Purified alpha 2-macroglobulin receptor/LDL receptor-related protein binds urokinase.plasminogen activator inhibitor type-1 complex. Evidence that the alpha 2-macroglobulin receptor mediates cellular degradation of urokinase receptor-bound complexes.

Complexes between 125I-labeled urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) bound to purified alpha 2-macroglobulin (alpha 2M) receptor (alpha 2MR)/low density lipoprotein receptor-related protein (LRP). No binding was observed when using uPA. The magnitude of uPA.PAI-1 binding was comparable with that of the alpha 2MR-associated protein (alpha 2MRAP). Binding of uPA.PAI-1 was blocked by natural and recombinant alpha 2MRAP, and about 80% inhibited by complexes between tissue-type plasminogen activator (tPA) and PAI-1, and by a monoclonal anti-PAI-1 antibody. In human monocytes, uPA.PAI-1, like uPA and its amino-terminal fragment, bound to the urokinase receptor (uPAR). Degradation of uPAR-bound 125I-uPA.PAI-1 was 3-4-fold enhanced as compared with uncomplexed uPAR-bound uPA. The inhibitor-enhanced uPA degradation was blocked by r alpha 2MRAP and inhibited by polyclonal anti-alpha 2MR/LRP antibodies. This is taken as evidence for mediation of internalization and degradation of uPAR-bound uPA.PAI-1 by alpha 2MR/LRP.     

Biological control of tissue plasminogen activator-mediated fibrinolysis

Fibrinolysis, the body's ability to degrade fibrin, is an integrated part of hemostasis. Overactivity in the fibrinolytic system causes bleeding and underactivity causes thrombosis. Tissue plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), alpha 2-antiplasmin (alpha 2-AP) and plasminogen are definitely involved in fibrinolysis because: (1) these components can be assigned a fibrinolytic role in purified systems, i.e. in vitro, and (2) abnormal structural variants and abnormal levels of these components give rise to bleeding or to thrombosis. The biological control of tPA-mediated fibrinolysis is both cellular and humoral. The cellular regulation compasses synthesis of tPA and PAI-1 and release/uptake of these components. The humoral regulation involves: (1) the reaction between tPA and PAI-1; (2) the fibrin-stimulated plasminogen activation; (3) the reaction between plasmin and alpha 2-AP and (4) plasmin degradation of fibrin. The highly developed biological control of tPA-mediated fibrinolysis is indicative of its physiological importance.     

Acute phase protein alpha 1-acid glycoprotein interacts with plasminogen activator inhibitor type 1 and stabilizes its inhibitory activity.

alpha(1)-Acid glycoprotein, one of the major acute phase proteins, was found to interact with plasminogen activator inhibitor type 1 (PAI-1) and to stabilize its inhibitory activity toward plasminogen activators. This conclusion is based on the following observations: (a) alpha(1)-acid glycoprotein was identified to bind PAI-1 by a yeast two-hybrid system. Three of 10 positive clones identified by this method to interact with PAI-1 contained almost the entire sequence of alpha(1)-acid glycoprotein; (b) this protein formed complexes with PAI-1 that could be immunoprecipitated from both the incubation mixtures and blood plasma by specific antibodies to either PAI-1 or alpha(1)-acid glycoprotein. Such complexes could be also detected by a solid phase binding assay; and (c) the real-time bimolecular interactions monitored by surface plasmon resonance indicated that the complex of alpha(1)-acid glycoprotein with PAI-1 is less stable than that formed by vitronectin with PAI-1, but in both cases, the apparent K(D) values were in the range of strong interactions (4.51 + 1.33 and 0.58 + 0.07 nm, respectively). The on rate for binding of PAI-1 to alpha(1)-glycoprotein or vitronectin differed by 2-fold, indicating much faster complex formation by vitronectin than by alpha(1)-acid glycoprotein. On the other hand, dissociation of PAI-1 bound to vitronectin was much slower than that from the alpha(1)-acid glycoprotein, as indicated by 4-fold lower k(off) values. Furthermore, the PAI-1 activity toward urokinase-type plasminogen activator and tissue-type plasminogen activator was significantly prolonged in the presence of alpha(1)-acid glycoprotein. These observations suggest that the complex of PAI-1 with alpha(1)-acid glycoprotein can play a role as an alternative reservoir of the physiologically active form of the inhibitor, particularly during inflammation or other acute phase reactions.     

