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PubMed1998

Expression of sialylated Lewisx gangliosides in cultured lens epithelial cells from rhesus monkey.

Monkey and human lenses contain essentially the same glycosphingolipids, and Lewisx and sialylated Lewisx epitopes are expressed on the terminal structure of neolactotetraosylceramide. However, monolayer cultures of lens epithelial cells from rhesus monkey expressed gangliosides GM3, GD3 and a small amount of GM1, but not sialylated Lewisx epitopes. Eight-week-old cultures on various extracellular matrices resulted in morphological changes in lens epithelial cells. Monolayer of cells cultured on vitronectin or polylysine assembled into aggregates after 4 weeks of culture. Cells cultured on vitronectin expressed sialyl-Lewisx gangliosides and did not exhibit GD3. On collagens, fibronectin and laminin elongated cells were observed in cells cultured for 8 weeks. Thus, the interaction between cells and extracellular matrices influenced morphology and glycosphingolipid composition in lens epithelial cells.

Animals↗
PubMed2001

Different structural requirements for plasminogen activator inhibitor 1 (PAI-1) during latency transition and proteinase inhibition as evidenced by phage-displayed hypermutated PAI-1 libraries.

Plasminogen activator inhibitor type 1 (PAI-1) is a member of the serine protease inhibitor (serpin) superfamily. Its highly mobile reactive-center loop (RCL) is thought to account for both the rapid inhibition of tissue-type plasminogen activator (t-PA), and the rapid and spontaneous transition of the unstable, active form of PAI-1 into a stable, inactive (latent) conformation (t(1/2) at 37 degrees C, 2.2 hours). We determined the amino acid residues responsible for the inherent instability of PAI-1, to assess whether these properties are independent and, consequently, whether the structural basis for inhibition and latency transition is different. For that purpose, a hypermutated PAI-1 library that is displayed on phage was pre-incubated for increasing periods (20 to 72 hours) at 37 degrees C, prior to a stringent selection for rapid t-PA binding. Accordingly, four rounds of phage-display selection resulted in the isolation of a stable PAI-1 variant (st-44: t(1/2) 450 hours) with 11 amino acid mutations. Backcrossing by DNA shuffling of this stable mutant with wt PAI-1 was performed to eliminate non-contributing mutations. It was shown that the combination of mutations at positions 50, 56, 61, 70, 94, 150, 222, 223, 264 and 331 increases the half-life of PAI-1 245-fold. Furthermore, within the limits of detection the stable mutants isolated are functionally indistinguishable from wild-type PAI-1 with respect to the rate of inhibition of t-PA, cleavage by t-PA, and binding to vitronectin. These stabilizing mutations constitute largely reversions to the stable "serpin consensus sequence" and are located in areas implicated in PAI-1 stability (e.g. the vitronectin-binding domain and the proximal hinge). Collectively, our data provide evidence that the structural requirements for PAI-1 loop insertion during latency transition and target proteinase inhibition can be separated.

Animals↗
PubMed2001

Developmental regulation of alphav integrins produces functional changes in astrocyte behavior.

To examine the role of the extracellular matrix in regulating astrocyte behavior we previously characterized alphav integrin expression on postnatal astrocytes in vitro and found that they express alphavbeta5 and alphavbeta8. Here we show that differentiation of immature cells into astrocytes is accompanied by developmental regulation of alphav integrins, downregulation of alphavbeta1 and alphavbeta8, and upregulation of alphavbeta5. In addition, using two previously described astrocyte cell lines, we found that the neurite-permissive A7 cell line expressed high levels of alphavbeta1 in addition to alphavbeta5 and alphavbeta8, while the neurite-inhibitory Neu7 cell line expressed only alphavbeta5. To examine integrin function we generated clones of the Neu7 cell line expressing alphavbeta1 or alphavbeta3 in addition to alphavbeta5. This showed that the parent Neu7 cells migrated more slowly than the A7 cells on fibronectin and vitronectin, but that Neu7 cells expressing alphavbeta1 or alphavbeta3 integrins showed enhanced migration on fibronectin and vitronectin, respectively. These results show that alphav integrin expression is regulated during astrocyte development and confirm an instructive role in cell migration for alphavbeta1 in embryonic cells and alphavbeta3 in astroglial tumors.

Animals↗
PubMed1994

Cellular and substrate adhesion molecules (integrins) and their ligands in cerebral amyloid plaques in Alzheimer's disease.

