Characterization of Monoclonal Antibodies to Human Tissue-Type Plasminogen Activator: Catalytic Inhibition and One-Two Chain Discriminatory Reactivities

1989 ◽  
Vol 62 (02) ◽  
pp. 742-747 ◽  
Author(s):  
Torgny Stigbrand ◽  
Lars Frängsmyr ◽  
Nils Bergsdorf ◽  
Per Wallen
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Conformational Epitope ◽  
Tissue Type ◽  
Type Plasminogen Activator ◽  
Tissue Plasminogen ◽  
A Chain ◽  
The One ◽  
Chain Form

SummaryA new set of monoclonal antibodies was generated against the tissue plasminogen activator (t-PA). One of the antibodies, 1:3 C5, was found to be able to distinguish between the one- and two-chain form of t-PA and also exerted significant amidolytic inhibitory activity. Several of the antibodies, as judged from their binding properties in immunosorbent tests, were found to be suitable for immunoaffinity purification purposes, i.e. 1:3C5, 1:3 G5, and 1:2 B9. Three of the mabs, 1:2 B9, 1:3 G5 and 2:2 BIO, were selectively reactive with the A-chain of t-PA, whereas indirect evidences indicated 1:3 C5 to be reactive with a conformational epitope on the B-chain. Three of the antibodies were reactive with porcine t-PA. This new set of antibodies should prove useful for structure-function investigations of t-PA.

Characterization of Two Monoclonal Antibodies Against Human Tissue-Type Plasminogen Activator

1985 ◽  
Vol 53 (02) ◽  
pp. 170-175 ◽  
Author(s):  
Raymond R Schleef ◽  
Manjula Sinha ◽  
David J Loskutoff
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Human Tissue ◽  
Association Constant ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
High Affinity ◽  
Standard Procedures ◽  
Type Plasminogen Activator

SummaryA series of hybridoma clones, each producing monoclonal antibodies to human tissue-type plasminogen activator (t-PA), were prepared from mice by standard procedures. Two of these clones were selected for further study. One HI72A1, produced antibodies that bound to t-PA and strongly inhibited its activity, whereas another, LI72D1, produced antibodies that bound to t-PA but did not affect its activity. The specificity of these antibodies was assessed in immunoabsorption experiments. Both immunoprecipitated 125I-labeled t-PA, and both were specific since only t-PA was recognized in conditioned media collected from Bowes melanoma cells cultured in the presence of 3H-leucine. Neither antibody recognized urokinase. t-PA was desorbed from antibody HI72A1-Sepharose columns with 0.5 M NaCl, consistent with its relatively low association constant (Ka = 9.37 × 107 M-1). In contrast, a strong chaotropic agent (i.e., 2 M KI) was required to elute t-PA from antibody LI72D1 columns (Ka = 2.08 × 109 M-1). This latter high affinity antibody was employed to develop an immunoradiometric assay for t-PA having a sensitivity of 0.5 ng/ml.

Characterization of a Panel of Monoclonal Antibodies Against Human Tissue-Type Plasminogen Activator

Hybridoma ◽  
1988 ◽  
Vol 7 (2) ◽  
pp. 177-184 ◽  
Author(s):  
THOMAS M. REILLY ◽  
SANDRA K. FLINT ◽  
BERNIE G. McHUGH ◽  
KATHLEEN M. WILSBACH-VOLCHECK ◽  
PIETER B.M.W.M. TIMMERMANS
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Human Tissue ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

scholarly journals Two-Chains Tissue Plasminogen Activator Unifies Met and NMDA Receptor Signalling to Control Neuronal Survival

2021 ◽  
Vol 22 (24) ◽  
pp. 13483
Author(s):  
Elodie Hedou ◽  
Sara Douceau ◽  
Arnaud Chevilley ◽  
Alexandre Varangot ◽  
Audrey M Thiebaut ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Neuronal Death ◽  
Neuronal Survival ◽  
Tissue Type ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Receptor Signalling ◽  
Type Plasminogen Activator ◽  
Tissue Plasminogen ◽  
Chain Form

Tissue-type plasminogen activator (tPA) plays roles in the development and the plasticity of the nervous system. Here, we demonstrate in neurons, that by opposition to the single chain form (sc-tPA), the two-chains form of tPA (tc-tPA) activates the MET receptor, leading to the recruitment of N-Methyl-D-Aspartate receptors (NMDARs) and to the endocytosis and proteasome-dependent degradation of NMDARs containing the GluN2B subunit. Accordingly, tc-tPA down-regulated GluN2B-NMDAR-driven signalling, a process prevented by blockers of HGFR/MET and mimicked by its agonists, leading to a modulation of neuronal death. Thus, our present study unmasks a new mechanism of action of tPA, with its two-chains form mediating a crosstalk between MET and the GluN2B subunit of NMDARs to control neuronal survival.

