Plasminogen activation by t-PA on the surface of human melanoma cells in the presence of alpha 2-macroglobulin secretion.

Several human melanoma cell lines produced tissue-type plasminogen activator (t-PA), as detected by zymography and immunocapture assay of culture media and cell lysates. Urokinase (u-PA) was found at only less than or equal to 1% the level of t-PA. Acid eluates of the cell surface indicated that the melanoma cells had t-PA bound on their surface, but no u-PA, and also had a very low capacity to bind exogenous u-PA. After incubation of the melanoma cells with 10% plasminogen-depleted fetal calf serum and human plasminogen, bound plasmin activity could be eluted from the cell surface with tranexamic acid, an analogue of lysine. This indicated that plasminogen was activated on the cell surface. The cell-surface plasmin formation was inhibited by an anti-catalytic monoclonal antibody to human t-PA, and not by an anti-catalytic antibody to u-PA. The melanoma cells also synthesized and secreted alpha 2-macroglobulin (alpha 2M), as shown by alpha 2M-specific mRNA in Northern blotting and detection of alpha 2M protein in conditioned cell culture media. The media were found to inhibit u-PA but not t-PA. This inhibition was related to their alpha 2M content, and immunoabsorption of alpha 2M removed the inhibitory activity. These studies suggest that t-PA can bind to the surface of melanoma cells and generate surface-bound plasmin. Because t-PA and cell-bound plasmin are unaffected by alpha 2M, t-PA may, in the case of melanoma cells, serve an analogous function to u-PA in supporting tumor cell invasion.     

Down-regulation of plasmin receptors on human sarcoma cells by glucocorticoids

After incubation of confluent monolayer cultures of human HT-1080 fibrosarcoma cells with purified native human plasminogen in plasminogen-depleted serum-containing medium, bound plasmin activity could be specifically eluted from the cells with tranexamic acid, an analogue of lysine. Dexamethasone reduced the amount of recoverable bound plasmin activity in a dose-dependent manner. Dexamethasone was also found to induce a time- and dose-dependent decrease in the ability of the cells to bind added plasmin. Untreated HT-1080 cells bound added plasmin with a high capacity (600,000 molecules bound per cell), and this decreased to an undetectable level after treatment with 100 nM dexamethasone. The kinetics of the loss of plasmin binding by the dexamethasone-treated sarcoma cells, a clear decrease after 4 h, correlated with those for the loss of cell-bound urokinase (u-PA) activity. Plasmin was not, however, bound to the active site of u-PA: an anti-catalytic monoclonal antibody to u-PA had no effect on plasmin binding. Other glucocorticoids, such as hydrocortisone and corticosterone, had a similar effect to dexamethasone on plasmin binding to HT-1080 cells. The effect of glucocorticoids on the plasmin receptor seemed to occur at least partly via a decrease in the affinity for plasmin, since the Kd for plasmin with untreated cells was 5.4 x 10(-9) M, and with cells treated with 5 nM dexamethasone, the Kd value for plasmin was 1.2 x 10(-7) M. These results show that glucocorticoids induce down-regulation of plasmin receptors on the surface of HT-1080 cells: a novel mechanism, in addition to the known effects of glucocorticoids on u-PA and PA inhibitors, by which human tumor cells may be disarmed of their pericellular proteolytic activity.     

Lipopolysaccharide-induced modulation of human monocyte urokinase production and activity

