Cell-associated Plasminogen Activator Research Articles

Plasminogen activation on the cell surface.

The plasminogen activation system appears to be widely involved in many biological processes in health and disease, but the regulation of plasmin generation or the mechanisms of stimulation by cell surface receptors are not well understood. Cell surface plasminogen activation requires binding sites for plasminogen substrate and activator enzyme before enhancement of plasmin generation rate is observed. The cell surface moieties involved in binding these reactants appear to be a mixed group of proteins and other molecules, many of which have been extensively investigated. The binding of plasminogen in particular is characterized by heterogeneous receptor molecules, present in high number but generally with low affinity for plasminogen. The low affinity of the interaction, with Kd values around 10(-6) M, presents considerable technical difficulties when studying and quantitating plasminogen binding to cells or isolated receptors. Studying plasminogen activation kinetics in the presence of cells also presents technical difficulties and raises difficult questions on interpretation of results. However, approaches developed to study enzyme activation systems in other areas of hemostasis may also be applied to the problems associated with pericellular proteolysis. Models should be developed that match In vitro experimental data and help us understand the meaning of kinetic constants derived from these systems. In this way it should be possible to better understand the regulation of plasminogen activation around the cell under normal conditions and in a variety of disease states where cell-associated plasminogen activation is believed to be up-regulated. Ultimately, a sound understanding of theses regulatory mechanisms will enable us to devise strategies for modulating proteolytic activity, test these approaches in well designed In vitro systems and relate these results to the in vivo situation.

Structure and function analysis of urinary trypsin inhibitor (UTI): identification of binding domains and signaling property of UTI by analysis of truncated proteins

The binding of urinary trypsin inhibitor (UTI) to its binding sites/receptors on tumor cells inhibits cell invasion in a number of experimental systems and that UTI downregulates constitutive and phorbol ester-induced urokinase production by certain tumor cells. To determine whether the carbohydrate moieties and core protein are required for urokinase suppression, we obtained UTI derivatives that contained O-glycoside-linked N-terminal glycopeptide (UTIm1), N-glycoside-linked C-terminal tandem Kunitz domains (UTIm2), UTI lacking O-glycoside (UTIc), asialo UTI (UTIa), UTI lacking N-glycoside (UTIn), purified Kunitz domain II of UTI (HI-8), and recombinant Kunitz domain II of UTI (R-020). The IC 50 of inhibiting binding of 125I-labeled UTI to cells was indistinguishable for UTIa, UTIn and intact UTI, whereas the IC 50 for inhibiting binding of 125I-labeled UTI to cells was 2.5-, 25- and 29-fold greater for UTIm1, UTIm2 and UTIc than for native UTI. We next looked at the suppression of the urokinase expression by UTI derivatives. An enzyme-linked immunosorbent assay was carried out to measure secreted and cell-associated urokinase. Intact UTI, UTIa, or UTIn effectively suppressed urokinase expression, but UTIm1, UTIm2, UTIc, HI-8 and R-020 had no significant effect. These data show that UTI requires either the N-terminal extension with the O-linked carbohydrate moiety (chondroitin 4-sulfate sugar side chain; Ala1 to Lys21 residues) or the Kunitz domain I (Lys22 to Arg77 residues) of UTI to bind to cells, but the urokinase expression was inhibited only by the O-glycoside-linked core protein without the N-glycoside side chain.

Cyclo19,31[D-Cys19]-uPA19-31 is a potent competitive antagonist of the interaction of urokinase-type plasminogen activator with its receptor (CD87).

Urokinase-type plasminogen activator (uPA) represents a central molecule in pericellular proteolysis and is implicated in a variety of physiological and pathophysiological processes such as tissue remodelling, wound healing, tumor invasion, and metastasis. uPA binds with high affinity to a specific cell surface receptor, uPAR (CD87), via a well defined sequence within the N-terminal region of uPA (uPA19-31). This interaction directs the proteolytic activity of uPA to the cell surface which represents an important step in tumor cell proliferation, invasion, and metastasis. Due to its fundamental role in these processes, the uPA/uPAR-system has emerged as a novel target for tumor therapy. Previously, we have identified a synthetic, cyclic, uPA-derived peptide, cyclo19,31uPA19-31, as a lead structure for the development of low molecular weight uPA-analogues, capable of blocking uPA/uPAR-interaction [Burgle et al., Biol. Chem. 378 (1997), 231-237]. We now searched for peptide variants of cyclo19,31uPA19-31 with elevated affinities for uPAR binding. Among other tasks, we performed a systematic D-amino acid scan of uPA19-31, in which each of the 13 L-amino acids was individually substituted by the corresponding D-amino acid. This led to the identification of cyclo19,31[D-Cys19]-uPA19-31 as a potent inhibitor of uPA/uPAR-interaction, displaying only a 20 to 40-fold lower binding capacity as compared to the naturally occurring uPAR-ligands uPA and its amino-terminal fragment. Cyclo19,31[D-Cys19]-uPA19-31 not only blocks binding of uPA to uPAR but is also capable of efficiently displacing uPAR-bound uPA from the cell surface and to inhibit uPA-mediated, tumor cell-associated plasminogen activation and fibrin degradation. Thus, cyclo19,31[D-Cys19]-uPA19-31 represents a promising therapeutic agent to significantly affect the tumor-associated uPA/uPAR-system.