Plasminogen activator inhibitor-1 and -3 increase cell adhesion and motility of MDA-MB-435 breast cancer cells

Plasminogen activator inhibitor-1 (PAI-1), an inhibitor of urokinase plasminogen activator, is paradoxically associated with a poor prognosis in breast cancer. PAI-1 is linked to several processes in the metastatic cascade. However, the role of PAI-1 in metastatic processes, which may be independent of protease inhibitory activity, is not fully understood. We report herein that PAI-1, when added exogenously to or stably transfected in human MDA-MB-435 breast carcinoma cells, had disparate effects on adhesion to extracellular matrix proteins and motility in vitro. Specifically, exogenously added PAI-1 inhibited cell adhesion to vitronectin but not fibronectin, in agreement with the literature. By contrast, stably transfected PAI-1 stimulated adhesion to both proteins. Wild-type PAI-1 was required for this stimulation, because expression of a non-protease inhibitory P14 (T333R) PAI-1 mutant failed to enhance adhesion. Compared with non-inhibitory PAI-1, wild-type PAI-1 also increased cell motility in chemotaxic assays. Furthermore, stable transfection of a related serine protease inhibitor, plasminogen activator inhibitor-3 (PAI-3, or protein C inhibitor) gave results similar to wild-type PAI-1. The stimulatory activity of PAI-3 was not seen with a non-protease inhibitory P14 PAI-3 mutant (T341R). We show that a downstream effect of endogenous wild-type PAI-1 and PAI-3 overexpression, but not their non-inhibitory counterparts, was the altered expression of alpha(2), alpha(3), alpha(4), alpha(5), and beta(1) integrin subunits. Additionally, blocking antibodies to beta(1) integrin inhibited PAI-1-induced adhesion. Our data provide experimental support for the stimulatory and inhibitory effects of PAI-1 in metastasis and introduce PAI-3 as another serpin potentially important in malignant disease.     

The effect of IL-1alpha on the expression of matrix metalloproteinases, plasminogen activators, and their inhibitors in osteoblastic ROS 17/2.8 cells

Interleukin-1 (IL-1) plays key roles in altering bone matrix turnover. This turnover is regulated by matrix metalloproteinases (MMPs), tissue inhibitor of matrix metalloproteinases (TIMPs), and the plasminogen activation system, including tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA) , and plasminogen activator inhibitor type-1 (PAI-1). In this study, we examined the effect of IL-1alpha on the expression of the MMPs, TIMPs, tPA, uPA, and PAI-1 genes in osteoblasts derived from the rat osteosarcoma cell line ROS 17/2.8. The cells were cultured in alpha-minimum essential medium containing 10% fetal bovine serum with 0 or 100 U/ml of IL-1alpha for up to 14 days. The levels of MMPs, TIMPs, uPA, tPA, and PAI-1 expression were estimated by determining the mRNA levels using real-time RT-PCR and by determining protein levels using ELISA. In IL-1alpha cultures, the expression levels of MMP-1, -2, -3, -13, and -14 exceeded that of the control through day 14 of culture, and the expression of MMPs increased markedly from the proliferative to the later stages of culture. The TIMP-1, -2, and -3 expression levels increased from the initial to the proliferative stages of culture. The expression of tPA increased greatly during the proliferative stage of culture, and uPA expression increased throughout the culture period, increasing markedly from the proliferative to the later stages of culture. In contrast, PAI-1 expression decreased in the presence of IL-1alpha through day 14. These results suggest that IL-1alpha stimulate bone matrix turnover by increasing MMPs, tPA, and uPA production and decreasing PAI-1 production by osteoblasts, and incline the turnover to the resolution.     

