8 weeks, respectively, of the treatment. 8. After 8 weeks of treatment, the kidney of treated rabbits was extracted with 2 M KCl. The activity of tissue plasminogen activator (free-type TPA) was revealed to be enhanced significantly (P less than 0.001) in the extracts. 9. The fibrinolytic enzyme increased in the blood was recognized by zymography to be mainly the UK type plasminogen activator with mol. wt of 53,000.

Possible identity of kallikrein binding protein with protein C inhibitor.

Protein C inhibitor (PCI) inhibits tissue kallikrein by forming stable 1:1 complexes (k1 = 2.3 x 10(4)M-1s-1). Heparin inhibits the tissue kallikrein/PCI-interaction and complex formation of 125I-tissue kallikrein in serum. 125I-tissue kallikrein complexes formed in plasma can be immunoprecipitated with monoclonal anti-PCI IgG suggesting that PCI might be identical to the kallikrein binding protein described previously (J. Chao et al. 1986, Biochem. J. 239, 325-331).

Type-1 inhibitor of plasminogen activators. Distinction between latent, activated and reactive centre-cleaved forms with thermal stability and monoclonal antibodies.

Type-1 inhibitor of plasminogen activators (PAI-1) occurs in purified preparations in a latent form that can be activated with denaturants; in vivo, latency is prevented by binding to vitronectin. We have compared latent, denaturant-activated and reactive centre-cleaved human PAI-1 with respect to thermal stability and affinity to monoclonal antibodies. By both criteria, latent and cleaved PAI-1 are very similar or indistinguishable, and clearly different from active PAI-1. Our findings suggest that the conformations of latent and reactive centre-cleaved PAI-1 are similar and resemble the so-called relaxed (R) serpin conformation, while that of active PAI-1 is different and resembles the stressed (S) serpin conformation.

Plasminogen activators t-PA, u-PA and its inhibitor (PAI) in normal males and females.

We determined the plasma levels of tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PAI) activity and their antigen levels including urokinase plasminogen activator (u-PA) in 33 male and 27 female normal subjects. Males had mean t-PA activity of 0.50 iu/ml which was significantly lower (p less than 0.01) than the females 0.64 iu/ml. Males had higher (p less than 0.001) mean PAI activity (15.5 AU/ml) as compared to females 10.3 AU/ml. The respective mean levels of t-PA and PAI antigen were significantly higher (p less than 0.01) in males (8.1 ng/ml and 17.6 ng/ml) than in females (6.2 ng/ml and 12.1 ng/ml). The mean u-PA level in males was 1.54 ng/ml which was significantly higher (p less than 0.01) than in females with 1.02 ng/ml. In post-venous occlusion studies, females had a greater mean response of 8.6 fold in t-PA activity as compared to males with a mean of 4.5 fold increase. The mean t-PA antigen response in males was 2.0 fold increase as compared to 2.6 fold increase in the females. No significant responses were seen in both sexes in either PAI activity or antigen levels when compared with the resting state. In zymography studies, free t-PA, its inhibitor complexes and u-PA were demonstrated in the euglobulin fractions of stored plasma. This study demonstrates that significant differences in t-PA, u-PA and PAI exist between male and female subjects which should be taken into account when determining their levels in clinical conditions.

Plasminogen activator inhibitor 1 in human carcinoma tissues.

The content of PAI-I was measured in carcinoma tissues from the stomach and colorectum divided macroscopically into 3 portions: the central part of the carcinoma, the marginal part of the carcinoma containing some normal mucosa, and the normal mucosa. Among these tissues, the highest levels of PAI-I antigen were found in the central part of the carcinoma. On the other hand, no PAI-I antigen or activity was observed in the normal mucosae. The PAI-I produced in the stomach and colorectal carcinoma tissues showed a non-lytic zone with a molecular weight of 54 kDa by reverse fibrin autography, and this 54-kDa band reacted with anti-PAI-I IgG on an immunoblotted nitrocellulose membrane by the avidin-biotin complex method. The contents of PAI-2 in the carcinoma tissues were not significantly different from those in the normal mucosa of the stomach and colorectum. In both the stomach and colorectal carcinomas, the highest value of u-PA/total PA (sum of u-PA and t-PA) was observed in the central part of the carcinoma, followed by the marginal part of the carcinoma, and was lowest in the normal mucosa. We conclude that increased levels of PAI-I in malignant tissue of the stomach and colorectal tract may serve to modulate extra-cellular proteolysis by u-PA.

