Characterisation of Epitopes on Human Tissue Plasminogen Activator Recognised by a Group of Monoclonal Antibodies

1985 ◽  
Vol 53 (01) ◽  
pp. 045-050 ◽  
Author(s):  
Ian R MacGregor ◽  
Lisel R Micklem ◽  
Keith James ◽  
Duncan S Pepper
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Active Site ◽  
Amidolytic Activity ◽  
Binding Properties ◽  
Tissue Plasminogen ◽  
Site Blocking ◽  
Melanoma Tissue ◽  
Active Site Histidine

SummarySeven mouse monoclonal antibodies have been produced against human melanoma tissue plasminogen activator (t-PA). They were specifically bound to 125I t-PA but not 125I urokinase (u-PA) and inhibited t-PA, but not u-PA, activity in plasminogen- rich 125I fibrin wells. Three of the antibodies directly inhibited the amidolytic activity of t-PA and the two most effective also bound near the active site histidine residue as determined by competition experiments using active site blocking agents. Several antibodies interfered with the fibrin binding properties of t-PA. One antibody neither interacted with the active site nor inhibited fibrin binding but still effectively quenched t-PA activity in fibrin wells suggesting that it masks another region of the molecule necessary for effective biological activity.

scholarly journals Catabolism of human tissue plasminogen activator in mice

Blood ◽  
1985 ◽  
Vol 65 (3) ◽  
pp. 539-544 ◽  
Author(s):  
HE Fuchs ◽  
H Jr Berger ◽  
SV Pizzo
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Active Site ◽  
Human Tissue ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Organ Distribution ◽  
Tissue Plasminogen ◽  
Iodine 125 ◽  
Distribution Studies

The catabolism of human tissue plasminogen activator (t-PA) was studied in mice. The clearance of t-PA labeled with iodine 125 was rapid (t1/2). The clearance of phenylmethylsulfonyl-125I-t-PA, which is active site-inhibited, was identical to the active enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the vast majority of 125I-t-PA injected into the circulation was present as free enzyme and not in a complex with inhibitors. The clearance of 125I-t-PA was unaltered by large molar excesses of several ligands of known clearance specificities, including macroalbumin, asialoorosomucoid, and diisopropylphosphorylthrombin and was also not altered in the presence of a 1,000-fold molar excess of unlabeled t-PA. Organ distribution studies demonstrated that the early rapid clearance of 125I-t-PA occurred in hepatocytes, followed by a later renal phase of clearance. The clearance of 125I-urokinase (UK) also was studied and was very similar in all aspects to the clearance of 125I-t-PA. These results suggest that both t-PA and UK are cleared from the circulation by unique nonsaturable processes localized in the liver that are independent of the proteinase active site.

Bispecific Monoclonal Antibodies Produced by Somatic Cell Fusion Increase the Potency of Tissue Plasminogen Activator

1990 ◽  
Vol 64 (02) ◽  
pp. 260-266 ◽  
Author(s):  
Elizabeth E Branscomb ◽  
Marschall S Runge ◽  
Christopher E Savard ◽  
Keith M Adams ◽  
Gary R Matsueda ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Somatic Cell ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Cell Fusion ◽  
Bispecific Antibody ◽  
Solid Phase ◽  
Bispecific Antibodies ◽  
Somatic Cell Fusion ◽  
Tissue Plasminogen

SummaryBispecific monoclonal antibodies that bind simultaneously to human fibrin and tissue plasminogen activator (tPA) enhance the fibrinolytic potency of tPA. Two bispecific antibodies (F36.23 and F32.1) were generated by somatic cell fusion. Antibody F36.23 derives its tPA binding from monoclonal anti-tPA antibody TCL8 and its fibrin binding from monoclonal antifibrin antibody 59D8. After purification from cell supernatants and ascites by two steps of affinity chromatography, hybrid-hybridoma bispecific antibody F36.23 simultaneously bound tPA and fibrin in solution and in solid-phase assays. In an assay for the lysis of human fibrin monomer, F36.23 increased the fibrinolytic potency of tPA by 5 to 10 fold, regardless of whether the bispecific antibody had been combined with the tPA before or during the assay. Bispecific F36.23 F(ab′)2 also bound tPA and fibrin simultaneously, and the enhancement in fibrinolysis in the presence of F36.23 F(ab′)2 was identical to that in the presence of intact F36.23. The second bispecific antibody, F32.1, was produced by an alternative strategy that has a wider potential for applicaton in other systems. Hybridoma bispecific antibody F32.1 was derived from the fusion of immune splenocytes (in mice immunized with a synthetic oligopeptide representing the amino terminus of the α-chain of human fibrin) with the anti-tPA cell line TCL8. The properties of hybridoma bispecific antibody F32.1 and its F(ab′)2 were indistinguishable from those of hybrid-hybridoma bispecific antibody F36.23 in solid-phase binding assays and in assays of fibrinolysis. Bispecific antibodies produced by somatic cell fusion, particularly in the form of F(ab′)2, may have potential for use in clinical thrombolysis.

