Monoclonal Antibodies Against Human High Molecular Weight Urinary Urokinase: Application for Affinity Purification of Urinary Prourokinase

Wojta, Johann; Kirchheimer, Johannes C.; Turcu, L; Christ, Günter; Binder, Bernd R.

doi:10.1055/s-0038-1661561

Cited by 25 publications

(6 citation statements)

References 10 publications

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“…1 mg human plasmin was coupled to 1 ml CNBr activated Sepharose to obtain plasmin Sepharose (26). Monoclonal antibodies against u-PA were produced and charac terized as described previously (27). MPW 5 U K was finally selected and peroxidase labelled according to the method of Nakane and Kawaoi (28).…”

Section: Methodsmentioning

confidence: 99%

Evaluation of Fibrinolytic Capacity in Plasma during Thrombolytic Therapy with Single (scu-PA) or Two-chain Urokinase Type Plasminogen Activator (tcu-PA) by a Combined Assay System for Urokinase Type Plasminogen Activator Antigen and Function

et al. 1989

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SummaryA combined assay for urokinase type plasminogen activator (u-PA) activity and antigen determination in plasma samples is described. This assay is based on binding of u-PA to an antibody immobilized on a microtiter plate followed by determination of the enzymatic activity of the bound u-PA. Thereafter bound u-PA antigen can be quantified by means of a specific peroxidase labelled monoclonal antibody against u-PA. By use of this assay system u-PA activity and antigen can be determined with lower detection limits of 0.08 IU/ml and 1.0 ng/ml, respectively, and intraassay as well as interassay coefficients of variation of 10% and 12% for activity and 5% and 7% for antigen determinations, respectively. Normal plasma levels of u-PA antigen could be determined to be 1.88 nglml ± 0.61. Furthennore, this assay system allows specific quantification of u-PA antigen and activity during thrombolytic therapy.

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Section: Methodsmentioning

confidence: 99%

Evaluation of Fibrinolytic Capacity in Plasma during Thrombolytic Therapy with Single (scu-PA) or Two-chain Urokinase Type Plasminogen Activator (tcu-PA) by a Combined Assay System for Urokinase Type Plasminogen Activator Antigen and Function

et al. 1989

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“…The monoclonal antibodies MPW4UK and MPW5UK (22), both belonging to the IgG1 subclass and directed against u-PA, reacted with high as well as with low molecular weight u-PA. MPW5UK additionally interfered with plasminogen activation by u-PA. Radiolabeling of MPW5UK was performed as described for u-PA, resulting in a specific activity of 3.79 x 107 cpm//g.…”

mentioning

confidence: 99%

“…Preparation and Radiolabeling of u-PA. u-PA (double chain; Mr 54,000) was isolated from human urine by affinity chromatography on anti-u-PA monoclonal antibody (MPWSUK)-Sepharose followed by affinity chromatography on agmatine-Sepharose (22). SDS/PAGE followed by silver staining showed a single band in the unreduced samples, corresponding to Mr 54,000. u-PA (50 ,ug) was inactivated by incubation with 0.05 M DFP in 0.01 M potassium phosphate buffer (pH 7.4) for 2 hr at room temperature followed by excessive dialysis to remove free DFP; inactivated u-PA was found to be <0.5% as active as the native enzyme on plasminogen-rich fibrin/agarose plates (16).…”

mentioning

confidence: 99%

Functional inhibition of endogenously produced urokinase decreases cell proliferation in a human melanoma cell line.

Kirchheimer

Wojta

Christ

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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Binding of urokinase-type plasminogen activator (u-PA) to its receptor has been shown not only to focus proteolytic activity to the cell surface but also to exert a mitogenic effect on the human epidermal tumor cell line CCL 20.2. This report shows that u-PA is an autocrine mitogen in the human melanoma cell line GUBSB and that inhibition of receptor-bound u-PA by specific anti-u-PA antibodies causes a significant suppression of cell proliferation in this system. The GUBSB cell line secretes 70-80% of the u-PA in its active form and expresses high-affinity u-PA receptors with a Kd of 5.2 X 10-10 M and 2.8 x 104 binding sites per cell. Approximately 70% of the u-PA receptors on these cells are occupied by endogenously secreted u-PA. Addition of the monoclonal antiu-PA antibody MPW5UK (10 nM), directed against the active site of u-PA, twice daily to the cell cultures resulted in a significant decrease of [3H~thymidine incorporation by the tumor cells, whereas a 10 times higher concentration of the monoclonal antibody MPW4UK, which does not inhibit plasminogen activator activity of u-PA, was necessary to achieve the same effect. In addition, diisopropyl fluorophosphateinactivated u-PA, in a concentration 50-fold higher than the concentration necessary to saturate the u-PA receptor (250 pM), decreased [3H]thymidine incorporation similarly to the specific antibody, proving that active u-PA is required for the mitogenic effect. Inhibition of endogenous u-PA production by cycloheximide reduced [3H]thymidine incorporation significantly; after addition of exogenous u-PA, [3H]thymidine incorporation increased again in the cycloheximide-treated cells. Therefore, inhibition of receptor-bound u-PA might represent a tool not only to inactivate cell-bound proteolytic activity, necessary for invasion, but also to exert a specific antiproliferative effect on certain tumor cells.Malignant cells contain and secrete plasminogen activators (PAs; EC 3.4.21.31), trypsin-like serine proteases, to a much higher extent than their respective normal counterparts (1-3). Immunological characterization has shown that most malignant cells produce urokinase-type PA (u-PA) rather than tissue-type PA (t-PA) (2). u-PA contains not only an activesite domain consistent with its serine protease character and involved in the activation ofplasminogen but also a "kringle" domain, found in several other seine proteases, and a growth-factor domain homologous to the epidermal growth factor (4).