Cells of the monocyte/macrophage lineage are known to produce urokinase type plasminogen activator (u-PA) and are active participants in the inflammatory response. Modulation of cellular u-PA production, for instance in response to LPS, may have an important impact on the evolution of inflammatory lesions. A definitive picture of how monocyte u-PA production and activity are regulated by LPS is lacking. We addressed this issue directly by measuring u-PA Ag and activity in mononuclear cell cultures. By using a competition ELISA to quantitate u-PA Ag, we found that LPS-stimulated mononuclear cells in culture increased u-PA production in a dose-dependent manner and that all the u-PA detected was attributable to the monocytes therein. Increasing amounts of u-PA were secreted into the medium, bound to the cell surface, and found intracellularly. Although the absolute amounts of u-PA varied from donor to donor, the increases seen with LPS stimulation were a consistent and statistically significant finding. Only the cell-surface-bound u-PA was fibrinolytically active, however, with this activity increasing upon LPS stimulation. All monocyte cell-surface-associated fibrinolytic activity was attributed to u-PA, as shown by plasminogen dependence, neutralization by antibodies to u-PA, and identification of fibrinolytically active molecules eluted from the cell surface. The surface bound u-PA was not inhibited by its physiologic inhibitors, PAI-1 or PAI-2, whereas free u-PA was. Hence LPS stimulation results in monocytes exhibiting increased cell-surface-associated u-PA Ag and fibrinolytic activity, in spite of concomitant high levels of plasminogen activator inhibitor type 2 production. This surface-bound enzymatic activity may influence the ability of monocytes to migrate in and interact with an inflammatory microenvironment.     

Generation of cell surface-bound plasmin by cell-associated urokinase-type or secreted tissue-type plasminogen activator: a key event in melanoma cell invasiveness in vitro.

Recently, we have shown that plasminogen activators (PAs) of both types, urokinase-type (uPA) as well as tissue-type (tPA), are involved in the in vitro invasiveness of human melanoma cells. The present study is focused on the generation and importance of cell surface-bound plasmin in this process. The human melanoma cell lines MelJuso and MeWo expressed plasminogen binding sites on the cell surface. Plasminogen binding was saturable and not species-specific, since human and bovine plasminogen bound to the cells with comparable efficiency. The activation of the proenzyme plasminogen bound on MelJuso cells, which expressed surface-associated uPA activity, occurred almost synchronously with binding to the cell surface. Removal of cell-associated uPA considerably reduced plasmin generation on these cells. In contrast, plasminogen activation on MeWo cells, which secreted tPA into the culture supernatant and which were devoid of surface-associated PA activity, was by far less effective. The efficiency of the activation process could be increased by addition of exogenous tPA. With both cell lines, plasmin generation on the cell surface was suppressed by inhibitory monoclonal antibodies specific for the respective PA type. Selective inhibition of cell surface-associated plasmin by preincubating the cells with an inhibitory monoclonal antibody or with aprotinin, as well as removal of plasmin from the cell surface, led to a significant decrease in cellular invasiveness of both cell lines into various biological substrates such as fibrin gel, the basement membrane extract Matrigel, or intact extracellular matrix. Both cell lines were able to penetrate an intact cell layer of the human keratinocyte line HaCaT, a process, which also proved to be dependent on cell-associated plasmin. In conclusion, these data provide evidence that plasminogen activation associated with the surface of human melanoma cells is catalyzed much more efficiently by cell-associated uPA (MelJuso) than by secreted tPA (MeWo). Cell-associated plasmin, which is protected from inactivation by serum inhibitors, represents the essential component of the proteolytic cascade of plasminogen activation during in vitro invasiveness of human melanoma cells.     

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.     

Generation of cell surface-bound plasmin by cell-associated urokinase-type or secreted tissue-type plasminogen activator: A key event in melanoma cell invasiveness in vitro

Recently, we have shown that plasminogen activators (PAs) of both types, urokinase-type (uPA) as well as tissue-type (tPA), are involved in the in vitro invasiveness of human melanoma cells. The present study is focused on the generation and importance of cell surface-bound plasmin in this process. The human melanoma cell lines MelJuso and MeWo expressed plasminogen binding sites on the cell surface. Plasminogen binding was saturable and not species-specific, since human and bovine plasminogen bound to the cells with comparable efficiency. The activation of the proenzyme plasminogen bound on MelJuso cells, which expressed surface-associated uPA activity, occurred almost synchronously with binding to the cell surface. Removal of cell-associated uPA considerably reduced plasmin generation on these cells. In contrast, plasminogen activation on Me Wo cells, which secreted tPA into the culture supernatant and which were devoid of surface-associated PA activity, was by far less effective. The efficiency of the activation process could be increased by addition of exogenous tPA. With both cell lines, plasmin generation on the cell surface was suppressed by inhibitory monoclonal antibodies specific for the respective PA type. Selective inhibition of cell surface-associated plasmin by preincubating the cells with an inhibitory monoclonal antibody or with aprotinin, as well as removal of plasmin from the cell surface, led to a significant decrease in cellular invasiveness of both cell lines into various biological substrates such as fibrin gel, the basement membrane extract Matrigel, or intact extracellular matrix. Both cell lines were able to penetrate an intact cell layer of the human keratinocyte line HaCaT, a process, which also proved to be dependent on cell-associated plasmin. In conclusion, these data provide evidence that plasminogen activation associated with the surface of human melanoma cells is catalyzed much more efficiently by cell-associated uPA (MelJuso) than by secreted tPA (MeWo). Cell-associated plasmin, which is protected from inactivation by serum inhibitors, represents the essential component of the proteolytic cascade of plasminogen activation during in vitro invasiveness of human melanoma cells.     

