DIFFERENCE IN EXPRESSION OF THE PLASMINOGEN ACTIVATION SYSTEM IN SYNOVIAL TISSUE OF PATIENTS WITH RHEUMATOID ARTHRITIS AND OSTEOARTHRITIS

Protcolytic joint destruction in inflammatory and non-inflammatoryarthropathy is believed to be mediated, at least in part, bythe plasminogen activation (PA) system. To further investigatepossible involvement of the PA system, we quantified immunoreactiveurokinase-type plasminogen activator (u-PA), tissue-type plasminogenactivator (t-PA), both plasminogen activator inhibitors (PAI-1and PAI-2) and u-PA-receptor (u-PAR) in synovial tissue extractsof 14 patients with rheumatoid arthritis (RA) and 12 with osteoarthritis(OA). u-PA, PAI-1, PAI-2 and u-PAR concentrations were significantlyhigher in RA than in OA patients. t-PA antigen levels were significantlylower in RA than in OA synovial tissue extracts. Immunohistochemistrywas performed to compare the distribution and staining intensityof these components in samples of RA and OA synovial tissue.Intense immunostaining of u-PA, u-PAR, PAI-1 and, to a lesserdegree, PAI-2 was observed predominantly in the synovial liningof RA patients. In OA patients, u-PA, PAI-1, PAI-2 and u-PARwere barely detectable. t-PA immunostaining was restricted tothe endothelial side of vascular walls in both groups. We concludethat the observed increase of u-PA, u-PAR and PAI expression,distributed mainly in the synovial lining area of proliferativeand invasively growing synovial tissue in RA patients, supportsa pathogenic role for the PA system in destructive arthritis.Depressed t-PA-mediated plasminogen activation might contributeto delayed intra-articular fibrin removal. KEY WORDS: Urokinase, Plasminogen activation, Immunohistochemistry, Rheumatoid arthritis, Osteoarthritis     

Comparison of the inhibitory effects of two types (90 kDa and 190 kDa) of hyaluronic acid on the expression of fibrinolytic factors in human synovial fibroblasts

 Hyaluronic acid (HA) has been shown to be clinically effective, and is currently used for the treatment of arthropathy. We previously reported that HA of molecular weight 90 kDa (90-HA) inhibits the fibrinolytic factors in human synovial fibroblasts. In the present study, we investigated the effect of high molecular weight (190 kDa) HA (190-HA) compared with 90-HA on the pericellular fibrinolytic system of human synovial fibroblasts in osteoarthritis (OA) and rheumatoid arthritis (RA). Human synovial fibroblasts were obtained from synovial tissues of OA and RA, and were cultured in the presence and absence of 90-HA and 190-HA. Antigens of urokinase-type plasminogen activator (u-PA) and PA inhibitor-1 (PAI-1) were measured by ELISA, and the u-PA activity of the cell surface fraction was evaluated by electrophoretic enzymography. The binding assay of u-PA and the immunocytochemical analysis of u-PA were performed to detect u-PA receptor (u-PAR). HA inhibited the secretion of both u-PA and PAI-1 antigens from the synovial fibroblasts of OA to their conditioned medium, and the degree of inhibition was more effective in OA than in RA. The u-RA binding assay to these cells showed that both 90-HA and 190-HA slightly decreased the maximal number of binding sites (Bmax) in OA. However, in RA, stimulation with 90-HA and 190-HA decreased Bmax by a half and a quarter, respectively. Immunohistochemical analysis showed that u-PAR was constitutively expressed in both synovial fibroblasts, but if these cells were treated with HA, the decrease in the staining of u-PAR was more pronounced in RA than in OA. Furthermore, the degree was more effective with 190-HA than with 90-HA. HA inhibited the pericellular fibrinolytic activity mediated by the u-PA/u-PAR system in synovial fibroblasts of OA and RA, and 190-HA inhibited it more effectively than 90-HA. Received: August 17, 2001 / Accepted: December 13, 2001     

Functions of the fibrinolytic system in human Ito cells and its control by basic fibroblast and platelet-derived growth factor