Identity between the placental protein PP10 and the specific plasminogen activator inhibitor of placental type PAI-2.

The highly specific plasminogen activator inhibitor of placental type, PAI-2, occurs in the placenta in a low molecular mass form of 46.6 kDa, and in pregnancy plasma in a (possibly glycosylated) high molecular mass form of 60 kDa. Extensive knowledge is available about the functional properties of PAI-2 as a plasminogen activator inhibitor and about its molecular biology and regulation. Of the several placenta proteins (PP) isolated, one of them, PP10, has a molecular mass of 48 kDa and its occurrence in malignancy and in complications during pregnancy has been the topic of a number of studies, though its properties and physiological significance are unknown. The present findings constitute evidence of immunological identity between PP10 and PAI-2. The sections of the amino acid sequence of PP10 analysed here were found to have identical counterparts in the sequence of the low molecular mass form of PA1-2, but in several preparations PP10 was found to occur in an inactive two-chain form due to cleavage of an Arg-Thr bond, the two peptide chains being linked to each other by a disulphide bridge. The cleavage site is identical to that observed in the reaction between PAI-2 and urokinase. The results make it possible to coordinate and correlate the findings of many separate studies and our own observations on PP10 and PAI-2.

Rat alveolar epithelial cells concomitantly express plasminogen activator inhibitor-1 and urokinase.

There is considerable evidence to suggest that intra-alveolar plasminogen activation is instrumental in many aspects of inflammatory lung injury and subsequent tissue repair. Rat alveolar epithelial cells produce large quantities of urokinase-type plasminogen activator (uPA) in vitro, and uPA expression is modulated in association with cellular differentiation and exposure to inflammatory mediators. We now report that these cells also secrete heat-stable PA inhibitory activity having the characteristics of PA inhibitor type 1 (PAI-1). In particular, immunoreactive PAI-1 was demonstrable in conditioned media, cell lysates, and extracellular matrix from epithelial cell cultures. As alveolar epithelial cells differentiated in vitro, secreted PA inhibitor activity increased significantly from 104 +/- PAI U/ml (n = 5, mean +/- SE) on day 2 to 442 +/- 150 on day 7 in parallel with increases in secreted and matrix-associated immunoreactive PAI-1. PAI-1 mRNA expression decreased over this same period suggesting posttranscriptional regulation. The levels of both newly synthesized antigen and PAI-1 mRNA were increased by exposure to lipopolysaccharide and tumor necrosis factor-alpha. Thus, by the coexpression of uPA and PAI-1, the alveolar epithelium may actively regulate the generation of plasmin in both the normal and injured alveolus.

Purification and characterisation of plasminogen activator inhibitor 2 produced in Saccharomyces cerevisiae.

Expression of plasminogen activator inhibitor 2 (PAI-2) under the control of the protease B gene promoter in a mutant strain of Saccharomyces cerevisiae, DS569, resulted in its accumulation intracellularly at up to 20% of the soluble cell protein. Provision of an N-terminal signal sequence resulted in the secretion of a hyperglycosylated molecule. The intracellularly produced PAI-2 was purified by copper-chelate and anion-exchange chromatography to greater than 95% pure and was fully active. The recombinant PAI-2 formed SDS-stable complexes with urokinase and tissue-type plasminogen activator and inhibited the proteases with similar reaction kinetics to placental PAI-2 (second-order rate constant for uPA, 2.4 x 10(6) M-1 s-1, and for two-chain tPA, 0.7 x 10(5) M-1 s-1). As is the case for placental PAI-2, the N-terminus of the yeast-derived recombinant PAI-2 was blocked. The high productivity and consequent ease of purification mean that S. cerevisiae provides an excellent source of recombinant PAI-2 for investigation of its therapeutic potential in the treatment of neoplastic and inflammatory diseases.

Isolation of plasminogen activator inhibitor-2 (PAI-2) from human placenta. Evidence for vitronectin/PAI-2 complexes in human placenta extract.