Plasminogen activation initiated by single-chain urokinase-type plasminogen activator. Potentiation by U937 monocytes

The binding of urokinase-type plasminogen activators (u-PA) to receptors on various cell types has been proposed to be an important feature of many cellular processes requiring extracellular proteolysis. We have investigated the effect of single-chain u-PA binding to the monocyte-like cell line U937 on plasminogen activation. A 16-fold acceleration of the activation of plasminogen was observed at optimal concentrations of single-chain u-PA. This potentiation was abolished by the addition of either 6-aminohexanoic acid or the amino-terminal fragment of u-PA, thus demonstrating the requirement for specific binding of both single-chain u-PA and plasminogen to the cells. The mechanism of the enhancement of plasmin generation appears to be due primarily to an increase in the rate of feedback activation of single-chain u-PA to the more active two-chain u-PA by cell-bound plasmin, initially generated by single-chain u-PA. This increased activity of the plasminogen activation system in the presence of U937 cells provides a mechanism whereby u-PAs may exert their influence in a variety of cell-associated proteolytic events.     

Alpha 2-macroglobulin restricts plasminogen activation to the surface of RC2A leukemia cells.

Human RC2A myelomonocytic leukemia cells are able to activate the prourokinase (pro-u-PA) they secrete so that active u-PA is present both in serum-free conditioned medium from these cells, as well as on the cell surface. When the cells are grown in serum-containing medium, no u-PA activity can be found in the medium but active u-PA is found bound to the cell surface where it can generate bound plasmin. This distribution of u-PA activity was shown to be, first, the net result of slow inactivation of free active u-PA by serum inhibitor(s) and simultaneous rapid uptake of u-PA onto the cell surface. Binding to cells was at least six times faster than inactivation by 10% serum. The principal serum inhibitor of u-PA was identified as alpha 2-macroglobulin (alpha 2M), and prior inactivation of u-PA by purified human alpha 2M was also shown to prevent uptake of u-PA activity onto cells. Second, although endogenous u-PA could form covalent complexes with purified alpha 2M in the culture medium of RC2A cells, covalent alpha 2M complexes were not formed by u-PA on the cell surface; the u-PA taken up in this compartment was protected against alpha 2M inhibition. u-PA anchored to plastic surfaces via monoclonal antibodies to the amino-terminal region of u-PA was also protected against alpha 2M, suggesting that the protection of cell surface u-PA results from a steric effect. These results provide evidence as to how the active u-PA produced by leukemia cells can contribute to proteolytic activity on their cell surface in the presence of serum inhibitors.     

