Localization of Urokinase Type Plasminogen Activator to Focal Adhesions Requires Ligation of Vitronectin Integrin Receptors

We have recently shown that urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 are both found extracellularly beneath cultured human skin fibroblasts and HT-1080 sarcoma cells, but in distinct localizations. Here, the ultrastructural distribution of u-PA was studied using immunoferritin electron microscopy. In HT-1080 cells, u-PA on the extracellular aspect of the plasma membrane was detected at sites of direct contact of the cell with the growth substratum beneath all parts of the ventral cell surface. The ferritin-labeled adhesion plaques, which were enriched in submembraneous microfilaments, were frequently seen at the leading lamellae of the cells as well as in lamellipodia and microspikes. Besides the cell-substratum adhesion plaques, ferritin label was detected at cell-cell contact sites. Double-label immunofluorescence showed a striking colocalization of u-PA and vinculin in both HT-1080 cells and WI-38 lung fibroblasts, which is consistent with u-PA being a focal contact component. The u-PA-containing focal contacts of WI-38 cells had no direct codistribution with fibronectin fibrils. In WI-38 cells made stationary by cultivation in a medium containing 0.5% FCS, vinculin plaques became highly elongated and more centrally located, whereas u-PA immunolabel disappeared from such focal adhesions. These findings show that plasma membrane-associated u-PA is an intrinsic component of focal contacts, where, we propose, it enables directional proteolysis for cell migration and invasion.

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Urokinase type plasminogen activator and inhibitor type-1 plasma levels in colorectal cancer

Gastroenterology ◽

10.1016/s0016-5085(01)82981-4 ◽

2001 ◽

Vol 120 (5) ◽

pp. A599-A600 ◽

Cited By ~ 1

Author(s):

L HERSZENYI ◽

F FARINATI ◽

G ISTVAN ◽

M PAOLI ◽

G ROVERONI ◽

...

Keyword(s):

Colorectal Cancer ◽

Plasminogen Activator ◽

Plasma Levels ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Inhibitor Type

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Urokinase-type plasminogen activator receptor

AfCS-Nature Molecule Pages ◽

10.1038/mp.a004089.01 ◽

2009 ◽

Author(s):

Nicolai Sidenius ◽

Francesco Blasi

Keyword(s):

Plasminogen Activator ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Plasminogen Activator Receptor

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Expression and Characterization of Clustered Charge-to-Alanine Mutants of Low Mr Single-Chain Urokinase-Type Plasminogen Activator

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642397 ◽

1994 ◽

Vol 71 (01) ◽

pp. 134-140 ◽

Cited By ~ 6

Author(s):

S Ueshima ◽

P Holvoet ◽

H R Lijnen ◽

L Nelles ◽

V Seghers ◽

...

Keyword(s):

Plasminogen Activator ◽

Solvent Accessibility ◽

Site Directed Mutagenesis ◽

Clot Lysis ◽

Wild Type ◽

Single Chain ◽

Charged Amino Acids ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Pharmacokinetic Properties

SummaryIn an effort to modify the fibrinolytic and/or pharmacokinetic properties of recombinant low M r single-chain urokinase-type plasminogen activator (rscu-PA-32k), mutants were prepared by site-directed mutagenesis of clusters of charged amino acids with the highest solvent accessibility. The following mutants of rscu-PA-32k were prepared: LUK-2 (Lys 212, Glu 213 and Asp 214 to Ala), LUK-3 (Lys 243 and Asp 244 to Ala), LUK-4 (Arg 262, Lys 264, Glu 265 and Arg 267 to Ala), LUK-5 (Lys 300, Glu 301 and Asp 305 to Ala) and LUK-6 (Arg 400, Lys 404, Glu 405 and Glu 406 to Ala).The rscu-PA 32k moictic3 were expressed in High Five Ttichoplasiani cells, and purified to humugciicily from the conditioned cell culture medium, with recoveries of 0.8 to 3.7 mg/1. The specific fibrinolytic activities (220,000 to 300,000 IU/mg), the rates of plasminogen activation by the single-chain moieties and the rates of conversion In lwo chain moieties by plasmin were comparable for mutant and wild-type rscu PA 32k moieties, with the exception of LUK-5 which was virtually inactive. Equi-effective lysis (50% in 2 h) of 60 pi 125I-fibrin labeled plasma clots submerged in 0.5 ml normal human plasma was obtained with 0.7 to 0.8 μg/ml of wild-type or mutant rscu-PA-3?.k, except with LUK-5 (no significant lysis with 16 pg/ml). Following bolus injection in hamsters, all rscu-PA-32k moieties had a comparably rapid plasma clearance (1.3 to 2.7 ml/min), as a result of a short initial half-life (1.4 to 2.5 min). In hamsters with pulmonary embolism, continuous intravenous infusion over 60 min at a dose of 1 mg/kg, resulted in 53 to 72% clot lysis with the mutants, but only 23% with LUK-5, as compared to 36% for wild-type rscu-PA-32k.These data indicate that clustered charge-to-alanine mutants of rscu-PA-32k, designed to eliminate charged regions with the highest solvent accessibility, do not have significantly improved functional, fibrinolytic or pharmacokinetic properties.