Transforming Growth Factor Beta-1 Stimulates Invasivity of Hepatic Stellate Cells by Engagement of the Cell-associated Fibrinolytic System

The activation of hepatic stellate cells (HSC) during liver fibrogenesis has been shown to be mediated by paracrine and autocrine loops involving transforming growth factor-β1 (TGF-β1) as the main fibrogenic mediator secreted by activated macrophages, endothelial cells and liberated by disintegrated platelets. The cell-associated plasminogen activation system regulates extracellular matrix (ECM) catabolism and cell movement. We have studied whether TGF-β1 could modulate the plasminogen activation system in human HSC and the role of such protease system in the activity of TGF-β1 on HSC. Urokinase plasminogen activator receptors (u-PAR), u-PA and plasminogen activator inhibitor type 1 (PAI-1) were determined by immunoassay and RNase protection assay. Cell migration, evaluated either as chemotaxis or as chemoinvasion, was studied in Boyden chambers after addition of TGF-β1, and inhibition with anti-u-PA and anti-u-PAR antagonists [antibodies against u-PA and u-PAR and antisense oligonucleotides (aODN) against u-PAR mRNA]. We have shown that TGF-β1 is not mitogenic for HSC, while it is a powerful motogen either in chemotaxis or chemoinvasion assays. TGF-β1 up-regulates the synthesis and expression of PAI-1, as well as u-PAR expression and exposure at the cell membrane, while it does not affect u-PA levels. TGF-β1-dependent chemoinvasion of reconstituted basement membrane exploits the cell-associated plasminogen activation system, since it is blocked by monoclonal antibodies against u-PA and against various u-PAR domains, as well as by anti-u-PAR aODN. We have also observed a cumulative effect of TGF-β1, b-FGF and PDGF in the invasion assay of HSC: in the presence of low amounts of TGF-β1 the chemoinvasive activity of PDGF and bFGF is dramatically increased. Also this cooperation requires u-PAR and is inhibited by monoclonal antibodies against u-PAR domains I, II and III.

Vascular and cellular proteolytic activity in mice with α2-antiplasmin gene inactivation

Objective: To study the role of α2-antiplasmin (α2-AP), the main physiological plasmin inhibitor, in controlling vascular and cellular proteolytic activity.Materials: Arteries, organs and cell cultures derived from α2-AP-deficient (α2-AP–/–) mice or from their wild-type littermates (α2-AP+/+).Results: In serum-free conditioned medium of α2-AP+/+or α2-AP–/–skin fibroblasts, the time course (0–72 h) of PAI-1 antigen and of t-PA or u-PA antigen and activity production was similar. Activation of proMMP-9 (gelatinase B) upon addition of plasmin(ogen) to serum-free conditioned medium of fibroblasts was consistently detectable with α2-AP–/–but not with α2-AP+/+cells. In aorta and femoral arterial extracts of α2-AP+/+or α2-AP–/–mice, t-PA and u-PA activity levels were comparable, and fibrin zymography with cryosections did not reveal significant differences in fibrinolytic activity. In liver or kidney extracts of α2-AP+/+or α2-AP–/–mice, t-PA, u-PA, PAI-1 and plasminogen antigen levels were comparable; t-PA or u-PA activity was not detected in liver extracts and was present at comparable levels in kidney extracts.Activation of plasminogen to plasmin in solution by cell-associated plasminogen activator, and activation of cell-bound plasminogen by tcu-PA was comparable for fibroblasts of both genotypes.Conclusions: α2-AP does not play a crucial role in controlling vascular or cellular proteolytic activity in mice.

Tumour cell thrombospondin-1 regulates tumour cell adhesion and invasion through the urokinase plasminogen activator receptor.