Matrix Metalloproteinases and Cellular Fibrinolytic Activity

Several molecular interactions between the matrix metalloproteinase (MMP) and the plasminogen/plasmin (fibrinolytic) system may affect cellular fibrinolysis. MMP-3 (stromelysin-1) specifically hydrolyzes urokinase (u-PA), yielding a 17 kD NH₂-terminal fragment containing the functionally intact receptor (u-PAR)-binding sequence and a 32 kD COOH-terminal fragment containing the intact serine proteinase domain. MMP-3 generates an angiostatin like fragment (containing kringles 1-4 with the cellular binding domains) from plasminogen. Treatment with MMP-3 of monocytoid THP-1 cells saturated with bound plasminogen, resulted in a dose-dependent reduction of the amount of u-PA-activatible plasminogen. Treatment with MMP-3 of cell-bound u-PA, in contrast, did not alter cell-associated u-PA activity. These data thus indicate that MMP-3 may downregulate cell-associated plasmin activity by decreasing the amount of activatible plasminogen, with out affecting cell-bound u-PA activity. MMP-3 also specifically interacts with the main inhibitors of the fibrinolytic system. Thus, MMP-3 specifically hydrolyzes human ₂-antiplasmin (₂-AP), the main physiological plasmin inhibitor. ₂-AP cleaved by MMP-3 no longer forms a stable complex with plasmin and no longer interacts with plasminogen. Cleavage and inactivation of ₂-AP by MMP-3 may constitute a mechanism favoring local plasmin-mediated proteolysis. Furthermore, MMP-3 specifically hydrolyzes and inactivates human plasminogen activator inhibitor-1 (PAI-1). Stable PAI-1 bound to vitronectin is cleaved and inactivated by MMP-3 in a comparable manner as free PAI-1; the cleaved protein, however, does not bind to vitronectin. Cleavage and inactivation of PAI-1 by MMP-3 may thus constitute a mechanism decreasing the antipro teolytic activity of PAI-1 and impairing the potential inhibitory effect of vitronectin-bound PAI-1 on cell adhesion and/or migration. These molecular interactions of MMP-3 with enzymes, substrates and inhibitors of the fibrinolytic system may thus play a role in the regulation of (cellular) fibrinolysis. Furthermore, the temporal and topographic expression pattern of MMP components, as well as studies in gene-deficient mice, suggest a functional role in neointima formation after vascular injury.     

Thrombin increases proliferation and decreases fibrinolytic activity of kidney glomerular epithelial cells.

Human glomerular epithelial cells (GECs) in culture synthesize single-chain, urokinase-type plasminogen activator (SC-uPA), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor 1 (PAI-1) and possess specific membrane-binding sites for u-PA. Using purified 125I-alpha thrombin, we demonstrate here the presence of two populations of specific binding sites for thrombin on GECs (1.Kd = 4.3 +/- 1.0 x 10(-10) M, 5.4 +/- 1.4 x 10(4) M sites per cell, 2. Kd = 1.6 +/- 0.5 x 10(-8) M, 7.9 +/- 1.8 x 10(5) sites per cell). Purified human alpha thrombin promoted the proliferation of GECs and induced a time- and dose-dependent increase of SC-uPA, t-PA, and PAI-1 antigens released by GECs. Thrombin-mediated increase in antigen was paralleled by an increase in the levels of corresponding u-PA and PAI-1 messenger RNA. In contrast, thrombin decreased u-PA activity in conditioned medium. This discrepancy between u-PA antigen and u-PA activity was explained by a limited proteolysis of SC-uPA by thrombin, leading to a two-chain form detected by immunoblotting and that could not be activated by plasmin. Thrombin also decreased the number of u-PA binding sites on GECs (p less than 0.05) without changing receptor affinity. Hirudin inhibited the binding and the cellular effects of thrombin, whereas thrombin inactivated by diisopropylfluorophosphate had no effect, indicating that both membrane binding and catalytic activity of thrombin were required. We conclude that thrombin, through specific membrane receptors, stimulates proliferation of GECs and decreases the fibrinolytic activity of GECs both at the cell surface and in the conditioned medium. These results suggest that thrombin could be involved in the pathogenesis of extracapillary proliferation and persistency of fibrin deposits in crescentic glomerulonephritis.     

Binding and activation of plasminogen at the surface of Staphylococcus aureus. Increase in affinity after conversion to the Lys form of the ligand.