During liver fibrogenesis, hepatic stellate cells (HSC) proliferate and migrate under the influence of growth factors, including platelet-derived growth factor (PDGF) and basic-fibroblast growth factor (b-FGF). The plasminogen activation system regulates extracellular matrix (ECM) catabolism and cell movement. We evaluated the expression and biological functions of the plasminogen activation system in human HSC and its interaction with PDGF and b-FGF. Urokinase-plasminogen activator receptors (u-PAR) were measured by radioligand binding, cell cross-linking, immunoassay, and RNAse protection assay. u-PA and plasminogen activator inhibitors (PAIs) expression and activities were analyzed by zymography, immunoassay, and RNase protection assay. Cell migration and proliferation, studied in Boyden chambers and by microscopic counting, were evaluated after the addition of PDGF, b-FGF, and blockade with anti-u-PA, anti-u-PAR antibodies, and antisense oligodeoxynucleotides (aODN) against u-PAR mRNA. We have shown that HSC produce u-PAR, u-PA, and PAI-1. PDGF and b-FGF up-regulate u-PA and u-PAR, but not PAI-1, and exogenous addition of u-PA stimulates HSC proliferation, chemotaxis, and chemoinvasion. Inhibition of u-PA/u-PAR with antibodies against u-PA or u-PAR and with u-PAR aODN inhibit the proliferative, chemotactic, and chemoinvasive activity of PDGF and b-FGF. These findings indicate that u-PA and u-PAR are required for the mitogenic and chemoinvasive activity of PDGF and b-FGF on HSC.     

Interleukin-1beta regulates urokinase plasminogen activator (u-PA), u-PA receptor, soluble u-PA receptor, and plasminogen activator inhibitor-1 messenger ribonucleic acid expression in cultured human endometrial stromal cells

The interleukin-1 (IL-1) system plays an integral role in local intercellular interactions during implantation. In addition, the plasminogen activator system, especially urokinase plasminogen activator (u-PA), plasminogen activator inhibitor (PAI-1), and u-PA receptor (u-PAR), are crucial during embryo implantation. Decidualization and implantation are complex processes dependent upon several proteases, including u-PA, and IL-1 is known to affect PA activity in several cell types. We investigated the role of IL-1beta in regulating u-PA, PAI-1, u-PAR, and soluble u-PAR messenger ribonucleic acid (mRNA) expression in cultured human endometrial stromal cells using quantitative competitive PCR. For confirmation of the mRNA data, we measured PAI-1 and u-PAR protein by enzyme-linked immunosorbent assay. Confluent stromal cell cultures treated with progesterone and estradiol for 9 days were stimulated with IL-1beta, and IL-1beta plus IL-1beta antibody for an additional 24 h. Total RNA was extracted, reverse transcribed, and coamplified using quantitative and competitive PCR with internal standards. IL-1beta increased PAI-1, u-PAR, and soluble u-PAR expression in a dose-dependent manner, and this result was reversed by anti-IL-1beta antibody treatment. u-PA mRNA expression was not dependent on IL-1beta. These results suggest that IL-1 may be important in regulating PAI-1 and u-PAR during stromal cell decidualization before implantation.     

Antithrombotic regulation in human endothelial cells by fibrinolytic factors

Vascular endothelial cells (ECs) modulate the blood fibrinolytic system by secreting tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and their inhibitor, type-1 plasminogen activator inhibitor (PAI-1). ECs also express t-PA receptors (t-PAR) and u-PA receptors (u-PAR) on their cell surfaces, assembling both enzymes to regulate the cellular fibrinolytic activity. In addition, ECs modulate these factors in response to several stimuli. Fibrin clots on ECs induce the up- and downregulation of t-PA and PAI-1 production, respectively, thus causing an effective lysis of the fibrin clot. Heat shock (43 degrees C) increases the expression of u-PA, t-PA, PAI-1, and u-PAR by which ECs become more fibrinolytic around the cells. Furthermore, because ECs possess t-PAR and u-PAR on their cell surfaces, the binding of t-PA and u-PA is a critical event, which affords ECs the localized and condensed fibrinolytic potential. Therefore, ECs play a central role in antithrombotic activity by regulating the levels of these fibrinolytic factors.     