We have previously shown that platelet-produced thrombospondin-1 up-regulates the urokinase plasminogen activator and its receptor and promotes tumour cell invasion. Although tumour cells produce thrombospondin-1 in vivo, they produce only minimal amounts of thrombospondin-1 in vitro. To determine the effect of tumour cell-produced thrombospondin-1 in the regulation of the plasminogen/plasmin system and tumour cell invasion, we studied THBS-1 -transfected MDA-MB-435 breast cancer cells that overexpress thrombospondin-1. The role of urokinase plasminogen receptor in thrombospondin-1-mediated adhesion and invasion was studied by antisense inhibition, enzymatic cleavage and antibody neutralization. Tumour cell adhesion to collagen and laminin was evaluated. Tumour cell invasion was studied in a modified Boyden chamber collagen invasion assay. Tumour cell thrombospondin-1 induced a 2–7 fold increase in urokinase plasminogen activator receptor and cell-associated urokinase plasminogen activator expression and a 50–65% increase in cell-associated urokinase plasminogen activator and plasmin activities. Furthermore, tumour cell thrombospondin-1 promoted tumour cell invasion and decreased tumour cell adhesion through up-regulation of urokinase plasminogen activator receptor-controlled urokinase plasminogen activator and plasmin activities. We conclude that tumour cell-produced thrombospondin-1 may play a critical role in the regulation of tumour cell adhesion and tumour cell invasion. © 2000 Cancer Research Campaign

Lysophosphatidylcholine induces urokinase-type plasminogen activator and its receptor in human macrophages partly through redox-sensitive pathway.

Urokinase-type plasminogen activator (uPA) and its cell surface receptor (uPAR) have been shown to be expressed in macrophages in atherosclerotic arterial walls, but the regulatory mechanisms of their expression remain unclear. The present study was performed to examine the effects of lysophosphatidylcholine (lysoPC), an important atherogenic lipid, on the expression of uPA and uPAR in human monocyte-derived macrophages. LysoPC upregulated the mRNA expression of uPA and uPAR, and it increased the protein expression of uPA in the culture medium and bound to the cell surface and of uPAR in the particulate fraction of the cells. LysoPC significantly increased the binding of the amino-terminal fragment of uPA to the treated cells and the cell-associated plasminogen activator activity. LysoPC stimulated superoxide anion production and increased intracellular oxidant levels in the cells. The combined incubation with reduced glutathione diethyl ester or N-acetylcysteine, antioxidants, suppressed the upregulation of uPA and uPAR mRNA and the increase in plasminogen activator activity by lysoPC. uPA and uPAR mRNA expression was also induced by the incubation with xanthine and xanthine oxidase, a superoxide anion-generating system. The results suggest that lysoPC increased the expression of uPA and uPAR and their functional activities in human monocyte-derived macrophages, at least in part through a redox-sensitive mechanism. This coordinate increase in the expression of uPA and uPAR in human macrophages by lysoPC could play an important role in plaque formation and disruption, arterial remodeling, and angiogenesis in atherosclerotic arterial walls.

Characterization of Cell-Associated Plasminogen Activation Catalyzed by Urokinase-Type Plasminogen Activator, but Independent of Urokinase Receptor (uPAR, CD87)

The 55-kD urokinase (uPA) receptor (uPAR, CD87) is capable of binding uPA and may be involved in regulating cell-associated plasminogen activation and pericellular proteolysis. While investigating the relationship between uPAR levels and plasmin generation, we found that uPA-catalyzed plasminogen activation is stimulated by cells which do not express uPAR. This uPAR-independent mechanism appears to be at least as effective in vitro as uPAR-dependent stimulation, such that stimulation on the order of 30-fold was observed, resulting from improvements in both apparent kcat and apparent Km. The mechanism depends on simultaneous binding of both uPA and plasminogen to the cell and requires the presence of the amino-terminal fragment (ATF), available in single chain and two chain high-molecular-weight uPA, but not low-molecular-weight uPA. Stimulation was observed in all leukemic cell lines investigated at similar optimum concentrations of 106to 107 cells/mL and may be more general. A mechanism is proposed whereby uPA can associate with binding sites on the cell surface of lower affinity, but higher capacity than uPAR, but these are sufficient to stimulate plasmin generation even at subphysiologic uPA concentrations. This mechanism is likely to operate under conditions commonly used for in vitro studies and may have some significance in vivo.