Characterization of Human Tissue-Type Plasminogen Activator with Monoclonal Antibodies: Mapping of Epitopes and Binding Sites for Fibrin and Lysine

1992 ◽  
Vol 67 (01) ◽  
pp. 088-094 ◽  
Author(s):  
Ute Zacharias ◽  
Bernhard Fischer ◽  
Franz Noll ◽  
Horst Will
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Human Tissue ◽  
Tissue Type ◽  
Reduced Form ◽  
Amidolytic Activity ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Catalytically Active ◽  
A Chain

SummaryThe study defines interactions between human tissue-type plasminogen activator (t-PA) and 21 mouse monoclonal antibodies (mAb). Characterization includes epitope distribution, reactivity of different forms of t-PA with antibodies, and modification of t-PA function by antibody binding.Eighteen antibodies are directed against t-PA A-chain. These antibodies recognize four distinct epitopes (A, B, C, D) and one partially overlapping epitope (D’). The remaining three antibodies are directed against two different epitopes (E, F) on catalytically active t-PA B-chain. A-chain reactive antibodies do not bind to the reduced form of t-PA, while B-chain reactive antibodies bind to reduced and deglycosylated t-PA forms. The latter antibodies associate more tightly with sc t-PA than with tc t-PA and have a higher affinity for t-PA-PAI 1 complex as compared to free t-PA.The analysis of functional effects of antibodies reveals that antibodies directed against all above defined epitopes inhibit interactions between t-PA and fibrin: a) binding of t-PA to fibrin, b) fibrinolytic activity of t-PA, and c) fibrin activation of sc t-PA amidolytic activity. The observations support the assumption that several sites of t-PA are involved in fibrin binding and that fibrin-bound t-PA is closely surrounded by the fibrin mesh. Many antibodies quench also binding of t-PA to lysine-Sepharose. Experiments with free, non-fixed lysine confirm strong competition between lysine and mAb 16 and 18, directed against epitope A, and mAb 29, binding to epitope F. Weak inhibition is exerted on association of mAb 2, 21, and 25 to epitope D. Amidolytic activity is suppressed only by B-chain specific antibody 22.

In Vitro Stability of a Tissue-Type Plasminogen Activator Mutant, BM 06.022, in Human Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 906-911 ◽  
Author(s):  
D C Rijken ◽  
E Groeneveld ◽  
M M Barrett-Bergshoeff
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tissue Type ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Chain Form

SummaryBM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated.Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, α2-antiplasmin and α1-antitrypsin.During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a2-anti-plasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i. e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher.In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, a2-antiplasmin and a j-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.

Purification and Characterization of Tissue-Type Plasminogen Activator in Maxillary Mucosa with Chronic Inflammation

1985 ◽  
Vol 54 (02) ◽  
pp. 485-489 ◽  
Author(s):  
Yukiyoshi Hamaguchi ◽  
Masuichi Ohi ◽  
Yasuo Sakakura ◽  
Yasuro Miyoshi
Keyword(s):  
Plasminogen Activator ◽  
Chronic Inflammation ◽  
Gel Filtration ◽  
Potassium Acetate ◽  
Tissue Type ◽  
Purification And Characterization ◽  
Tissue Type Plasminogen Activator ◽  
Urokinase Inhibitor ◽  
Type Plasminogen Activator

SummaryTissue-type plasminogen activator (TPA) was purified from maxillary mucosa with chronic inflammation and compared with urokinase. Purification procedure consisted of the extraction from delipidated mucosa with 0.3M potassium acetate buffer (pH 4.2), 66% saturation of ammonium sulfate, zinc chelate-Sepharose, concanavalin A-Sepharose and Sephadex G-100 gel filtration chromatographies.The molecular weight of the TPA was approximately 58,000 ± 3,000. Its activity was enhanced in the presence of fibrin and was quenched by placental urokinase inhibitor, but not quenched by anti-urokinase antibody. The TPA made no precipitin line against anti-urokinase antibody, while urokinase did.All these findings indicate that the TPA in maxillary mucosa with chronic inflammation is immunologically dissimilar to urokinase and in its affinity for fibrin.

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