Integrins belonging to different subfamilies can be identified immunohistochemically in cerebral amyloid plaques. Monoclonal antibodies against the VLA family beta 1-integrins show staining of the corona of classical amyloid plaques for beta 1, alpha 3 and alpha 6. Immunostaining reveal also the presence of collagen and laminin in the corona. Activated microglial cells in classical plaques strongly express receptors belonging to the LeuCAM family (beta 2 integrins). The ligands ICAM and activated complement C3 are found in both amorphous and classical plaques. Vitronectin receptor (alpha v) is found in glial cells in classical plaques but its ligand vitronectin is seen in both amorphous and classical plaques. The data presented here demonstrate the presence of different cellular and substrate adhesive molecules (integrins) and their ligands in classical plaques. The findings suggest that amyloid plaques show signs of regeneration and tissue remodelling.

Aged↗
PubMed1991

Extracellular matrix receptor and platelet antigens on osteoclasts and foreign body giant cells.

Osteoclasts (OCs) and other cells of the mononuclear phagocyte system possess receptors for adhesive proteins present in the extracellular matrix. The antigenic phenotype of OCs and foreign body giant cells (FBGCs) was investigated for the presence of several integrin molecules and other largely platelet-associated antigens involved in cell adhesion reactions. Both OCs and FBGCs expressed the alpha-chains of the vitronectin receptor (CD51) and of the VLA-2 (CDw49b) and VLA-4 (CDw49d) molecules as well as their respective beta-chains, gpIIIa (CD61) and CD29. OCs and FBGCs also expressed CD9 and CD55 (DAF-Decay Accelerating Factor) and strongly reacted with antibodies directed against fibrinogen, fibronectin and vitronectin; the latter are ligands for several of the above matrix protein receptors. The data suggest that cell-cell and cell-matrix interactions involving adhesive proteins may be important in OC and FBGC function.

Antigens, Human Platelet↗
PubMed1996

Extracellular matrix components of the placental extravillous trophoblast: immunocytochemistry and ultrastructural distribution.

Invasive extravillous trophoblast cells of the human placenta are embedded in a self-secreted extracellular matrix, the matrix-type fibrinoid. The ultrastructure and molecular composition of the matrix-type fibrinoid of the term human placenta were studied by transmission electron microscopy and immunogold labelling. We used antibodies directed against different matrix proteins such as collagen type IV, laminin, vitronectin, heparan sulfate, various fibronectin isoforms, and against the oncofetal blood group antigen, "i". Immunogold labelling patterns of matrix proteins are the basis for the subdivision of the trophoblast-derived matrix-type fibrinoid into mosaic-like patches of structurally and immunocytochemically different compartments. Firstly, fine granular patches with structural similarities to basal lamina material are composed solely of collagen type IV and laminin. Secondly, an ultrastructurally amorphous glossy substance shows reactivity with antibodies against heparan sulfate and vitronectin. A third type of patches, fine fibrillar networks embedded in the above-mentioned glossy matrix, are reactive with antibodies against normal fibronectin isoforms (IST-4, IST-6, IST-9) and oncofetal isoforms (BC-1, FDC-6). The blood group precursor antigen "i" was not only expressed on the surfaces of the extravillous trophoblast cells but was associated with the fibronectin-positive fibrils. In conclusion, within this extracellular matrix, clear compartments of different composition can be distinguished from each other. Glycosylation with "i" in this matrix may be involved in immunological masking, thus preventing rejection of placenta and fetus.

Antibodies↗
PubMed1999

Extracellular matrix-induced cell migration from glioblastoma biopsy specimens in vitro.