Plasminogen activator inhibitor-2 (PAI-2), found in human placenta and pregnancy plasma, was prepared in a highly purified and functionally active form from human placenta. The purification was achieved by a combination of Rivanol and ammonium sulfate precipitation, followed by chromatography on DEAE Affigel Blue, hydroxylapatite and phenylalanine-Sepharose. PAI-2, which is precipitated by low Rivanol concentrations, can be selectively redissolved from the pellet by increasing the Rivanol concentration in the presence of a reducing agent, i.e. dithiothreitol. The purified protein shows a molecular mass of 45 kDa in SDS PAGE, cross-reacts with monoclonal antibodies against PAI-2 (Mab'PAI-2), and inhibits the amidolytic activity of urokinase-type plasminogen activator (u-PA) towards the chromogenic substrate Glu-Gly-Arg-pNA (S-2444). The specific activity of the purified inhibitor was 52,300 units/mg, attaining 71,000 units/mg in peak fractions. In the immunopurification of placental extract on anti-PAI-2 Sepharose, the eluate showed the expected reaction with Mab' PAI-2, and it also cross-reacted with anti-vitronectin serum. In order to complement these results, anti-vitronectin Sepharose was used for immunopurification of placenta extract. In Western Blot experiments the eluates of anti PAI-2 Sepharose and anti-vitronectin Sepharose both showed a heterogeneous pattern of high molecular weight bands recognized by either polyclonal antiserum against vitronectin or Mab'PAI-2. In either case, reduction of the eluates releases mainly a 45-kDa band, which is recognized by Mab'PAI-2, or 80-kDa and 76-kDa bands recognized by anti-serum against vitronectin. These data suggest that the predominant form of PAI-2 in placenta extract is heterogeneous and of high molecular mass, containing complexes in which vitronectin is covalently bound to PAI-2 by disulfide bridges.

Lysosomal degradation of receptor-bound urokinase-type plasminogen activator is enhanced by its inhibitors in human trophoblastic choriocarcinoma cells.

We have studied the effect of plasminogen activator inhibitors PAI-1 and PAI-2 on the binding of urokinase-type plasminogen activator (u-PA) to its receptor in the human choriocarcinoma cell line JAR. With 125I-labeled ligands in whole-cell binding assays, both uncomplexed u-PA and u-PA-inhibitor complexes bound to the receptor with a Kd of approximately 100 pM at 4 degrees C. Transferring the cells to 37 degrees C led to degradation to amino acids of up to 50% of the cell-bound u-PA-inhibitor complexes, whereas the degradation of uncomplexed u-PA was 15%; the remaining ligand was recovered in an apparently intact form in the medium or was still cell associated. The degradation could be inhibited by inhibitors of vesicle transport and lysosomal hydrolases. By electron microscopic autoradiography, both 125I-u-PA and 125I-u-PA-inhibitor complexes were located over the cell membrane at 4 degrees C, with the highest density of grains over the membrane at cell-cell interphases, but, after incubation at 37 degrees C, 17 and 27% of the grains for u-PA and u-PA-PAI-1 complexes, respectively, appeared over lysosomal-like bodies. These findings suggest that the u-PA receptor possesses a clearance function for the removal of u-PA after its complex formation with a specific inhibitor. The data suggest a novel mechanism by which receptor-mediated endocytosis is initiated by the binding of a secondary ligand.

Plasminogen activator inhibitor type I stabilizes vitronectin-dependent adhesions in HT-1080 cells.

Polyclonal antibodies against plasminogen activator inhibitor type-I (PAI-1) caused rapid retraction and rounding of substrate-attached HT-1080 cells. The kinetics and extent of antibody-mediated cell rounding were not dependent on either urokinase or plasmin activity. Cells adherent to vitronectin-coated substrates detached within 2 h of antibody addition. Cells adherent to fibronectin were unaffected by the antibodies. Immunoblotting of substrate-attached material indicated that HT-1080 cells deposited PAI-1 into vitronectin, but not fibronectin, dependent contacts. These data suggest that the antibody-mediated cell rounding resulted from a steric disruption of vitronectin-dependent adhesions, indicating that the binding site on vitronectin for PAI-1 is near, but does not overlap, the binding site for vitronectin receptor. The accumulation of PAI-1 into vitronectin-dependent adhesion sites correlated temporally with the preferential degradation of fibronectin from the substrate. HT-1080 cells adherent to either fibronectin or vitronectin were able to activate exogenous plasminogen to plasmin. Plasmin levels were increased 200% on cells adherent to fibronectin and 100% on cells adherent to vitronectin. In the presence of a neutralizing antibody against PAI-1, vitronectin adherent cells activated plasminogen to the same extent as fibronectin adherent cells. Plasmin levels of 200% above baseline were associated with retraction of cells from the substrate. The ability of vitronectin adherent cells to activate exogenous plasmin was completely blocked in the presence of neutralizing antibodies against urokinase. These data represent the first demonstration that vitronectin-associated PAI-1 regulates urokinase in focal contact areas.