Detection of both type 1 and type 2 plasminogen activator inhibitors in human cells

This report describes the development and use of functional immunoradiometric assays that distinguish the activity of beta-migrating endothelial-type plasminogen activator inhibitor (PAI-1) from that of placental-type plasminogen activator inhibitor (PAI-2). These assays are based upon the binding of PAI-1 and PAI-2 to immobilized single-chain tissue-type plasminogen activator (tPA) and to immobilized urokinase (UK), respectively. The extent of binding of each PAI is quantified by incubating the PAI-PA complex first with rabbit antiserum specific for the individual PAI and then with 125I-labeled goat antirabbit IgG. In control experiments, the assays were shown to be sensitive, dose-dependent over a wide range, and specific for each PAI. These assays were employed to establish the PAI profile of a variety of human cells. Neither PAI-1 nor PAI-2 could be detected in Bowes melanoma cells or in a renal adenocarcinoma cell line (ACHN), while the histiocytic lymphoma cell (U-937) produced only PAI-2. Five cell lines, including two that were previously shown to contain one or the other PAI (e.g., umbilical vein endothelial cells and a fibrosarcoma cell line, HT-1080) in fact contained both PAIs. The cells containing both PAIs were studied in more detail. In each case, SDS treatment of CM was shown to enhance PAI-1 activity (by converting the latent form of this inhibitor into its active form) and to destroy PAI-2 activity. Various compounds including interleukin 1, dexamethasone, and phorbol myristate acetate were found to selectively influence the cellular production of one PAI without concomitantly affecting the production of the other, suggesting that the synthesis of these inhibitors is not coordinately regulated.     

Modulation of plasminogen activator and plasminogen activator inhibitor expression in the human U373 glioblastoma/astrocytoma cell line by inflammatory mediators.

The human U373 glioblastoma/astrocytoma cell line was found to constitutively produce and secrete a plasminogen activator and a plasminogen activator inhibitor. The plasminogen activator was identified as urokinase based on apparent molecular weight, immunoblotting with anti-urokinase antibodies, and Northern blotting with a human urokinase cDNA probe. The inhibitor secreted by U373 cells was found to be related to the PAI-1 molecule based on reactivity with anti-human PAI-1 antibodies, apparent molecular weight, and Northern blot analysis with a human PAI-1 cDNA probe. The expression of both urokinase and the PAI-1-like molecule by U373 cells could be modulated by phorbol myristate acetate or by inflammatory mediators such as interferon-gamma and interleukin-1. In the case of interleukin-1, the alpha form exhibited no detectable effect while the beta form not only elevated inhibitor levels, it also appeared to induce the production of tissue plasminogen activator. Thus, in these cells interleukin-1 beta induces alterations in PA and PAI expression and interleukin-1 alpha does not, even though the two forms are reported to utilize the same cellular receptor.     

Structure-function studies of the SERPIN plasminogen activator inhibitor type 1. Analysis of chimeric strained loop mutants.

Three chimeric mutants of plasminogen activator inhibitor 1 (PAI-1) have been constructed where the strained loop of wild type PAI-1 (wtPAI-1) has been replaced with a 19-amino acid region from either plasminogen activator inhibitor 2 (PAI-2), antithrombin III, or with an artificial serine protease inhibitor superfamily consensus strained loop. The inhibitors were expressed in Escherichia coli, and the purified proteins had specific activities toward urokinase-type plasminogen activator (uPA) or the single- and two-chain forms of tissue type plasminogen activator (tPA) that were similar to wtPAI-1. Experiments suggest that the strained loop of PAI-1 is not responsible for the transition between the latent and the active conformations or for binding to vitronectin. Second-order rate constants for the interactions with uPA and single- or two-chain tPA were similar to those of wtPAI-1. Values range from a low of 1.8 x 10(5) M-1 s-1 for the interaction of the PAI-2 chimera with single-chain tPA to a high value of 1.6 x 10(7) M-1 s-1 for the consensus mutant with two-chain tPA. This former value is 200 times higher than the reported rate constant for the interaction between PAI-2 and single-chain tPA, suggesting that structures outside of the strained loop are responsible for the major differences in specificity between PAI-1 and PAI-2.     