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Inhibition in Purified Systems and in Human Plasma of Chimaeric Plasminogen Activators Consisting of the NH2-Terminal Region of Tissue-Type Plasminogen Activator and the COOH-Terminal Region of UrokinaseType Plasminogen Activator

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647039 ◽

1988 ◽

Vol 60 (02) ◽

pp. 247-250 ◽

Cited By ~ 1

Author(s):

H R Lijnen ◽

L Nelles ◽

B Van Hoef ◽

F De Cock ◽

D Collen

Keyword(s):

Plasminogen Activator ◽

Rate Constants ◽

Plasminogen Activators ◽

Second Order ◽

Tissue Type ◽

Single Chain ◽

Chain Molecules ◽

Order Rate ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator

SummaryRecombinant chimaeric molecules between tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator (scu-PA) or two chain urokinase-type plasminogen activator (tcu-PA) have intact enzymatic properties of scu-PA or tcu-PA towards natural and synthetic substrates (Nelles et al., J Biol Chem 1987; 262: 10855-10862). In the present study, we have compared the reactivity with inhibitors of both the single chain and two chain variants of recombinant u-PA and two recombinant chimaeric molecules between t-PA and scu-PA (t-PA/u-PA-s: amino acids 1-263 of t-PA and 144-411 of u-PA; t-PA/u-PA-e: amino acids 1-274 of t-PA and 138-411 of u-PA). Incubation with human plasma in the absence of a fibrin clot for 3 h at 37° C at equipotent concentrations (50% clot lysis in 2 h), resulted in significant fibrinogen breakdown (to about 40% of the normal value) for all two chain molecules, but not for their single chain counterparts. Preincubation of the plasminogen activators with plasma for 3 h at 37° C, resulted in complete inhibition of the fibrinolytic potency of the two chain molecules but did not alter the potency of the single chain molecules. Inhibition of the two chain molecules occurred with a t½ of approximately 45 min. The two chain variants were inhibited by the synthetic urokinase inhibitor Glu-Gly-Arg-CH2CCl with apparent second-order rate constants of 8,000-10,000 M−1s−1, by purified α2-antiplasmin with second-order rate constants of about 300 M−1s−1, and by plasminogen activator inhibitor-1 (PAI-1) with second-order rate constants of approximately 2 × 107 M−1s−1.It is concluded that the reactivity of single chain and two chain forms of t-PA/u-PA chimaers with inhibitors is very similar to that of the single and two chain forms of intact u-PA.

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Physical Exercise Induces Enhancement of Urokinase-Type Plasminogen Activator (u-PA) Levels in Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647459 ◽

1991 ◽

Vol 65 (01) ◽

pp. 082-086 ◽

Cited By ~ 24

Author(s):

G Dooijewaard ◽

A de Boer ◽

P N C Turion ◽

A F Cohen ◽

D D Breimer ◽

...

Keyword(s):

Physical Exercise ◽

Plasminogen Activator ◽

Maximal Exercise ◽

Normal Values ◽

Bicycle Ergometer ◽

Tissue Type ◽

Healthy Male ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Fibrinolytic Potential

SummaryThe enhancement of the blood fibrinolytic potential by physical exercise is generally attributed to the release of tissue-type plasminogen activator (t-PA) from the vessel wall. In this study we have investigated the possible contribution of urokinase-type plasminogen activator (u-PA).Six healthy male volunteers (age 21–25 years) were screened for their ability to perform maximal exercise for their age-group for 12 min on a bicycle ergometer. Subsequently, on one occasion they were required to remain supine for 2 h (from 8.30 a. m. onwards) and on another they performed maximal exercise (from 9.00 a.m. onwards). During exercise an increase in u-PA antigen and plasmin-activatable pro-urokinase (proUK) activity, concurrent with t-PA antigen and euglobulin t-PA activity, was observed in all six volunteers, while at rest these parameters remained unaffected. Mean u-PA- and t-PA antigen increased, respectively, from 4.2 ± 1.0 ng/ml and 5.8 ± 2.1 ng/ml before exercise to 9.8 ± 3.0 ng/ml and 18.3 ± 3.8 ng/ml (peak). Mean plasminactivatable proUK activity and t-PA activity increased, respectively, from 2.1 ± 0.4 ng/ml and 0.3 ± 0.2 ng/ml before exercise to 4.3 ± 1.7 ng/ml and 7.2 ± 4.0 ng/ml (peak). The increases were statistically significant throughout (paired t-test, pre vs post, antigen P <0.005 and activity P <0.02). After cessation of exercise u-PA and t-PA declined concurrently to normal values with a 50"/" decay in about 5 min. In conclusion, we found that both u-PA antigen and plasmin-activatable proUK activity are, concurrently with t-PA, enhanced upon exercise and, therefore, we consider that u-PA also contributes to – and co-operates in – the enhancement of the blood fibrinolytic potential and activity under these conditions.