Characterization of Cell-Associated Plasminogen Activation Catalyzed by Urokinase-Type Plasminogen Activator, but Independent of Urokinase Receptor (uPAR, CD87)

AbstractThe 55-kD urokinase (uPA) receptor (uPAR, CD87) is capable of binding uPA and may be involved in regulating cell-associated plasminogen activation and pericellular proteolysis. While investigating the relationship between uPAR levels and plasmin generation, we found that uPA-catalyzed plasminogen activation is stimulated by cells which do not express uPAR. This uPAR-independent mechanism appears to be at least as effective in vitro as uPAR-dependent stimulation, such that stimulation on the order of 30-fold was observed, resulting from improvements in both apparent kcat and apparent Km. The mechanism depends on simultaneous binding of both uPA and plasminogen to the cell and requires the presence of the amino-terminal fragment (ATF), available in single chain and two chain high-molecular-weight uPA, but not low-molecular-weight uPA. Stimulation was observed in all leukemic cell lines investigated at similar optimum concentrations of 106to 107 cells/mL and may be more general. A mechanism is proposed whereby uPA can associate with binding sites on the cell surface of lower affinity, but higher capacity than uPAR, but these are sufficient to stimulate plasmin generation even at subphysiologic uPA concentrations. This mechanism is likely to operate under conditions commonly used for in vitro studies and may have some significance in vivo.

Role of Urokinase Plasminogen Activator Receptor in Thrombospondin 1-Mediated Tumor Cell Invasion

We previously showed that thrombospondin 1 (TSP-1) upregulates the plasminogen/plasmin system and promotes breast tumor cell invasion. Preliminary data from our laboratory using neutralizing antibodies suggested that the upregulation in breast tumor cell invasion seen in response to TSP-1 involved the urokinase plasminogen activator receptor (uPAR). To confirm these findings in MDA-MB-231 breast cancer cells, we developed three other strategies to study the role of uPAR in tumor cell adhesion and TSP-1-mediated tumor cell invasion: (a) enzymatic cleavage of uPAR with glycosylphosphatidylinositol-specific phospholipase C; (b) inhibition at the mRNA level with a uPAR antisense construct (cells named LKAS-MDA); (c) inhibition of plasminogen binding with the lysine analogue ϵ-aminocaproic acid. Adhesion to laminin and type I and type IV collagen with and without the addition of ϵ-aminocaproic acid was studied. Tumor cell invasion was studied in a modified Boyden chamber collagen invasion assay. Antisense uPAR inhibition decreased uPAR expression by 48–66% and cell-associated urokinase plasminogen activator (uPA) by 30–68%. Additionally, antisense uPAR inhibition induced a 68–70% reduction in uPA and plasmin activities. Antisense uPAR transfection increased tumor cell adhesion by 46–53%. A similar effect was observed in ϵ-aminocaproic acid-treated MDA-MB-231 cells. TSP-1-mediated tumor cell invasion was almost completely inhibited by either antisense uPAR inhibition or treatment with phospholipase C or ϵ-aminocaproic acid. We conclude that uPAR plays a crucial role in the regulation of tumor cell adhesion and TSP-1-mediated tumor cell invasion.

Modulation of Cell-associated Plasminogen Activation by Stromelysin-1 (MMP-3)

Stromelysin-1 (MMP-3) cleaves a 55 kDa kringle 1-4 fragment, containing the lysine-binding site(s) involved in cellular binding, from 92 kDa plasminogen and removes a 17 kDa NH2-terminal fragment, containing the cellular receptor-binding site, from 45 kDa urokinase (u-PA), but a potential role of MMP-3 in the regulation of cellular fibrinolytic activity by affecting binding and/or activation of plasminogen and/or single-chain u-PA has not been established. Human plasminogen (input concentration 100 nM for 4x10(6) cells per ml) was shown to bind specifically to human monocytoid THP-1 cells, to murine MMP-3 deficient smooth muscle cells (SMC) and fibroblasts (1.9, 0.92 and 1.0x10(6) molecules per cell, respectively). Treatment with MMP-3 (final concentration 0-50 nM) of cells saturated with bound plasminogen (about 25 nM), overnight at 37 degrees C, resulted in a dose-dependent reduction of the amount of u-PA activatable plasminogen (reduction to 25-40% of the value in the absence of MMP-3). Immunoblotting with specific monoclonal antibodies and autoradiography of eluates of the cells treated with MMP-3 revealed cleavage of plasminogen into the 55 kDa fragment and miniplasminogen (kringle 5 plus the proteinase domain). Binding of human single chain u-PA (scu-PA) to human THP-1 and HT 1080 cells amounted to 2.5x10(6) and 7.1x10(6) molecules per cell, respectively. Treatment with MMP-3 (final concentration 0-25 nM) of cell-bound u-PA (about 17 nM for THP-1 and 47 nM for HT1080 cells), overnight at 37 degrees C, did not alter cell-associated u-PA activity, measured in a direct chromogenic substrate assay or in a plasminogen-coupled chromogenic substrate assay (residual u-PA activity always > or =85% of that without MMP-3 treatment). Autoradiography of 125I-labeled u-PA moieties, removed from the cells by treatment with acid or with phosphatidylinositol phospholipase C, confirmed that u-PA remained essentially intact after MMP-3 treatment. These data indicate that MMP-3 may downregulate cell-associated plasmin activity by decreasing the amount of activatible plasminogen, without affecting cell-bound u-PA activity.