Transforming growth factor-beta1 produced by ovarian cancer cell line HRA stimulates attachment and invasion through an up-regulation of plasminogen activator inhibitor type-1 in human peritoneal mesothelial cells

The processes of ovarian cancer dissemination are characterized by altered local proteolysis, cellular proliferation, cell attachment, and invasion, suggesting that the urokinase-type plasminogen activator (uPA) and its specific inhibitor (plasminogen activator inhibitor type-1 (PAI-1)) could be involved in the pathogenesis of peritoneal dissemination. We showed previously that expression of uPA and PAI-1 in the human ovarian cancer cell line HRA can be down-regulated by exogenous bikunin (bik), a Kunitz-type protease inhibitor, via suppression of transforming growth factor-beta1 (TGF-beta1) up-regulation and that overexpression of the bik gene can specifically suppress the in vivo growth and peritoneal dissemination of HRA cells in an animal model. We hypothesize that the plasminogen activator system in mesothelial cells can be modulated by HRA cells. To test this hypothesis, we used complementary techniques in mesothelial cells to determine whether uPA and PAI-1 expression are altered by exposure to culture media conditioned by HRA cells. Here we show the following: 1) that expression of PAI-1, but not uPA, was markedly induced by culture media conditioned by wild-type HRA cells but not by bik transfected clones; 2) that by antibody neutralization the effect appeared to be mediated by HRA cell-derived TGF-beta1; 3) that exogenous TGF-beta1 specifically enhanced PAI-1 up-regulation at the mRNA and protein level in mesothelial cells in a time- and concentration-dependent manner, mainly through MAPK-dependent activation mechanism; and 4) that mesothelial cell-derived PAI-1 may promote tumor invasion possibly by enhancing cell-cell interaction. This represents a novel pathway by which tumor cells can regulate the plasminogen activator system-dependent cellular responses in mesothelial cells that may contribute to formation of peritoneal dissemination of ovarian cancer.     

大鼠卵巢细胞分泌纤溶酶原激活因子抑制因子（PAI—1）的...

Rat ovarian cells produce not only plasminogen activator (tPA) but also plasminogen activator inhibitor type 1 (PAI-1), and their coordinated geneexpression induced by gonadotropins are thought to be responsible for follicular rupture. In this study, it was demonstrated that (1) theca-interstitial compartment synthesizes the majority of PAI-1 activity in the ovary before ovulation, the follicular wall may therefore serve as a specific barrier to prevent the secretion of PA into the extrafollicular compartment; (2) Granulosa cells contribute only small amount of ovarian PAI-1 activity, but synthesize most of tissue-type plasminogen activator activity involved in the process leading to ovulation: (3) Since only matured cumulus-oocyte complexes secrete high level of tPA and PAI-1, both tPA and PAI-1 activity in the conditioned medium may be used as reliable markers for evaluating oocyte quality for in vitro fertilization.     

The THP-1 cell line is a urokinase-secreting mononuclear phagocyte with a novel defect in the production of plasminogen activator inhibitor-2