Untreated Staphylococcus aureus cells, strain Cowan I, specifically bound 125I-Glu-plasminogen. The binding was inhibited by both unlabeled Glu-plasminogen and Glu-plasmin. The Lys form of plasminogen, which lacks the 8-kDa amino-terminal activation peptide, was approximately 100-fold more effective than the Glu form in competing with the binding of 125I-labeled Glu-plasminogen. This suggests an increase in binding affinity upon removal of the activation peptide. Fibronectin, fibrinogen and IgG, plasma components known to bind to the staphylococcal surface, did not significantly interfere with the binding. The competing activity in plasma was abolished by specifically absorbing plasminogen from the plasma sample. L-Lysine and a fragment of plasminogen containing three of the first five protein attachment domains present in the molecule (kringle structures) also competed with plasminogen for binding suggesting that the lysine-binding sites of plasminogen were involved in its interaction with staphylococci. Scatchard analysis revealed high- and low-affinity binding sites. Kd and the number of high-affinity binding sites were 1.7 nM and 780 binding sites/bacterial cell, respectively. 125I-Glu-plasminogen bound to staphylococcal surface was converted to plasmin by tissue-type plasminogen activator. The conversion took place also in the presence of plasma. If the conversion was carried out in the absence of low-molecular-mass plasmin inhibitors such as aprotinin, the bound Glu-plasmin was further converted to Lys-plasmin. The surface-bound plasmin was enzymically active, as judged by digestion of the synthetic substrate, S-2251. The plasminogen conversion shown by the present experiments not only leads to the surface-bound plasmin but seems to considerably increase the affinity of plasmin for its binding site. This may represent a physiologically relevant method for a bacterial cell to retain surface-bound active plasmin which is also protected from its soluble plasma inhibitors. This novel mechanism for staphylococci to adopt surface-bound proteolytic activity, without the interference of plasma components, may have some role in the tissue penetration and invasion of microbes during infection.     

The inactivation of single-chain urokinase-type plasminogen activator by thrombin on cultured human endothelial cells

Single-chain urokinase-type plasminogen activator (scu-PA) is cleaved by thrombin, resulting in an inactive molecule called thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T). There is no knowledge about cell-mediated inactivation of scu-PA. We have studied whether scu-PA bound to cultured human umbilical vein endothelial cells (HUVEC) could be inactivated by thrombin. High molecular weight scu-PA was bound to HUVEC and incubated with increasing amounts of thrombin for 30 min at 37 degrees C. Cell-bound urokinase-type plasminogen activator (u-PA) was released and levels of scu-PA, tcu-PA/T and active two-chain u-PA were measured using sensitive bioimmunoassays. Cell-bound scu-PA was efficiently inactivated by thrombin. Fifty percent inactivation of scu-PA occurred at about 0.2 nM thrombin. In the presence of monoclonal anti-urokinase receptor IgG, at least 50% of the binding of scu-PA to HUVEC was inhibited. The relative amount of tcu-PA/T that was generated by thrombin was not affected by the monoclonal antibody. These results indicated that scu-PA bound to HUVEC via the urokinase receptor can be inactivated by thrombin. The efficient inactivation of cell-bound scu-PA suggests that a cofactor for thrombin may be involved, like thrombomodulin or glycosaminoglycans. It is concluded that scu-PA bound to the urokinase receptor on a cell surface can be inactivated by thrombin, which may have profound effects on u-PA-mediated local fibrinolysis and extracellular proteolysis during processes in which thrombin is also involved.     

Interaction of plasminogen activator inhibitor (PAI-1) with vitronectin

Immobilized vitronectin was found to bind both purified plasminogen activator inhibitor type 1 (PAI-1) and the PAI-1 in conditioned culture medium of human sarcoma cells. Similarly, immobilized PAI-1 bound both purified vitronectin and vitronectin from normal human serum. These interactions were demonstrated using both enzyme immunoassay and radioiodinated proteins. Solid-phase vitronectin bound PAI-1 with Kd 1.9 x 10(-7) M, and the reverse interaction gave a Kd 5.5 x 10(-8) M. Evidence was also found for a second type of binding with a Kd below 10(-10) M. The molar ratios of the two proteins in the complex at the saturation levels were approximately one molecule of soluble PAI-1 bound per three molecules of immobilized vitronectin and approximately one molecule of soluble vitronectin being bound per one molecule of immobilized PAI-1. Binding of PAI-1 to vitronectin did not lead to an irreversible loss of the ability of PAI-1 to inhibit urokinase (u-PA) and tissue-type plasminogen activator (t-PA). Active u-PA released vitronectin-bound 125I-labeled PAI-1 radioactivity, suggesting that u-PA interacts with the complex. The Mr 50,000 urokinase cleavage product of PAI-1 also bound to vitronectin, but this bound fragment did not inhibit u-PA. Binding of PAI-1 to vitronectin did not interfere with the ability of vitronectin to promote the adhesion and spreading of cells. These results suggest that the interaction between vitronectin and PAI-1 may serve to confine pericellular u-PA activity to focal contact sites where cells use proteolysis in regional detachment.     