PAI-1 secretion and matrix deposition in human peritoneal mesothelial cell cultures: Transcriptional regulation by TGF-β1

Plasminogen activator inhibitor-1 (PAI-1), the main inhibitor of plasminogen activators in plasma and in peritoneum, impairs plasmin formation that is essential for the repair processes of the mesothelium damaged by peritoneal dialysis fluids and peritonitis. The fibrogenetic cytokine transforming growth factor-beta (TGF-beta) displays variable effects on extracellular matrix remodeling enzymes and their inhibitors depending on tissues and cell lines. We previously found an unexpected stimulating effect of TGF-beta 1 on matrix metalloproteinase-9 in peritoneal mesothelial cells. In this study, we analyzed the effects of TGF-beta 1 on PAI-1 production and deposition in extracellular matrix. We used primary cultured mesothelial cells and a recently established human peritoneal mesothelial cell line (HMrSV5). Cell-associated and secreted plasminogen activators and their inhibitors were detected and characterized by substrate gel zymography. PAI-1 was identified by reverse zymography and by Western blotting, and total PAI-1 was measured by ELISA. Secreted and cell-associated PA activity was measured by its ability to activate plasminogen into plasmin, that is, by the release of paranitroaniline from the plasmin synthetic substrate S-2251. PAI-1 mRNA accumulation was assessed by Northern blot. In vitro nuclear run-on assays were carried out to determine whether TGF-beta 1 had transcriptional effects on PAI-1 expression. Finally, the subcellular distribution of PAI-1 was analyzed by immunofluorescence and by immunogold silver staining. TGF-beta 1 increased PAI-1 antigen in the conditioned media of HMrSV5 cells, in a time- and concentration-dependent manner. This induced a dramatic decrease of free tPA in the cell medium and of membrane-bound uPA, and a parallel increase of high molecular weight PA-PAI complexes. Consequently, secreted and cell-associated plasminogen activator activities were considerably reduced. In primary cultured peritoneal mesothelial cells, TGF-beta 1 also induced PAI-1 secretion and the shift of tPA toward high molecular weight complexes. TGF-beta 1 increased PAI-1 mRNA in a time- and concentration-dependent manner. This effect was at least in part transcriptional since an approximately threefold increase in the rate of PAI-1 gene transcription was observed in nuclei sampled after a four-hour cell exposure to 5 ng/ml TGF-beta 1. Finally, TGF-beta 1 substantially increased the amount of intracellular and matrix-associated PAI-1. These results suggest that excessive TGF-beta 1 stimulated PAI-1 could prevent appropriate peritoneal healing by impairing the degradation of fibrin and of unorganized matrix components, and by interfering with cell migration.

Inhibition of angiogenesis on glycated collagen lattices.

Advanced glycation endproduct (AGE) accumulation in extracellular matrix proteins has been demonstrated in diabetic patients with a significant correlation with the severity of diabetic complications. AGE accumulation induces matrix protein cross-link formation, resulting in an increased stiffness of matrix fibres and the reduction of the susceptibility of matrix proteins to proteolytic degradation. We examined whether glycation-induced collagen cross-linking may affect vascular endothelial cell behaviours such as invasion, proliferation and differentiation, using the in vitro angiogenesis model of capillary-like structure formation in three-dimensional matrices of collagen type I. Endothelial cells cultured on collagen gel with angiogenic factors (the combination of fibroblast growth factor-2 and vascular endothelial growth factor) invaded the underlying collagen matrix, and organized capillary-like cord structures in the gel. We found that endothelial cell invasion into glycated collagen gel was significantly attenuated without any effect on proteinase activity including cell-associated plasminogen activator and matrix metalloproteinase in the conditioned medium. In addition, subsequent capillary-like cord formation was also inhibited in glycated collagen gel. In contrast, endothelial cell proliferation was enhanced on glycated collagen gel with or without angiogenic factors compared with control collagen gel. These results suggest that the structural alterations of extracellular matrix proteins through the glycation-induced cross-link formation affect the interaction between endothelial cell and extracellular matrix, resulting in the impairment of an adequate neovascularization in diabetic patients.