Mononuclear phagocytes regulate the generation of plasmin by secreting urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-2 (PAI-2). We investigated the production of plasminogen activator (PA) and PA inhibitor by the human monocytic leukemia cell line, THP-1. Similar to U937 monoblast-like cells and peripheral blood monocytes (PBM), THP-1 cells produce a PA that is specifically neutralized by anti-uPA antibody and comigrates with human high molecular mass uPA (54 kDa) on casein-plasminogen zymogaphy. PA activity could be dissociated from intact THP-1 cells by brief treatment with a weak acid-glycine buffer, indicating that the uPA is secreted and bound to receptors on the plasma membrane. Regulation of uPA proceeds normally in THP-1 cells, with cell-associated PA activity increasing from 77 +/- 20 to 163 +/- 26 and 325 +/- 30 mPU/10(6) cells in response to PMA and LPS, respectively; parallel increases in steady state levels of uPA mRNA were observed. In contrast to normal expression of uPA activity, functional PAI-2 could not be demonstrated in either the conditioned media or cell lysates of THP-1 under basal or stimulated conditions. Both U937 and PBM secrete low levels of PA inhibitor activity that increase substantially in response to stimulation with PMA and LPS. Immunoreactive PAI-2, measured by ELISA, was undetectable in THP-1 lysates or conditioned medium, but was consistently present in U937 and PBM, paralleling the presence of PA inhibitor activity. THP-1 cells express low levels of an abnormally sized mRNA for PAI-2 and demonstrate a regulatory defect whereby steady state levels of PAI-2 mRNA are markedly reduced upon stimulation with PMA or LPS. By contrast, U937 and PBM respond to identical stimulation with increases in PAI-2 mRNA. We conclude that THP-1 cells express a structurally abnormal species of PAI-2 mRNA, with complete loss of inhibitory activity as well as altered function of PMA- and LPS-responsive regulatory elements.     

Cytoplasmic and nuclear interaction between Rb family proteins and PAI-2: a physiological crosstalk in human corneal and conjunctival epithelial cells

Extracellular plasminogen activator inhibitor type-2 (PAI-2) is a potent inhibitor of urokinase-type plasminogen activator (u-PA) and also acts as a multifunctional protein. However, the biological activity of intracellular PAI-2, as well as its intracellular targets, until now remain an enigma. Here, we show that pRb2/p130 and Rb1/p105, but not p107, interact with PAI-2 in both the cytoplasm and nucleus of normal primary human corneal and conjunctival epithelial cells. We provided the first in vivo evidence that a specific fragment of the PAI-2 promoter is bound simultaneously by pRb2/ p130, PAI-2, E2F5, histone deacetylase 1 (HDAC1), DNA methyltransferase 1 (DNMT1), and histone methyltransferase (SUV39H1), in normal primary human corneal epithelial cells, and by pRb2/p130, PAI-2, E2F5, HDAC1, and DNMT1, in normal primary human conjunctiva epithelial cells. Our results strongly indicate a physiological interaction between pRb family members and PAI-2, suggesting the hypothesis that pRb2/p130 and PAI-2 may cooperate in modulating PAI-2 gene expression by chromatin remodeling, in normal corneal and conjunctival cells.     

Bezafibrate attenuates the overexpression of plasminogen activator inhibitor-1 messenger RNA by a combination of mono-unsaturated fatty acid and insulin in hepG2 cells

The effects of bezafibrate (PPAR alpha activator) and troglitazone (PPAR gamma activator) on the expression of plasminogen activator inhibitor type-1 (PAI-1) in HepG2 cells were investigated. Exposure of the cells for 24 hours to either oleic acid or insulin showed no obvious effects on PAI-1 synthesis, whereas the combination of the two agents induced a 2.3-fold increase in PAI-1 synthesis, which was accompanied by a 3-fold increase in both the 2.2 kb and 3.2 kb forms of PAI-1 mRNA. This up-regulation of PAI-1 synthesis was attenuated by bezafibrate in a dose-dependent manner (1-100 microM) with 30% reversal at 100 microM. In contrast, troglitazone further stimulated PAI-1 synthesis to 140% of the level obtained in the presence of both oleic acid and insulin. This attenuation by bezafibrate and enhancement by troglitazone required the presence of both oleic acid and insulin. It is interesting that PAI-1 expression was affected so differently by these two PPAR activators.     