Evidence for a novel binding protein to urokinase-type plasminogen activator in platelet membranes

Endogenous urokinase-type plasminogen activator (u-PA) has been identified in platelet membrane, and platelets have been shown to take up exogenous high molecular weight u-PA from the ambient medium. In this report, the mechanism of the association of u-PA with platelets was investigated using recombinant, single chain u-PA. When gel filtered human platelets were incubated with radiolabeled u-PA, the u- PA was found to specifically and saturably bind to the resting platelets in a dose-dependent manner. Unlabeled u-PA and the amino terminal fragment of u-PA inhibited 125I-u-PA binding to platelets with a mean IC50 of 65 and 58 nmol/L, respectively. A single saturable binding site in intact resting platelets was found with a mean kd of 43 +/- 25 nmol/L and 2263 +/- 809 sites per platelet. In contrast to resting platelets, 125I-u-PA did not bind to thrombin-induced platelets. Western blotting studies, using a monoclonal or a polyclonal antibody specific for the u-PA cell-surface receptor (u- PAR), failed to show evidence of u-PAR in resting platelets, whereas, u-PAR was found at approximately 54 and approximately 48 kD on U937 monocytes, which served as a positive control. Ligand blotting of platelet membrane and of U937 cell proteins with 125I-u-PA revealed a u-PA binding protein of approximately 70 kD in the platelets and one of approximately 54 kD in the U937 cells. Complexion of u-PA with a platelet membrane protein was also shown by gel filtration of a mixture of u-PA and platelet membrane proteins. A u-PA complex was further shown by enzyme-linked immunosorbent assay when microtiter plates were coated with platelet membrane proteins, and this complex formation was shown to be dose-dependent and saturable with an apparent kd of 17 nmol/L. It was concluded that platelet membrane contains a specific, high affinity u-PA-binding protein that is distinct from u-PAR.     

Role of urokinase and its receptor in basal and stimulated colonic epithelial cell migration in vitro

BACKGROUND: Migration of colonic epithelial cells is important for mucosal repair following injury. The urokinase (u-PA) system regulates migration in other cell types. AIM: To examine the role of u-PA and its receptor (u-PAR) in colonic epithelial cell migration. METHODS: Migration was assessed over 24 hours in circular wounds made in confluent monolayers of LIM1215 and Caco-2 human colon cancer cells. The function of u-PA and u-PAR was ablated with antisense oligonucleotides to block expression, with synthetic u-PA peptides to block interaction, and with aprotinin to block u-PA mediated proteolysis. RESULTS: Migration was stimulated two to threefold by exogenous u-PA, an effect dependent on u-PAR binding but independent of u-PA mediated mitogenesis and proteolysis. Expression of u-PA and u-PAR was inhibited by 80% by the appropriate antisense oligonucleotide. Basal migration and the motogenic effects of butyrate, epidermal growth factor, and phorbol-12-myristate-13-acetate were suppressed by the u-PAR antisense oligonucleotide (40-60%) but were at best minimally affected following inhibition of u-PA expression and binding. CONCLUSIONS: In an in vitro model of wounded colonic epithelium, u-PAR promotes cell migration through mechanisms that are not exclusively dependent on u-PA binding. Therefore, u-PA and u-PAR may contribute to colonic mucosal repair in vivo.     

Plasminogen activators in synovial fluid and plasma from patients with arthritis.

The activity of plasminogen activators and inhibitors in the synovial fluid and plasma of patients with various forms of chronic arthritis was characterised. Tissue-type plasminogen activator antigen (t-PA:Ag), urokinase-type plasminogen activator antigen (u-PA:Ag), the proenzyme single chain u-PA (scu-PA), and plasminogen activator inhibitor (PAI) were measured in the synovial fluid and plasma of 22 patients with seropositive rheumatoid arthritis (RA), 13 with seronegative RA, and 23 patients with various forms of arthritis. In all patient groups the levels of t-PA:Ag in synovial fluid were lower and the levels of u-PA:Ag and PAI higher than plasma levels. Synovial fluid u-PA was more activated than plasma u-PA. Comparison of the patient groups showed that the largest differences between fibrinolytic parameters in synovial fluid and plasma were present in patients with seropositive RA followed by patients with seronegative RA and patients with various forms of arthritis. This order paralleled the functional and radiological scores of joint destruction in the patient groups studied. The results of this study indicate that suppression of t-PA production and enhancement of u-PA synthesis and activation in arthritic joints are associated with the clinical severity of arthritis.     