Role of Urokinase in the Fibrogenic Response of the Lung to Mineral Particles

The lung plasminogen activator (PA) response was examined in four different models of particle-induced pulmonary lesions in NMRI mice (single intratracheal administration, 0.75 to 5 mg/mouse). Sequential changes in cellular (total and differential counts) and biochemical markers of alveolitis (lactate dehydrogenase [LDH], total proteins) were monitored in bronchoalveolar fluid (BALF) and the fibrotic lung response was assessed histologically. An intense but spontaneously resolving alveolitis was produced by manganese dioxide (MnO2) and a fibrosing alveolitis was elicited by crystalline silica (DQ12). Minimal and noninflammatory responses were obtained after instillation of titanium dioxide (TiO2) and tungsten carbide (WC), respectively. The comparison between the resolving and the fibrosing alveolitis model was especially taken into consideration in an attempt to identify fibrinolytic changes associated with the development of fibrosis. At the alveolitis stage, similarly increased BALF PA activities were measured in both the resolving and the fibrosing alveolitis models whereas only slight and no PA modifications were noted after administration of TiO2 and WC, respectively. Persistently (up to 120 d) increased BALF PA activity was selectively associated with the progression to fibrosis (DQ12), suggesting that PA is involved in the fibrotic process. ELISA measurements demonstrated that the changes in BALF PA activity were exclusively related to changes in urokinase (uPA), not tissue-type PA. A rapid and persisting (up to Day 30) upregulation of cell-associated PA activity occurred after DQ12, MnO2, and TiO2 treatment only. Cellular PA activity was however significantly higher in fibrogenic inflammatory cells recovered from DQ12 than from MnO2-treated mice suggesting that the intensity of cellular PA upregulation may represent an early indicator of the progression to fibrosis. The implication of urokinase in the pathogenesis of silica-induced fibrosis was demonstrated by the use of a uPA knockout mice. The acceleration of the fibrotic process in uPA-deficient compared with the wild type animals demonstrated the contribution of uPA to limit the fibrotic process.

High Molecular Weight Kininogen Regulates Prekallikrein Assembly and Activation on Endothelial Cells: A Novel Mechanism for Contact Activation

AbstractThe consequences of assembling the contact system of proteins on the surface of vascular cells has received little study. We asked whether assembly of these proteins on the surface of cultured human endothelial cells (HUVECs) results in the activation of prekallikrein (PK) and its dependent pathways. Biotinylated PK binds specifically and reversibly to HUVECs in the presence of high molecular weight kininogen (HK) (apparent Kd of 23 ± 11 nmol/L,Bmax of 1.7 ± 0.5 × 107 sites per cell [mean ± SD, n = 5 experiments]). Cell-associated PK is rapidly converted to kallikrein. Surprisingly, the activation of cell-associated HK•PK complexes is entirely independent of exogenous factor XII (Km = 30 nmol/L,Vmax = 12 ± 3 pmol/L/min in the absencev Km = 20 nmol/L,Vmax = 9.2 ± 2.1 pmol/L/min in the presence of factor XII). Rather, kallikrein formation is mediated by an endothelial cell-associated, thiol protease. Cell-associated HK is proteolyzed during the course of prekallikrein activation, releasing kallikrein from the surface. Furthermore, activation of PK bound to HK on HUVECs promotes kallikrein-dependent activation of pro-urokinase, resulting in the formation of plasmin. These results indicate the existence of a previously undescribed, factor XII-independent pathway for contact factor activation on HUVECs that regulates the production of bradykinin and may contribute to cell-associated plasminogen activation in vivo.

High Molecular Weight Kininogen Regulates Prekallikrein Assembly and Activation on Endothelial Cells: A Novel Mechanism for Contact Activation

The consequences of assembling the contact system of proteins on the surface of vascular cells has received little study. We asked whether assembly of these proteins on the surface of cultured human endothelial cells (HUVECs) results in the activation of prekallikrein (PK) and its dependent pathways. Biotinylated PK binds specifically and reversibly to HUVECs in the presence of high molecular weight kininogen (HK) (apparent Kd of 23 +/- 11 nmol/L, Bmax of 1.7 +/- 0.5 x 10(7) sites per cell [mean +/- SD, n = 5 experiments]). Cell-associated PK is rapidly converted to kallikrein. Surprisingly, the activation of cell-associated HK.PK complexes is entirely independent of exogenous factor XII (Km = 30 nmol/L, Vmax = 12 +/- 3 pmol/L/min in the absence v Km = 20 nmol/L, Vmax = 9.2 +/- 2.1 pmol/L/min in the presence of factor XII). Rather, kallikrein formation is mediated by an endothelial cell-associated, thiol protease. Cell-associated HK is proteolyzed during the course of prekallikrein activation, releasing kallikrein from the surface. Furthermore, activation of PK bound to HK on HUVECs promotes kallikrein-dependent activation of pro-urokinase, resulting in the formation of plasmin. These results indicate the existence of a previously undescribed, factor XII-independent pathway for contact factor activation on HUVECs that regulates the production of bradykinin and may contribute to cell-associated plasminogen activation in vivo.