The present knowledge about the interaction between the extracellular matrix (ECM) and gliomas is mostly based on studies of permanent cell lines. Since such cultures have undergone an extensive clonal selection in vitro, the experimental results obtained may be quite different from those obtained from studies on true biopsy specimens. The present work demonstrates how different ECM components affect tumor cell migration from human glioblastoma specimens grown as biopsy sample spheroids. Biopsy specimens from 12 glioblastomas and 1 gemistocytic astrocytoma were included in this study. Spheroids were directly initiated from the biopsy specimens, and after 3-4 weeks in culture, they were used in a migration assay. A custom-made filtered medium, where the high molecular weight (>100 kDa) proteins were removed, was supplemented with the following ECM components: laminin, fibronectin, collagen type IV and vitronectin. The cell migration was negligible when spheroids were propagated in the filtered medium. The ECM components as well as complete DMEM evoked strong stimulatory effects on different biopsy specimens. Opposed to that observed earlier for permanent glioma cell lines, highly variable responses were observed between the different biopsy samples on the various ECM components. In general, correlation analyses revealed that specimens that were strongly stimulated by laminin were also stimulated strongly by fibronectin, collagen type IV and vitronectin. This suggests that the capacity to migrate as a response to ECM was confined more to each biopsy specimen than to any specific ECM component. Since biopsy sample spheroids, as original tumors, consist of different cell types, an immunohistochemical characterization of the migrating cells was also performed. Anti-glial fibrillary acidic protein (GFAP) staining revealed both GFAP-positive and -negative migrating cells. Immunostaining for von Willebrand factor and CD11b indicated that the migrating cells were neither endothelial nor microglial cells. This study, therefore, indicates that migratory responses of glioma biopsy specimens to different ECM components is much more heterogeneous than that observed earlier for cell lines. Furthermore, the presented findings support the notion that gliomas may utilize different cell surface receptors for their migration, depending on the cell substrates available.

Adult↗
PubMed1995

Disparity in blood activation by two different heparin-coated cardiopulmonary bypass systems.

BACKGROUND: Several studies have indicated reduced "blood activation" in heparin-coated cardiopulmonary bypass systems. The present study compares the effect of two different heparin coatings on different blood activation indices. METHODS: Low-risk patients (n = 40) were randomized to coronary artery bypass grafting using cardiopulmonary bypass with surfaces coated entirely by either the Duraflo II heparin coat or the Carmeda Biological Active Surface, or with identical uncoated equipment. In all cases, a standard systemic heparin dosage was used. Complement activation (C3 activation products C3bc and C3a and formation of fluid phase terminal SC5b-9 complement complex), neutrophil activation (lactoferrin and myeloperoxidase), and lytic inhibitors (vitronectin and clusterin) were quantified during cardiopulmonary bypass and 6 hours postoperatively. RESULTS: Heparin coating by either method reduced the formation of terminal SC5b-9 complement complex and the release of lactoferrin and myeloperoxidase compared with uncoated systems. Lactoferrin and myeloperoxidase levels increased significantly during cardiopulmonary bypass in the Duraflo II group, whereas no significant increase was observed in the Carmeda Biological Active Surface group. The least formation of terminal SC5b-9 complement complex and neutrophil activation was observed with the Maxima Carmeda Biological Active Surface-coated equipment. The vitronectin and clusterin concentrations were significantly less reduced in the Duraflo II compared with the control group. This study underlines the importance of terminal SC5b-9 complement complex as a suitable marker in the evaluation of complement activation during cardiopulmonary bypass. CONCLUSIONS: Both heparin coatings reduce blood activation, probably more so with Carmeda Biological Active Surface than with Duraflo II.

Anticoagulants↗
PubMed1994

Expression of the complement membrane attack complex and its inhibitors in Pick disease brain.

The immunohistochemical localization of the complement membrane attack complex (MAC) was examined in Pick disease brain and compared with the distribution of three of its inhibitors, vitronectin, protectin and clusterin. Pick bodies were stained intensely for both the MAC and protectin, weakly for vitronectin, but negatively for clusterin. However, the clusterin antibody intensely stained some pyramidal neurons in affected cortical areas, including ballooned neurons. The present study indicates that a complement-mediated attack is associated with the formation of Pick bodies, and provides further suggestive evidence that clusterin may be a marker for active neuronal degeneration.

Aged↗
PubMed1990

Inhibition of neural crest cell migration by aggregating chondroitin sulfate proteoglycans is mediated by their hyaluronan-binding region.