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“…Details of the purification have been described elsewhere [16]. Although it was shown that the sc-uPA preparation used only exhibited a single silver-stainable band in the reduced and non-reduced sample, trace contamination of the sc-uPA preparation by the active high-Mr urokinase cannot, however, be excluded.…”

Section: Discussionmentioning

confidence: 99%

“…Sc-uPA and high-M, urokinase were isolated from human urine by affinity chromatography on monoclonal, antiurokinase IgG (MPW5UK)-Sepharose followed by affinity chromatography on agmatine-Sepharose to separate active bound from inactive unbound urokinase as described previously [16]. The two products were analyzed by SDS/polyacrylamide gel electrophoresis followed by silver staining and could be shown to consist of a single stainable protein band in the unreduced samples corresponding to an M , of 54000.…”

Section: Urokinasesmentioning

confidence: 99%

Effect of the cyanogen-bromide-2 fragment of fibrinogen on plasminogen activation by single-chain urokinase-type plasminogen activator

et al. 1987

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Activation of Glu-plasminogen by single-chain urokinase-type plasminogen activator (sc-uPA), isolated from human urine, was studied in a purified system in the absence and presence of the cyanogen bromide fibrinogen fragment, FCB 2, and compared to plasminogen activation by two-chain high-M, urokinase. Plasminogen activation by sc-uPA was significantly increased by the FCB-2 fibrinogen fragment, an effect brought about by decrease of apparent K,,, and increase of apparent k,,,. During the course of plaminogen activation by scu-PA, two-chain urokinase was formed from '251-s~-~PA to a significant degree only when a concentration of 30 nM plasmin was reached in the incubation mixture; this was only the case in the system stimulated by FCB-2 fibrinogen fragment and only after 30 min. Formation of two-chain urokinase was not, however, related to the increase in the rate of plasmin formation induced by the FCB-2 fibrinogen fragment.Plasminogen activators are proteases which activate plasminogen to plasmin, an enzyme involved in fibrinolysis [ 11 and other processes in which extracellular proteolysis is required [2 -41. Two entities of plasminogen activators are generally recognized, the urokinase-type plasminogen activator (u-PA), and the tissue-type plasminogen activator (t-PA) [S, 61. Tissue-type plasminogen activator, normally found at very low concentrations in human plasma, exhibits thrombus-specific fibrinolysis by activating plasminogen on the surface of fibrin [I, 7-91. The other plasminogen activator, urokinase, originally isolated from urine, is a trypsin-like serine protease consisting of two polypeptide chains connected by a single disulfide bridge [lo, 1 I]. Evidence that urokinase is produced in a single-chain form (prourokinase, sc-uPA) which can become activated by plasmin to the two-chain form was provided as early as 1973 [12]. sc-uPA could also be isolated from human plasma and other sources [13 -161; it has the same apparent molecular mass as high-molecular-mass (high-M,) urokinase but differs in its single-chain structure, its low activity towards synthetic substrates and its slower reaction with diisopropylfluorophosphate [15]. However, scu-PA activates plasminogen efficiently [17] and sc-uPA can be converted to fully active two-chain high-Mr urokinase by limited proteolysis with plasmin [I81 or plasma kallikrein 1191.Furthermore, similarly as for t-PA, a fibrin specificity was found also for sc-uPA in studies in vitro and in vivo. This fibrin effect is, however, likely to be mediated by a different mechanism [18,20, 211 involving also fibrin-enhanced Gluplasminogen activation by two-chain urokinase [22].Besides the question of whether plasma urokinase contributes substantially to the fibrinolytic activity in blood or not, it remains unclear whether plasminogen activation by sc-uPA proceeds in the presence of fibrin via formation of two-chain urokinase. Furthermore there are no data available on whether fibrin exerts a direct effect on plasminogen activation by sc-uPA [17, 231. It was therefore ...

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Monoclonal Antibodies Against Human High Molecular Weight Urinary Urokinase: Application for Affinity Purification of Urinary Prourokinase

Cited by 25 publications

References 10 publications

Evaluation of Fibrinolytic Capacity in Plasma during Thrombolytic Therapy with Single (scu-PA) or Two-chain Urokinase Type Plasminogen Activator (tcu-PA) by a Combined Assay System for Urokinase Type Plasminogen Activator Antigen and Function

Evaluation of Fibrinolytic Capacity in Plasma during Thrombolytic Therapy with Single (scu-PA) or Two-chain Urokinase Type Plasminogen Activator (tcu-PA) by a Combined Assay System for Urokinase Type Plasminogen Activator Antigen and Function

Functional inhibition of endogenously produced urokinase decreases cell proliferation in a human melanoma cell line.

Effect of the cyanogen-bromide-2 fragment of fibrinogen on plasminogen activation by single-chain urokinase-type plasminogen activator

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