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An Enzyme-Linked Immunosorbent Assay for Urokinase-Type Plasminogen Activator (u-PA) and Mutants and Chimeras Containing the Serine Protease Domain of u-PA

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648387 ◽

1992 ◽

Vol 67 (01) ◽

pp. 095-100 ◽

Cited By ~ 18

Author(s):

Paul J Declerck ◽

Leen Van Keer ◽

Maria Verstreken ◽

Désiré Collen

Keyword(s):

Serine Protease ◽

Plasminogen Activator ◽

Active Site ◽

Enzyme Linked Immunosorbent Assay ◽

Single Chain ◽

Protease Domain ◽

Serine Protease Domain ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Normal Human

SummaryAn enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 ± 0.6 ng/ml (mean ± SD, n = 27).The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising A A 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with α2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity. When purified scu-PA-32k was added to pooled normal human plasma at final concentrations ranging from 20 to 1,000 ng/ml, recoveries in the ELISA were between 84 and 110%.The assay was successfully applied for the quantitation of pharmacological levels of scu-PA and t-PA(AA87_274)/scu-PA(AA138-411) in plasma during experimental thrombolysis in baboons.Thus the present ELISA, which is specifically dependent on the presence of the serine protease part of u-PA, is useful for measurement of a wide variety of variants and chimeras of u-PA which are presently being developed for improved thrombolytic therapy.

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Localization of Fibrinolytic Activators and Inhibitors in Normal and Atherosclerotic Vessels

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650397 ◽

1996 ◽

Vol 75 (06) ◽

pp. 933-938 ◽

Cited By ~ 21

Author(s):

Marten Fålkenberg ◽

Johan Tjärnstrom ◽

Per Örtenwall ◽

Michael Olausson ◽

Bo Risberg

Keyword(s):

Endothelial Cells ◽

Plasminogen Activator ◽

Plasminogen Activator Inhibitor ◽

Vasa Vasorum ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Tissue Plasminogen ◽

The Media ◽

Activator Inhibitor ◽

Pai 1

SummaryLocal fibrinolytic changes in atherosclerotic arteries have been suggested to influence plaque growth and promote mural thrombosis on ruptured or ulcerated plaques. Increased levels of plasminogen activator inhibitor (PAI-1) have been found in atherosclerotic arteries. In this study tissue plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA) and PAI-1 were localized in arterial biopsies of healthy and atherosclerotic vessels by immunohistochemis-try. The expression of fibrinolytic regulators was related to the distribution of endothelial cells (EC) and macrophages. Results: t-PA was expressed in vasa vasorum. PAI-1 was positive in endothelial cells, in the media and in the adventitia. Increased expression of t-PA, u-PA and PAI-1 was found in atherosclerotic vessels. t-PA, u-PA, PAI-1 and macrophages were co-localized in plaques. These results support the concept that macrophages can be important in the local regulation of fibrinolysis in atherosclerotic vessels.

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Evolution of Urokinase-Type Plasminogen Activator (u-PA) and Tissue-Type Plasminogen Activator (t-PA) in Orthotopic Liver Transplantation (OLT)

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651548 ◽

1993 ◽

Vol 69 (01) ◽

pp. 056-059 ◽

Cited By ~ 2

Author(s):

G Himmelreich ◽

G Dooijewaard ◽

P Breinl ◽

W O Bechstein ◽

P Neuhaus ◽

...