Distinct localizations of urokinase-type plasminogen activator and its type 1 inhibitor under cultured human fibroblasts and sarcoma cells

We studied the immunocytochemical localization of urokinase-type plasminogen activator (u-PA) and the type 1 plasminogen activator inhibitor (PAI-1) in human fibroblasts and sarcoma cells, using both polyclonal and monoclonal antibodies. The u-PA was found to be located at discrete cell-substratum contact sites, and also at areas of cell-cell contacts, whereas PAI-1 was distributed as a homogeneous carpet excluding strialike areas on the substrate under the cells. To confirm the extracellular localization of u-PA and PAI-1, we stained the cells live at 0 degree C before fixation. A double-labeling experiment showed different distribution of u-PA and PAI-1 under the cells, and especially their peripheral parts. The staining pattern of u-PA and PAI-1 resisted treatment with 0.2% saponin followed by mechanical removal of cells, a method previously reported to isolate focal contact membranes of fibroblasts. We further demonstrated the deposition of u-PA to the contact areas of cells obtained by saponin treatment by zymography, and that of PAI-1 by metabolic labeling, reverse zymography, immunoblotting, and immunoprecipitation. Fibronectin was also present in the preparations. The deposition of both PAI-1 and fibronectin by the sarcoma cells was enhanced, after treating the cells with 10(-6) M dexamethasone. The confinement of u-PA to discrete contact sites and the more uniform distribution of PAI-1 on the cell substratum may explain how cells producing large amounts of enzyme inhibitors can produce PA-mediated focal proteolysis.     

Matrix-bound plasminogen activator inhibitor type 1 inhibits the invasion of human monocytes into interstitial tissue.

Invasion of tissue by monocytes in the course of cellular immune reactions is a multistep process that is thought to be based on the action of urokinase type plasminogen activator (u-PA), an ubiquitous serine protease able to convert the zymogen plasminogen into the active protease plasmin. Expression and occupation of urokinase-type plasminogen activator receptors (u-PA-R) are known to be up-regulated by IFN-gamma and TNF-alpha, and endogenously occupied u-PA-R were found to be instrumental in monocyte invasiveness. We used the amnion invasion assay to investigate whether monocyte invasiveness is affected by matrix-bound plasminogen activator inhibitors (PAI) and by fluid phase u-PA. We show in this study that preincubation of amnion membranes with 1.5 U/cm2 PAI-1 decreases invasion of IFN-gamma activated monocytes by 70% compared with controls. Anti-vitronectin antibodies, which block PAI-1 binding to the matrix, abrogate the inhibitory effect of PAI-1 on monocyte invasiveness, indicating that active PAI-1 is bound via matrix-associated vitronectin. In contrast, preincubation of the amnion membrane with PAI-2 which does not bind to the extracellular matrix has no effect on monocyte invasiveness. Finally, the inhibitory action of matrix-bound PAI-1 can be abrogated by addition of 5 IU/ml u-PA to the monocytes in the invasion chamber. These findings indicate that monocyte invasiveness might be regulated not only by expression and occupation of u-PA-R but also by matrix-bound PAI-1.     