Structure, Function and Expression on Blood and Bone Marrow Cells of the Urokinase‐Type Plasminogen Activator Receptor, uPAR

Several important functions have been assigned to the receptor for urokinase-type plasminogen activator, uPAR. As implied by the name, uPAR was first identified as a high affinity cellular receptor for urokinase plasminogen activator (uPA). It mediates the binding of the zymogen, pro-uPA, to the plasma membrane where trace amounts of plasmin will initiate a series of events referred to as "reciprocal zymogen activation" where plasmin converts pro-uPA to the active enzyme, uPA, which in turn converts plasma membrane-associated plasminogen to plasmin. This is an efficient machinery to generate broad-spectrum proteolytic activity which is spatially restricted to the plasma membrane, since plasmin that diffuses away from the plasma membrane is rapidly inactivated by circulating inhibitors (i.e., alpha 2-antiplasmin). The system is controlled by a series of plasminogen activator inhibitors (PAIs), most importantly PAI-1 and PAI-2, providing means of temporally restricting the process of plasminogen activation. In addition to its role in plasminogen activation, compelling evidence has demonstrated a role for uPAR in cell-cell and cell-extracellular matrix adhesion, both directly and indirectly. uPAR is directly involved in binding to the extracellular matrix molecule, vitronectin, and the affinity of this binding is increased when uPAR is occupied by (pro-)uPA. A more indirect but presumably very important role of uPAR in cell adhesion seems to be mediated through interactions between uPAR and beta 1- or beta 2-integrins. It has been demonstrated that uPAR may bind physically to integrins in a reversible manner. The interaction seems to be of functional importance since the affinity of the integrin for its corresponding ligand is modulated by the association of integrin with uPAR. In some experimental setups uPAR has been shown to reduce the affinity of the associated integrin for certain ligands, while other experimental systems have demonstrated an increased affinity of the interaction between integrin and ligand after binding of uPAR to the integrin. Finally, uPAR has also been shown to participate in signal transduction events. Since uPAR is not a transmembrane molecule but belongs to the group of proteins that are tethered to the plasma membrane via a glycosyl-phosphatidylinositol anchor, association with a transmembrane adaptor is required for transmission of signals via uPAR. Integrins may serve as such signal transducers, and indeed uPAR has been shown to be associated in the plasma membrane with complexes of integrins and (phosphorylated) tyrosin kinases suggesting a role for these complexes in transmembrane transmission of signals via uPAR. In the hematopoietic system it has been shown that urokinase-type plasminogen activator (uPAR) is expressed as a differentiation antigen on cells of the myelomonocytic lineage and as an activation antigen on monocytes and T lymphocytes. Neutrophils contain intracellular reservoirs of uPAR that are translocated to the plasma membrane upon activation, and neutrophils from patients with the rare blood disease paroxysmal nocturnal hemoglobinuria (PNH) that fail to express glycosyl-phosphatidylinositol-anchored proteins including uPAR, show a very significantly reduced transmigration over an endothelial barrier. Cell-associated plasminogen activation by PNH-affected neutrophils is severely impaired, and it has been proposed that this may be causally related to the propensity for thrombosis in PNH. The pattern of expression of uPAR in hematological malignancies mirrors the expression by normal blood and bone marrow counterparts with some exceptions (differentiated myeloid leukemias are positive, undifferentiated myeloid may be negative and the majority of lymphoid leukemias and lymphomas are negative). The potential clinical relevance of uPAR expression in leukemias and lymphomas has not been determined.

Regulation of Single Chain Urokinase Binding, Internalization, and Degradation by a Plasminogen Activator Inhibitor 1-Derived Peptide