We have recently shown that the large hyaluronan-aggregating chondroitin sulfate proteoglycan from cartilage (PG-LA) is unfavorable as a substrate for neural crest cell migration in vitro and that this macromolecule inhibits cell dispersion on fibronectin substrates when included in the medium (R. Perris and S. Johansson, 1987, J. Cell Biol. 105, 2511-2521). In this study we present data on the specificity of the migration-repressing activity of PG-LA and data on the molecular mechanisms by which the proteoglycan might impair neural crest cell motility. Soluble PG-LA potently impaired cell migration on substrates of laminin/laminin-nidogen, vitronectin, and collagen types I, III, IV, and VI. When tested in solid-phase binding assays, PG-LA bound avidly to substrates of collagen types I-III and V. Conversely, minimal amounts of the proteoglycan bound to substrates of laminin-nidogen, vitronectin, collagen types IV and VI, and fibronectin or to a proteolytic fragment encompassing its cell-binding domain (105 kDa). Preincubation of these substrates with soluble PG-LA prior to plating of the cells had no effect on their locomotory behavior. These results indicate that PG-LA affects neural crest cell movement primarily through an interaction with the cell surface, rather than by association with the cell motility-promoting substrate molecules. The molecular interaction of soluble PG-LA with neural crest cells was further examined by analyzing the effects of isolated domains of the proteoglycan on cell migration on fibronectin. Addition of chondroitin sulfate chains, the core protein free of glycosaminoglycans, the isolated hyaluronan-binding region (HABr), or a proteolytic fragment corresponding to the keratan sulfate-enriched domain of the PG-LA to neural crest cells migrating on fibronectin or the 105-kDa fibronectin fragment had no significant effect on their motility. After reduction and alkylation, PG-LA was considerably less efficient in perturbing cell movement on fibronectin substrates and virtually ineffective in altering migration on the 105-kDa fragment. In the presence of hyaluronan fragments of 16-30 monosaccharides in length, or an antiserum against the HABr, the migration repressing activity of PG-LA was reduced in a dose-dependent fashion. Furthermore, the inhibitory action of PG-LA was significantly reduced by treatment of the cells with Streptomyces hyaluronidase.(ABSTRACT TRUNCATED AT 400 WORDS)

Ambystoma↗
PubMed1985

Hepatocyte adhesion on plastic. Different mechanisms for serum- and fibronectin-mediated adhesion.

Hepatocytes adhere well on plastic in the presence of serum or fibronectin and subsequent spreading is not prevented when protein synthesis was blocked by cycloheximide. Protein synthesis-independent spreading was also observed in cultures containing serum depleted of fibronectin by affinity chromatography. This indicates that serum-mediated adhesion is independent of fibronectin and suggests the existence of an adhesion factor other than fibronectin in serum. The involvement of different membrane components for fibronectin- and serum-mediated adhesion was demonstrated by experiments where the different adhesion-inhibiting activities of antisera raised against plasma membranes of rat liver and Morris hepatoma 7777 (Neumeier et al., FEBS lett 168 (1984) 241-244) were used. Whereas anti-liver antibodies inhibited both types of adhesion, anti-hepatoma antibodies were only able to prevent fibronectin-mediated adhesion. This indicates again that two different mechanisms are responsible for fibronectin- and serum-mediated adhesion. Fractionation of fetal calf serum (FCS) by size exclusion HPLC revealed that proteins of molecular weights of 60-80 kD promoted attachment and spreading of hepatocytes. Spreading was not perturbated by anti-hepatoma antibodies, indicating that an adhesion factor of 60-80 kD is responsible for serum-mediated adhesion. 'Serum-spreading factor', also called vitronectin, from human plasma has been described as having a similar molecular weight. The purified factor was found to mediate hepatocyte adhesion which was not inhibited by anti-hepatoma antibodies. This suggests that serum-mediated adhesion depends on an adhesion factor present in FCS, which is similar to or identical with vitronectin.

Animals↗
PubMed1988

Mechanisms of serum protein binding and cell anchorage to immobilized serotonin and indole analogs.

Bovine aortal endothelial cells, bovine smooth muscle cells, chick embryo fibroblasts, and baby hamster kidney cells all attached and grew on immobilized tryptamine or L-tryptophan as successfully as on immobilized serotonin. A detailed investigation employing different serum compositions combined with cell blotting and immunoblotting techniques revealed that adhesion of cells to each of the immobilized indole analogs was mediated by vitronectin and fibronectin. Quantitative analyses revealed major differences in the variety of serum proteins adsorbed to each of the immobilized indole analogs and in particular major differences in the amounts of adsorbed vitronectin. However, similar levels of adsorbed fibronectin and fibronectin fragments were found on each of the immobilized indole analogs. The results indicate that (i) different composites of surface-adsorbed proteins may be directed by chemical differences between the immobilized indole analogs and (ii) mammalian cells may still populate chemically different surfaces with equal success despite differences in the surface profiles of adsorbed serum proteins.

Animals↗
PubMed1988

Adsorption from fetal calf serum of collagen-like proteins which bind fibronectin and promote cell attachment.