Tissue-type plasminogen activator binding to human endothelial cells. Evidence for two distinct binding sites

The endothelium may contribute to fibrinolysis through the binding of plasminogen activators or plasminogen activator inhibitors to the cell surface. Using a solid-phase radioimmunoassay, we observed that antibodies to recombinant tissue-type plasminogen activator (rt-PA) and plasminogen activator inhibitor type 1 (PAI-1) bound to the surface of cultured human umbilical vein endothelial cells (HUVEC). HUVEC also specifically bound added radiolabeled rt-PA with apparent steady-state binding being reached by 1 h at 4 degrees C. When added at low concentrations (less than 5 nM), rt-PA bound with high affinity mainly via the catalytic site, forming a sodium dodecyl sulfate-stable 105-kDa complex which dissociates from the cell surface over time and which could be immunoprecipitated by a monoclonal antibody to PAI-1. rt-PA bound to this high affinity site retained less than 5% of its expected plasminogen activator activity. At higher concentrations, binding did not require the catalytic site and was rapidly reversible. rt-PA initially bound to this site retained plasminogen activator activity. These studies suggest that tissue-type plasminogen activator and PAI-1 are expressed on the surface of cultured HUVEC. HUVEC also express unoccupied binding sites for exogenous tissue-type plasminogen activator. The balance between the expression of plasminogen activator inhibitors and these unoccupied binding sites for plasminogen activators on the endothelial surface may contribute to the regulation of fibrinolysis.     

Identification and characterization of human endothelial cell membrane binding sites for tissue plasminogen activator and urokinase

Cultured human endothelial cells synthesize and secrete two types of plasminogen activator, tissue plasminogen activator (t-PA) and urokinase (u-PA). Previous work from this laboratory (Hajjar, K.A., Hamel, N. M., Harpel, P. C., and Nachman, R. L. (1987) J. Clin. Invest. 80, 1712-1719) has demonstrated dose-dependent, saturable, and high affinity binding of t-PA to two sites associated with cultural endothelial cell monolayers. We now report that an isolated plasma membrane-enriched endothelial cell fraction specifically binds 125I-t-PA at a single saturable site (Kd 9.1 nM; Bmax 3.1 pmol/mg membrane protein). Ligand blotting experiments demonstrated that both single and double-chain t-PA specifically bound to a Mr 40,000 membrane protein present in detergent extracts of isolated membranes, while high molecular weight, low molecular weight, and single-chain u-PA associated with a Mr 48,000 protein. Both binding interactions were reversible and cell-specific and were inhibitable by pretreatment of intact cells with nanomolar concentrations of trypsin. The relevant binding proteins were not found in subendothelial cell matrix, failed to react with antibodies to plasminogen activator inhibitor type 1 and interacted with their respective ligands in an active site-independent manner. The isolated t-PA binding site was resistant to reduction and preserved the capacity for plasmin generation. In contrast, the isolated u-PA binding protein was sensitive to reduction, and did not maintain the catalytic activity of the ligand on the blot. The results suggest that in addition to sharing a matrix-associated binding site (plasminogen activator inhibitor type 1), both t-PA and u-PA have unique membrane binding sites which may regulate their function. The results also provide further support for the hypothesis that plasminogen and t-PA can assemble on the endothelial cell surface in a manner which enhances cell surface generation of plasmin.     

Active transforming growth factor-β in human melanoma cell lines: No evidence for plasmin-related activation of latent TGF-β

Cultured human melanoma cells were found to secrete TGF-β mostly in latent biologically inactive form but in addition five of six melanoma cell lines studied produced in conditioned culture medium active TGF-β in the range from 370 to 610 pg per 10⁶ cells per 24 h. A distinct characteristic of these melanoma cell lines is that they form active surface-bound plasmin by the activation of plasminogen with surface-bound tissue-type plasminogen activator. The present study was performed to assess the role of plasmin in the process of latent TGF-β activation in the melanoma cell lines. No direct correlation was found between cell-associated plasmin activity and the amount of active TGF-β present in the conditioned medium of individual cell lines. The melanoma cell lines exhibited diverse responses to exogenous active TGF-β1; three cell lines were growth-stimulated, two were growth-inhibited, and one had a very low sensitivity to the growth factor. The active TGF-β produced by the melanoma cells was found to inhibit the natural killer cell function of peripheral blood lymphocytes, suggesting that it may have an immunosuppressive effect and a role in the development of melanomas. © 1996 Wiley-Liss, Inc.     

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.