丁酸对人结肠癌细胞尿激酶型纤溶酶原激活剂及受体表达的影响

目的探讨丁酸对人结肠癌细胞尿激酶型纤溶酶原激活剂(u-PA)及其受体(u-PAR)表达的影响。方法将人传代结肠癌细胞株SW1116接种于不含或含不同浓度丁酸(2、3、4、7、10mmol／L)的培养基中，培养6、24、48、72h后，收集细胞用ELISA法测定胞质中u-PA和u-PAR的表达情况。结果随着培养基中丁酸浓度的增加和培养时间的延长，经丁酸处理的SW1116细胞表达的u-PA和u-PAR与对照组相比显著减少，当丁酸浓度≥4mmol／L和培养时问≥48h时，两者表达不再增加，反而呈逐步下降趋势。结论丁酸可抑制SW1116细胞u-PA和u-PAR表达。     

Src-dependence and pertussis-toxin sensitivity of urokinase receptor-dependent chemotaxis and cytoskeleton reorganization in rat smooth muscle cells.

The catalytically inactive precursor of urokinase-type plasminogen activator (pro-u-PA) induced a chemotactic response in rat smooth muscle cells (RSMC) through binding to the membrane receptor of urokinase (u-PA receptor [u-PAR]). A soluble form of u-PAR activated by chymotrypsin cleavage as well as a peptide located between domain 1 and 2 of u-PAR reproduced the effect of pro-u-PA on cell migration. The chemotactic pro-u-PA effect correlates with a dramatic reorganization of actin cytoskeleton, of adhesion plaques, and with major cell shape changes in RSMC. Pro-u-PA induced a decrease in stress fiber content, membrane ruffling, actin ring formation, and disruption leading to the characteristic elongated cell shape of motile cells with an actin semi-ring located close to the leading edge of cells. u-PAR effects on both chemotaxis and cytoskeleton were sensitive to pertussis toxin and, hence, possibly require G proteins. u-PAR effects are accompanied by a relocation of u-PAR, vitronectin receptor (VNR) alphavbeta3, beta1 integrin subunit, and Src tyrosine kinase to the leading membrane of migrating cells. In conclusion, our data show that pro-u-PA, via binding to u-PAR, controls a signaling pathway, regulated by tyrosine kinases and possibly G proteins, leading to cell cytoskeleton reorganization and cell migration.     

Involvement of plasminogen activator and plasminogen activator inhibitor type 1 in spermatogenesis, sperm capacitation, and fertilization

Increasing evidence suggests that local fibrinolysis generated by plasminogen activator (PA) and modulated by plasminogen activator inhibitor type 1 (PAI-1) is essential for mammalian spermatogenesis, sperm capacitation, and fertilization. Tissue-type PA (t-PA), urokinase-type PA (u-PA), and PAI-1 have been reported in the testes of various animals. Sertoli cells within the seminiferous epithelium are believed to play a central role in the control and maintenance of spermatogenesis by producing regulatory factors, including PA/PAI-1. Fertilization is a unique and exquisitely choreographed cellular interaction between male and female gametes, in which some basic biochemical mechanisms remain unresolved. A key issue is the molecular basis of sperm-egg recognition, binding, and penetration. Sperm capacitation and the acrosomal reaction appear to rely on local fibrinolysis generated by the PA/PAI-1 system. Ejaculated spermatozoa from various species carry u-PA activity. The u-PA receptor (uPAR) and the inhibitor PAI-1 have also been reported to bind on the sperm membrane surface. Thus, it is possible that uPAR and PAI-1 function in a counterbalanced and coordinated way on the surface of spermatozoa to regulate the u-PA binding capacity. This review summarizes evidence for the involvement of PA/PAI-1 system in spermatogenesis, sperm capacitation, and fertilization.     