Baby hamster kidney cells were seeded onto Western blots of fetal serum proteins which had been extracted from several foreign surfaces. This revealed that the major cell adhesive proteins adsorbed onto these surfaces from fetal serum were (1) fibronectin of Mr 220,000 Da and (2) vitronectin of Mr 65,000 and 78,000 Da. Two minor bands of cell attachment were observed at Mr 153,000 and Mr 134,000 Da in the fetal serum proteins extracted from heparin-agarose and serotonin-agarose. However, by exposing the Western blots of separated proteins to a second round of serum proteins, prior to cell blotting, very strong cell adhesive bands were revealed at Mr 153,000, 134,000, and 120,000 Da. By (i) modifying the composition of the serum proteins used to treat the Western blots, (ii) using specific antibodies to fibronectin, and (iii) using radiolabeled fibronectin, it was conclusively demonstrated that the new cell adhesive bands owed their increased cell attachment activity to secondary binding of fibronectin. The new bands were shown (i) to be trypsin sensitive and collagenase sensitive and therefore to be collagen-like proteins and (ii) to react negatively in immunoblots using anti-fibronectin, anti-vitronectin, anti-fibrinogen, anti-fetuin or anti-thrombospondin. In SDS-PAGE (i) the Mr 120,000-Da protein comigrated with the alpha 2-chain of Type I collagen, (ii) the Mr 134,000-Da protein comigrated with the alpha 1-chain of Type I collagen, and (iii) the Mr 153,000-Da protein comigrated with the pN-alpha 1-chain of Type III collagen. Since the novel collagen-like proteins acted as strong sites of cell attachment on nitrocellulose blots by binding fibronectin, they might well promote cell attachment on the foreign surfaces from which they were extracted.

Adsorption↗
PubMed1995

Fluorescent liposomes as quantitative markers of phagocytosis by alveolar macrophages.

A new phagocytic assay based on liposome ingestion by alveolar macrophages (AMs) is described. Fluorescent microspheres were encapsulated in liposomes, which allowed rapid enumeration by fluorometry. Liposomes made in the presence of vitronectin had the protein exposed on their outer surfaces, as determined by immunolabelling. Liposomes and rat AMs were incubated under conditions favorable for phagocytosis. Observation by light and electron microscopy showed AMs engulfing liposomes, with gradual transfer of fluorescent label from liposome to cell interior. This transfer was due to bona fide phagocytosis, as evidenced by (1) fluorescence of liposome-encapsulated dihydrofluorescein (DHF)-zymosan exclusively within AMs and (2) triggering of the respiratory burst by zymosan-associated liposomes only under conditions that allowed phagocytosis. Phagocytic activity was expressed as liposomes/cell, the average number of liposomes phagocytosed per macrophage. We used this technique to follow phagocytosis over time and to measure the effects of lipopolysaccharide (LPS) and vitronectin on AM phagocytosis.

Animals↗
PubMed1992

Integrins and their accessory adhesion molecules in mammary carcinomas: loss of polarization in poorly differentiated tumors.

The integrins are alpha beta heterodimeric transmembrane proteins mediating cell-substratum as well as cell-cell interactions. To identify the pattern of expression of the beta 1, beta 3, and beta 4 integrins and their accessory adhesion molecules in relation to the malignant phenotype of invasive breast cancer, we performed an immunohistochemical study for the alpha 2 beta 1 (VLA-2), alpha 6 beta 1 (VLA-6), alpha v and alpha v beta 3 (vitronectin receptor), alpha 6 beta 4, carcinoembryonic antigen, and carcinoembryonic antigen-related molecules in a series of 37 invasive breast carcinomas. All integrin chains examined showed similar patterns in nonneoplastic breast tissue, with strong membrane staining of the myoepithelial cells and weak to moderate staining on the basolateral surfaces of the luminal cells. We found that downregulation of the alpha 2 chain of VLA-2 occurs more frequently in poorly differentiated grade III invasive ductal carcinomas (IDCs) (P = .048). Loss of alpha 6 beta 4 seems also to occur more frequently in grade III IDC (seven of 11 cases, 63.6%) than in grade I/II IDC (two of eight cases, 25%), although this did not reach statistical significance. Carcinoembryonic antigen and carcinoembryonic antigen-related antigens, which are known to function as accessory adhesion molecules, were found mainly in the cytoplasm of neoplastic cells and there was reduced membrane polarization in poorly organized tumors. In contrast the alpha v beta 3, vitronectin receptor heterodimer recognized by the 23C6 monoclonal antibody was weak or absent in normal breast epithelium, and was weakly expressed in two of 19 (10%) IDCs and in nine of 18 (50%) invasive lobular carcinomas (P = .008). However, the alpha v chain detected with the antibody 13C2 was weakly to moderately expressed on nonneoplastic epithelium and at a similar intensity in 13 of 19 IDCs and 15 of 17 invasive lobular carcinomas, suggesting that in IDC the alpha v chain may be associated with a different beta chain (possibly beta 1 or beta 5). No correlation between integrin expression and estrogen/progesterone receptor status was found. These data provide further evidence that in invasive breast carcinomas there is a widespread deregulated expression of integrins and their accessory adhesion molecules with loss of polarization. Changes in the expression and function of cell adhesion molecules, which control growth and differentiation, may have clinical relevance in the behavior of breast cancer.