Thrombin neutralizes plasminogen activator inhibitor 1 (PAI-1) that is complexed with vitronectin in the endothelial cell matrix

Vitronectin endows plasminogen activator inhibitor 1 (PAI-1), the fast-acting inhibitor of both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), with additional thrombin inhibitory properties. In view of the apparent association between PAI-1 and vitronectin in the endothelial cell matrix (ECM), we analyzed the interaction between PAI-1 and thrombin in this environment. Upon incubating 125I-labeled alpha-thrombin with endothelial cell matrix (ECM), the protease formed SDS-stable complexes exclusively with PAI-1, with subsequent release of these complexes into the supernatant. Vitronectin was required as a cofactor for the association between PAI-1 and thrombin in ECM. Metabolic labeling of endothelial cell proteins, followed by incubation of ECM with t-PA, u-PA, or thrombin, indicated that all three proteases depleted PAI-1 from ECM by complex formation and proteolytic cleavage. Proteolytically inactive thrombin as well as anticoagulant thrombin, i.e., thrombin in complex with its endothelial cell surface receptor thrombomodulin, did not neutralize PAI-1, emphasizing that the procoagulant moiety of thrombin is required for a functional interaction with PAI-1. A physiological implication of our findings may be related to the mutual neutralization of both PAI-1 and thrombin, providing a new link between plasminogen activation and the coagulation system. Evidence is provided that in ECM, procoagulant thrombin may promote plasminogen activator activity by inactivating PAI-1.     

Binding of type 1 plasminogen activator inhibitor to the extracellular matrix of cultured bovine endothelial cells

The binding of type 1 plasminogen activator inhibitor (PAI-1) to the extracellular matrix (ECM) of cultured bovine aortic endothelial cells was investigated using purified 125I-labeled or L-[35S]methionine-labeled PAI-1 as probes. Little specific binding of latent PAI-1 to ECM previously depleted of endogenous PAI-1 could be demonstrated. In contrast, the guanidine-activated form of PAI-1 bound to ECM in a dose- and time-dependent manner, and binding was saturable. The dissociation constant (Kd) for this interaction was estimated to be 60 nM by Scatchard analysis, and approximately 6 pmol of activated PAI-1 was bound per cm2 of ECM. Binding was relatively specific since unlabeled, activated PAI-1 competed with 35S-labeled PAI-1 for binding to ECM, but latent PAI-1 did not. Moreover, PAI-2, protein C inhibitor (i.e. PAI-3), protease nexin-1, and alpha 2-antiplasmin were not able to compete. Tissue-type plasminogen activator (tPA) also inhibited binding, but diisopropyl fluorophosphate-inactivated tPA did not. Pretreatment of ECM with tPA, urokinase-type PA, or thrombin had no effect on its ability to subsequently bind PAI-1, whereas trypsin, plasmin, and elastase pretreatment greatly reduced its ability to bind PAI-1. Guanidine-activated, radiolabeled PAI-1 resembled active endogenous PAI-1 since it was unstable in solution but stable when bound to ECM. In addition, it formed complexes with tPA that had a relatively low affinity for ECM. These data suggest that ECM of bovine aortic endothelial cells contains a protease-sensitive structure that binds active PAI-1 tightly and relatively selectively and that this association stabilizes PAI-1 against the spontaneous loss of activity that occurs in solution.     

Cross-linking of plasminogen activator inhibitor 2 and alpha 2-antiplasmin to fibrin(ogen)