Keyword(s):

Liver Transplantation ◽

Plasminogen Activator ◽

Orthotopic Liver Transplantation ◽

Tissue Type ◽

Bleeding Complications ◽

Activity Levels ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Anhepatic Phase

SummaryIn orthotopic liver transplantation (OLT) hyperfibrinolysis seems to be of causative importance for intra- and postoperative bleeding. Although recently hyperfibrinolysis has been successfully reduced by intraoperative aprotinin treatment, small increases of fibrinolysis still remain during OLT. Originally, tissue-type plasminogen activator (t-PA) was considered to be responsible for the increases, but the efficacy of aprotinin which inhibits besides plasmin also kallikrein and urokinase-type plasminogen activator (u-PA) suggested also a role for the intrinsic and contact system-dependent plasminogen activators. We investigated the role of u-PA. From 29 patients undergoing OLT with intraoperative aprotinin infusion arterial blood samples were taken at 7 different time points. The preoperative median values for u-PA antigen (u-PA Ag) and plasmin-activatable single-chain u-PA (scu-PA) levels, which were more than 2-fold above normal (both: p <0.01), decreased slightly during the preanhepatic phase and remained unchanged during the anhepatic phase. With reperfusion of the graft liver the two levels decreased significantly (p = 0.0003 and p = 0.006, respectively) to almost normal values, probably due to clearance by the graft liver. Active two-chain u-PA (tcu-PA) was preoperatively 2-fold above the detection limit, remained stable during the preanhepatic phase and increased 2-fold in the anhepatic phase (p = 0.0018). As expected tcu-PA also relapsed upon reperfusion, but to the preoperatively enhanced level, possibly caused by sustained activation of scu-PA by cathepsin B. t-PA activity levels were at the upper end of the normal range preoperatively, slightly increased during preanhepatic and anhepatic phases and decreased significantly with reperfusion. The increases in tcu-PA and t-PA activities during the anhepatic phase coincided with greatly increased fibrinolysis as demonstrated by thrombelastography, indicating that both u-PA and t-PA are involved in the development of fibrinolysis during OLT.One patient was excluded from statistical evaluations because preoperative u-PA Ag, scu-PA, tcu-PA and t-PA activity levels were much higher than in the other 28 patients. In the investigated group this patient was the only one with diffuse peritonitis intraoperatively and severe bleeding complications postoperatively which made retransplantation mandatory.

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The Role of the Low-Density Lipoprotein Receptor-Related Protein (LRP) in the Plasma Clearance and Liver Uptake of Recombinant Single-chain Urokinase-type Plasminogen Activator in Rats

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656039 ◽

1997 ◽

Vol 77 (04) ◽

pp. 710-717 ◽

Cited By ~ 5

Author(s):

Marieke E van der Kaaden ◽

Dingeman C Rijken ◽

J Kar Kruijt ◽

Theo J C van Berkel ◽

Johan Kuiper

Keyword(s):

Plasminogen Activator ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Single Chain ◽

Liver Uptake ◽

Type Plasminogen Activator ◽

Parenchymal Liver ◽

Urokinase Type Plasminogen Activator ◽

Density Lipoprotein Receptor

SummaryUrokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction. In vitro, recombinant single-chain u-PA (rscu-PA) expressed in E.coli is recognized by the Low-Density Lipoprotein Receptor-related Protein (LRP) on rat parenchymal liver cells. In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA in rats. A preinjection of the LRP inhibitor GST-RAP reduced the maximal liver uptake of 125I-rscu-PA at 5 min after injection from 50 to 30% of the injected dose and decreased the clearance of rscu-PA from 2.37 ml/min to 1.58 ml/min. Parenchymal, Kupffer and endothelial cells were responsible for 40, 50 and 10% of the liver uptake, respectively. The reduction in liver uptake of rscu-PA by the preinjection of GST-RAP was caused by a 91 % and 62% reduction in the uptake by parenchymal and Kupffer cells, respectively. In order to investigate the part of rscu-PA that accounted for the interaction with LRP, experiments were performed with a mutant of rscu-PA lacking residues 11-135 (= deltal25- rscu-PA). Deletion of residues 11-135 resulted in a 80% reduction in liver uptake and a 2.4 times slower clearance (0.97 ml/min). The parenchymal, Kupffer and endothelial cells were responsible for respectively 60, 33 and 7% of the liver uptake of 125I-deltal25-rscu-PA. Preinjection of GST-RAP completely reduced the liver uptake of delta 125-rscu-PA and reduced its clearance to 0.79 ml/min. Treatment of isolated Kupffer cells with PI-PLC reduced the binding of rscu-PA by 40%, suggesting the involvement of the urokinase-type Plasminogen Activator Receptor (u-PAR) in the recognition of rscu-PA. Our results demonstrate that in vivo LRP is responsible for more than 90% of the parenchymal liver cell mediated uptake of rscu-PA and for 60% of the Kupffer cell interaction. It is also suggested that u-PAR is involved in the Kupffer cell recognition of rscu-PA.

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