恶性肿瘤止凝血分子标志物转录的临床研究

目的　检测胃肠道恶性肿瘤止凝血分子标志物血浆含量及mRNA水平 ,探讨其与肿瘤浸润、播散的关系及检测的临床意义。方法　采用逆转录实时定量PCR技术检测 2 9例胃癌、2 3例肠癌患者组织中组织因子 (TF)、组织型纤溶酶原激活剂 (t PA)、尿激酶型纤溶酶原激活剂 (u PA)mRNA水平 ;用ELISA法同步检测患者血浆止凝血分子标志物含量 ,包括TF、凝血酶抗凝血酶复合物 (TAT)、t PA、u PA、u PA受体 (u PAR)、纤溶酶 抗纤溶酶复合物 (PAP)等。结果　术前恶性肿瘤组血浆TF、TAT、u PA、u PAR、PAP含量均较正常对照明显升高 (P <0 0 5 ) ,其中 ,有局部浸润、淋巴结肿大、远处脏器转移者u PA、u PAR升高更为显著 (P <0 0 1) ;TF、u PAmRNA在肿瘤细胞表达显著增高 (P <0 0 1) ,而t PAmRNA(P >0 0 5 )则减少。结论　凝血、纤溶功能亢进是胃肠道恶性肿瘤细胞易播散、浸润的主要原因之一。t PAmRNA的表达可能为组织分化较好的特征。逆转录实时定量PCR技术 ,使TF、u PAmRNA有望作为胃肠道恶性肿瘤病情监测指标而用于临床     

The effects of serine proteinase inhibitors on bone resorption in vitro

The aims of this study were to identify the role and sites of action of serine proteinases (SPs) in bone resorption, a process which involves a cascade of events, the central step of which is the removal of bone matrix by osteoclasts (OCs). This resorbing activity, however, is also determined by recruitment of new OCs to future resorption sites and removal of the osteoid layer by osteoblasts (OBs), which enables OCs to gain access to the underlying mineralized bone. The resorption systems we have studied consisted of (i) neonatal calvarial explants, (ii) isolated OCs cultured on ivory slices, (iii) mouse OBs cultured on either radiolabelled type I collagen films or bone-like matrix, (iv) bone marrow cultures to assess OC formation and (v) 17-day-old fetal mouse metatarsal bone rudiments to assess OC migration and fusion. Two separate SP inhibitors, aprotinin and alpha(2)-antiplasmin dose-dependently inhibited (45)Ca release from neonatal calvarial explants: aprotinin (10(-6) M) was the most effective SP inhibitor, producing a maximum inhibitory effect of 55.9%.Neither of the SP inhibitors influenced either OC formation or OC resorptive activity. In contrast, each SP inhibitor dose-dependently inhibited OB-mediated degradation of both type I collagen fibrils and non-mineralized bone matrix. In 17-day-old metatarsal explants aprotinin produced a 55% reduction in the migration of OCs from the periosteum to the mineralized matrix after 3 days in culture but after 6 days in culture aprotinin was without effect on OC migration. Primary mouse osteoblasts expressed mRNA for urokinase type plasminogen activator (uPA), tIssue type plasminogen activator (tPA), the type I receptor for uPA, plasminogen activator inhibitor types I and II and the broad spectrum serine proteinase inhibitor, protease nexin I. In situ hybridization demonstrated expression of tPA and uPA in osteoclasts disaggregated from 6-day-old mouse long bones. We propose that the regulation of these various enzyme systems within bone tIssue determines the sites where bone resorption will be initiated.     

Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of a cell surface-targeted plasmin inhibitor

OBJECTIVE: Joint destruction in rheumatoid arthritis (RA) is a result of degradation and invasion of the articular cartilage by the pannus tissue. The present study was undertaken to examine the role of the plasminogen activation system in cartilage degradation and invasion by synovial fibroblasts and investigate a novel gene therapeutic approach using a cell surface-targeted plasmin inhibitor (ATF.BPTI). METHODS: Adenoviral vectors were used for gene transfer. The effects of ATF.BPTI gene transfer on RA synovial fibroblast-dependent cartilage degradation were studied in vitro, and cartilage invasion was studied in vivo in the SCID mouse coimplantation model. RESULTS: The results indicate that cartilage matrix degradation by rheumatoid synovial fibroblasts is plasmin mediated and depends on urokinase-type plasminogen activator for activation. Targeting plasmin inhibition to the cell surface of the fibroblasts by gene transfer of a cell surface-binding plasmin inhibitor resulted in a significant reduction of cartilage matrix degradation in vitro and of cartilage invasion in vivo. Compared with uninfected rheumatoid synovial fibroblasts, the mean +/-SEM cartilage degradation in vitro was reduced to 87.9+/-0.9% after LacZ gene transfer versus a reduction to 24.0+/-1.6% after ATF.BPTI gene transfer (P<0.0001). The mean +/- SEM in vivo cartilage invasion score was 3.1+/-0.4 in the control-transduced fibroblasts and 1.8+/-0.4 in the ATF.BPTI-transduced fibroblasts (P<0.05). CONCLUSION: These results indicate a role of the plasminogen activation system in synovial fibroblast-dependent cartilage degradation and invasion in RA, and demonstrate an effective way to inhibit this by gene transfer of a cell surface-targeted plasmin inhibitor.     