Antigens, Surface↗
PubMed1994

Use of enhanced chemiluminescence to quantify protein adsorption to calcium phosphate materials and microcarrier beads.

The adsorption of serum proteins to calcium phosphate bone substitute materials, positively-charged dextrose and negatively-charged polystyrene microcarrier beads was compared by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Protein adsorption to hydroxyapatite (HA)-based materials was influenced by chemical composition. Surface charge sign, distribution and/or functional group affected protein adsorption to microcarrier beads. Enhanced chemiluminescence was used to quantify adsorption of fibronectin and vitronectin following Western blotting, and to monitor the kinetics of adsorption of these two proteins to HA and Biosilon. Relative to total protein, fluctuating levels of fibronectin were detected on both materials. In contrast, vitronectin adsorption increased over the course of the incubation period with maximal relative adsorption detected after 30 min on Biosilon and 60 min on HA.

Adsorption↗
PubMed2004

Pro-apoptotic signaling pathway activated by echistatin in GD25 cells.

Disintegrins, low molecular weight RGD-containing polypeptides isolated from snake venoms, have seen use as integrin antagonists in the field of tumor biology and angiogenesis. In this study, we investigated the molecular mechanism by which the disintegrin echistatin affects cell adhesion and signaling resulting in an apoptotic response in the GD25 cell system. Wild-type GD25 cells, which lack expression of the beta(1) family of integrin, and stable transfectants expressing the A isoform of beta(1) integrin subunit were used. Nanomolar concentrations of echistatin detached fibronectin- and vitronectin-adherent GD25 cells from immobilized substratum. However, prior to inducing detachment of adherent cells, echistatin caused apoptosis as measured by caspase-3 activation. Either cell detachment or apoptotic response induced by echistatin were more pronounced on fibronectin-adherent GD25 cells than on vitronectin-adherent ones. GD25 cell exposure to echistatin caused a reduction of tyrosine phosphorylation levels of pp125(FAK), whereas it didn't affect either Shc tyrosine phosphorylation levels or Shc-Grb2 functional association. The down-regulation of pp125(FAK) preceded apoptosis and cell detachment induced by echistatin. Our results indicate that pp125(FAK) and not Shc pathway is involved in echistatin-induced apoptotic response in the GD25 cell system.

Apoptosis↗
PubMed2004

Structural bases of the redox-dependent conformational switch in the serpin PAI-2.

Depending on the redox-status, the serpin plasminogen activator inhibitor type 2 (PAI-2) can exist in either a stable monomeric or polymerogenic form. The latter form, which spontaneously forms loop-sheet polymers, has an open beta-sheet A and is stabilized by a disulfide bond between C79 (in the CD-loop) and C161 (at the bottom of PAI-2). Reduction of this bond results in a closing of the beta-sheet A and converts PAI-2 to a stable monomeric form. Here we show that the stable monomeric and polymerogenic forms of PAI-2 are fully interconvertible, depending on redox-status of the environment. Our intramolecular distance measurements indicate that the CD-loop folds mainly on one side of the stable monomeric form of the inhibitor. However, the loop can translocate about 54A to the bottom of PAI-2 so that the C79-C161 disulfide bond can form under oxidizing conditions. We show also that the redox-active C79 can form a disulfide-link to the matrix protein vitronectin, suggesting that vitronectin can stabilize active PAI-2 in extracellular compartments. PAI-2 is therefore a rare example of a redox-sensitive protein for which the activity and polymerization ability are regulated by reversible disulfide bond formation leading to major translocation of a loop and significant conformational changes in the molecule.

Cysteine↗