The internalization and degradation of cell-associated urokinase type plasminogen activator (uPA) through the alpha2-macroglobulin receptor/low density lipoprotein-related receptor (alpha2MR/LRP) represent important steps in the control of plasmin formation. Complexes between two chain urokinase (tcuPA) and plasminogen activator type 1 are degraded rapidly whereas single chain urokinase (scuPA) is not, suggesting that alpha2MR/LRP requires specific epitopes in the serpin for effective function. We report an alternative mechanism that may contribute to this process. The binding of scuPA to LM-TK- cells that lack the uPA receptor was stimulated by the hexapeptide EEIIMD, corresponding to amino acids 350-355 of plasminogen activator type 1, which contacts the sequence RHRGGS, corresponding to amino acids 179-184 in uPA. EEIIMD increased the Bmax of scuPA binding 4-fold with the half-maximal effect achieved at a peptide concentration of 50 microM. Stimulation was dependent on the charge on the COOH-terminal amino acid but not on the NH2 terminus of the peptide. EEIIMD also stimulated the internalization and degradation of scuPA. Both the binding and internalization of scuPA in the presence of EEIIMD were blocked by recombinant, 39-kDa alpha2MR/LRP-associated protein as well as by an anti-alpha2MR/LRP antibody. EEIIMD also stimulated the binding of scuPA to purified alpha2MR/LRP. EEIIMD had no effect on the binding of tcuPA or of complexes between scuPA and its receptor. These results suggest that EEIIMD regulates the binding of scuPA with alpha2MR/LRP. These findings also suggest a potential mechanism by which scuPA can be cleared which is independent of activation by plasmin or binding to uPA receptor.

Upregulation of urokinase-type plasminogen activator by endogenous and exogenous HIV-1 Tat protein in tumour cell lines derived from BK virus/tat-transgenic mice.

To demonstrate that Tat modulates the plasminogen-dependent proteolytic activity of tumour cell lines derived from BK virus (BKV)/tat-transgenic mice by affecting the production of plasminogen activators (PA) and the PA inhibitor (PAI)-1 and to demonstrate that this occurs through mechanism(s) that are distinct from those responsible for transactivating activity of extracellular Tat. To assess whether endogenous Tat is responsible for PA activity in T53 adenocarcinoma cells, cell cultures were transfected with antisense Tat cDNA and evaluated for cell-associated PA activity by a plasmin chromogenic assay. The assay was also used to evaluate PA activity in T53 cells and T111 leiomyosarcoma cells stimulated by extracellular Tat. The type(s) of PA produced were identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis zymography. The levels of PAI-1 were evaluated by Western blotting. Tat transactivating activity was measured by a chloramphenicol acetyltransferase (CAT) enzyme-linked immunosorbent assay in HL3T1 cells containing integrated copies of an HIV-1 long terminal repeat (LTR)-CAT plasmid. Transfection of T53 cells with antisense Tat cDNA results in the decrease of Tat production and PA activity. Exogenously added Tat increases PA levels in T53 and in T111 cells. PA activity was identified as urokinase-type PA (uPA). Tat also increases the production of PAI-1 in T111 but not in T53 cells. Chloroquine and heparin have different affects on the LTR-CAT-transactivating and the PA-inducing activities of Tat. The fusion protein glutathione-S-transferase-Tat and the mutant Tat-1e, lacking the second Tat exon, cause LTR-CAT transactivation without stimulating uPA upregulation. Tat affects the fibrinolytic activity of tumour cell lines derived from BKV/tat-transgenic mice by modulating the production of both uPA and PAI-1 via autocrine and paracrine mechanisms of action. The capacity of Tat to modulate the plasminogen-dependent proteolytic activity of these tumour cell lines may contribute to their metastatic potential. The uPA-inducing activity of Tat depends upon specific biological and structural features of the Tat protein that are distinct from those responsible for its LTR-CAT-transactivating activity, suggesting distinct mechanisms of induction for the two biological responses.

Phorbol-ester-stimulated human lymphoid cell lines produce a plasminogen activator modulator inducing cell-bound urokinase-type plasminogen activator in malignant tumor cell lines

The importance of cell-associated plasminogen activation in tumor invasion and metastasis is becoming increasingly evident. To clarify the modulators of cell-associated plasminogen activation in malignant states, we have recently established an assay system utilizing endogenous plasminogen activators on the cell surface. In the present study using the assay system, we found that the conditioned medium from phorbol 12-myristate 13-acetate (PMA)-stimulated human lymphoid cell lines, HUT 78 and Raji, strongly enhanced plasminogen activator (PA) activity on the surface of human malignant tumor cell lines (WI-38 VAI3 2RA, A431, A549 and HT-1080). The enhancing effect was inhibited by the addition of actinomycin D. By gel filtration, the active substances in PMA-stimulated HUT 78- and Raji-conditioned media were eluted in similar fractions corresponding to molecular weights of 60 to 80 kDa. The active substance was heat-labile. The enhanced PA activities were completely inhibited by anti-urokinase-type plasminogen activator (uPA) IgG. Moreover, the active substance was found to increase in cell-bound uPA antigen. These findings suggest that a population of activated lymphocytes produces a plasminogen activator modulator that induces uPA on the surface of malignant tumor cells.