Urokinase-mediated fibrinolysis in the synovial fluid of rheumatoid arthritis patients may be affected by the inactivation of single chain urokinase type plasminogen activator by thrombin

BACKGROUND: Excessive fibrin deposition within the inflamed joints of rheumatoid arthritis (RA) patients suggests that local fibrinolysis is inefficient, which seems to be in contrast with the observed increased levels of urokinase type plasminogen activator (u-PA). Thrombin-mediated inactivation of single chain u-PA (scu-PA) into an inactive form called thrombin-cleaved two chain u-PA (tcu-PA/T) may provide a possible explanation for this contradiction. AIM: To assess the occurrence of tcu-PA/T in the synovial fluid of patients with RA and with osteoarthritis (OA), and in the synovial fluid of controls to find support for thrombin-mediated inactivation of scu-PA in RA. METHODS: Levels of scu-PA and tcu-PA/T were measured in the synovial fluid of 20 RA patients, nine OA patients and 14 controls using sensitive bioimmunoassays. Total urokinase antigen was quantified by a urokinase ELISA. RESULTS: tcu-PA/T was found in the synovial fluid of all RA and OA patients. Only in seven of 14 control samples, levels of tcu-PA/T could be measured above the detection limit of the assay (0.2 ng/ml). The concentrations of tcu-PA/T, scu-PA and u-PA:Ag were significantly higher in the synovial fluid of the RA and OA patients as compared with the controls, while the RA patients had significantly higher levels of tcu-PA/T and u-PA:Ag than the OA patients. In RA, tcu-PA/T seemed to account for more than 40% of total urokinase antigen, while the contribution of tcu-PA/T to total urokinase antigen was only minor in OA and the controls (9.0% and 6.6%, respectively). CONCLUSION: A significant part of the high total urokinase antigen in the synovial fluid of RA patients can be attributed to tcu-PA/T, implying that a large amount of scu-PA is not available for fibrinolysis because of its inactivation by thrombin. Thus, thrombin may promote the inflammation process in RA by inhibiting the fibrinolytic system and preventing the removal of fibrin.     

Fluorescent probes as tools to assess the receptor for the urokinase-type plasminogen activator on tumor cells.

Flow cytofluorometric protocols (FACScan) are described for the rapid and quantitative real-time analysis of binding of FITC-pro-u-PA to cell surface receptors (u-PAR) on living, resting, and also on PMA-stimulated human monocytic U937 cells. Binding of pro-u-PA was visualized by CLSM. This fairly new technique is superior over conventional fluorescence microscopy and is an alternative to electron microscopic approaches. Both flow cytofluorometry and confocal laser scanning microscopy allow the analysis quantitatively and with high-sensitivity binding of FITC-pro-u-PA to single suspended or adherent cells. By CLSM u-PA/u-PAR were found to be located in heterogeneously distributed discrete patches at the cell surface on U937 and not inside the cell. This is in agreement with previous studies by Hansen et al, who applied radioiodinated u-PA and electron microscopy to locate u-PAR on microvilli of fixed U937 cells. By flow cytofluorometry, it was possible to quantify the time-dependent and temperature-dependent binding of FITC-pro-u-PA to living single U937. Apparent saturation of u-PAR was achieved at 5 nM FITC-pro-u-PA for both nonstimulated and PMA-stimulated U937 cells. Half saturation of u-PAR was also determined. Nonstimulated U937 was 0.7 nM, and PMA-stimulated U937 was 1.1 nM of FITC-pro-u-PA. This increase in half-saturation concentration in PMA-stimulated cells is paralleled by a steep increase in binding sites (3.6-fold). The use of fluoresceinated reference beads is recommended to verify changes in affinity and binding sites. Using CLSM or flow cytofluorometry, it is also possible to study the structure relationship of u-PA/u-PAR in the presence of competitive binding analogues or inhibitors. Fluorescence techniques will also permit the identification of u-PAR-positive cells in blood, ascitic fluid, or biopsies obtained from cancer patients.