In this study, we identified lysine residues in the fibrinogen Aalpha chain that serve as substrates during transglutaminase (TG)-mediated cross-linking of plasminogen activator inhibitor 2 (PAI-2). Comparisons were made with alpha(2)-antiplasmin (alpha(2)-AP), which is known to cross-link to lysine 303 of the Aalpha chain. A 30-residue peptide containing Lys-303 specifically competed with fibrinogen for cross-linking to alpha(2)-AP but not for cross-linking to PAI-2. Further evidence that PAI-2 did not cross-link via Lys-303 was the cross-linking of PAI-2 to I-9 and des-alphaC fibrinogens, which lack 100 and 390 amino acids from the C terminus of the Aalpha chain, respectively. PAI-2 or alpha(2)-AP was cross-linked to fibrinogen and digested with trypsin or endopeptidase Glu-C, and the resulting peptides analyzed by mass spectrometry. Peptides detected were consistent with tissue TG (tTG)-mediated cross-linking of PAI-2 to lysines 148, 176, 183, 457 and factor XIIIa-mediated cross-linking of PAI-2 to lysines 148, 230, and 413 in the Aalpha chain. alpha(2)-AP was cross-linked only to lysine 303. Cross-linking of PAI-2 to fibrinogen did not compete with alpha(2)-AP, and the two proteins utilized different lysines in the Aalpha chain. Therefore, PAI-2 and alpha(2)-AP can cross-link simultaneously to the alpha polymers of a fibrin clot and promote resistance to lysis.     

The stratagem utilized by the plasminogen activator from the snake Trimeresurus stejnegeri to escape serpins

The plasminogen activator isolated from the venom of the snake Trimeresurus stejnegeri (TSV-PA) triggers plasmin production, along with tissue-type plasminogen activators (t-PA) and urokinase (u-PA). The half-life of TSV-PA in plasma is remarkable. We unveil in this paper two of the molecular mechanisms allowing TSV-PA to escape inhibition by plasma serpins. The first involves a phenylalanine at position 193 (chymotrypsinogen numbering system). Phe(193) distinguishes TSV-PA from nearly all trypsin-like proteinases, having glycine at this position. A mutant of TSV-PA (F193G), in which Phe(193) had been replaced by a glycine, was inactivated by plasminogen activator inhibitor 1 (PAI-1) and alpha(2)-antiplasmin 100-fold more rapidly than the wild-type enzyme. The second mechanism originates from the 37-loop of TSV-PA. Swapping the 37-loop of TSV-PA for either that of t-PA or that of u-PA also increased dramatically the rate of inactivation by PAI-1. Loop swapping and F193G mutations were additive, resulting in a rate of inactivation by PAI-1 that was 4 orders of magnitude higher than for the wild-type enzyme. The potential role of Phe(193) and of the 37-loop in the immunity of TSV-PA toward alpha(1)-antitrypsin and antithrombin is also discussed.     

Topological localization of plasminogen activator inhibitor type 2

BACKGROUND: Plasminogen activator inhibitor type 2 (PAI-2) is a member of the serine protease inhibitor (SERPIN) superfamily and forms stable complexes with urokinase type plasminogen activator (uPA). uPA can be found on the cell surface attached to its specific receptor (uPAR), allowing for controlled degradation of the extracellular matrix by the activation of plasminogen into plasmin. The aim of this study was to evaluate if PAI-2 could also be detected on the cell surface, providing a means of regulating the activity of cell surface uPA. METHODS: Intact or permeabilized cell lines or human peripheral blood leukocytes were assayed by flow cytometry for cell surface uPA or PAI-2. Plasma membrane-enriched preparations prepared from Jurkat, HaCaT, THP-1, U937, or MM6 cells were assayed by enzyme-linked immunosorbent assay (ELISA) or Western blotting for PAI-2 antigen. RESULTS: By flow cytometry, cell surface PAI-2 was not detected on monocytes from human peripheral blood, MM6, or HaCaT cells. Cell surface PAI-2 was only detected very weakly on the surface of U937 cells. In contrast, PAI-2 could be detected in all of these cells when fixed and permeabilized. By ELISA, PAI-2 was very abundant in the cytosol-enriched preparations of U937, MM6, and HaCaT cells, but was present in lower amounts in the plasma membrane-enriched preparations. By Western blotting, monomeric nonglycosylated PAI-2, but not uPA/PAI-2 complexes, could be detected in the cytosol and plasma membrane-enriched preparations. CONCLUSIONS: These results indicate that PAI-2 cannot be detected on the surface of PAI-2-expressing cells, and confirm that PAI-2 is predominantly a cytosolic protein.