Monoclonal Antibodies Towards the Fast Inhibitor of Tissue Plasminogen Activator from Human Plasma and Serum

1986 ◽  
Vol 55 (03) ◽  
pp. 383-387 ◽  
Author(s):  
G Urdén ◽  
Ulla Johansson ◽  
Joanna Chmielewska ◽  
J Brandt ◽  
B Wiman
Keyword(s):  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Hybridoma Cells ◽  
Spleen Cells ◽  
Inhibitor Complex ◽  
Tissue Plasminogen ◽  
Different Levels ◽  
Mouse Myeloma

SummaryHybridoma cells were produced by fusing mouse myeloma cells (SP 2/0 - Ag 14) with spleen cells from a Balb/c mouse, previously immunized with the partially purified complex between tissue plasminogen activator (t-PA) and its fast inhibitor from human plasma (serum). Screening with a radioimmunoassay revealed a number of hybridomas secreting antibodies directed towards the complex. Of these, about 1/3 reacted both with the complex and t-PA, whereas about 2/3 reacted only with the complex. Three of the latter hybridomas, producing antibodies directed towards the inhibitor-moiety in the complex have been cloned and the antibodies were studied in detail. PA-inhibitor activity in plasma or serum and t-PA/PA-inhibitor complex could be specifically adsorbed on all three insolubilized monoclonal antibodies (MCI, MC2 and MC3). None of the antibodies seems to be directed against structures of vital importance for the functional activity of the PA-inhibitor. In accordance with this finding the antibody with the highest avidity (MCI) reacts equally well with the PA-inhibitor alone or in complex with t-PA. A radioimmunoassay was devloped with this antibody and significant displacement was obtained with samples with PA-inhibitor concentrations above 2 AU/mL. In 13 plasma samples with different levels of PA-inhibitory activity a significant correlation was obtained when comparing this activity with the PA-inhibitor antigen as measured with the radioimmunoassay (r = 0.88).

scholarly journals Binding of tissue plasminogen activator to human endothelial cells. Importance of the B-chain as a ligand

1992 ◽  
Vol 287 (2) ◽  
pp. 407-413 ◽  
Author(s):  
X F Cheng ◽  
O Bäck ◽  
T K Nilsson ◽  
E Nylander Lundqvist ◽  
G Pohl ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Active Site ◽  
Binding Sites ◽  
Human Umbilical Cord ◽  
Cultured Endothelial Cells ◽  
Tissue Plasminogen ◽  
A Chain ◽  
Pai 1

The aim of the present study was to investigate the binding of tissue plasminogen activator (tPA) to cultured endothelial cells and to characterize binding structures present in the cultures. Studies on the binding of 125I-tPA to cultured endothelial cells from human umbilical-cord veins (HUVEC) indicated that the number of sites for specific binding of tPA is 8 x 10(5) per cell. Treatment with an excess of antibodies against plasminogen-activator inhibitor type 1 (PAI-1) caused an 80% decrease in the binding, leaving about 1.6 x 10(5) unoccupied binding sites per cell, which appeared to be different from PAI-1. About 1.9 x 10(5) binding sites/cell for tPA were found on the surface of HUVEC that had been detached from the matrix. This indicates that only minor amounts of PAI-1 occur on the surface of the cells. In addition, immunocytochemical analysis showed that PAI-1 antigen is present almost exclusively in the cytoplasm but was not observed on the surface of the cells, whereas tPA antigen is abundant on the plasma membrane of tPA-treated cells as well as intracellularly. Competition studies using unlabelled compounds showed that native tPA and tPA B-chain (the proteinase domain), as well as the inactive derivatives, B-chain inactivated with D-Phe-Pro-Arg-chloromethane and tPA-PAI-1 complex, caused a considerable quenching of the binding of 125I-tPA to HUVEC, whereas the isolated A-chain had no demonstrable effect. Two components (apparent molecular masses 38 kDa and 56 kDa) reacting with tPA but lacking PAI-1 antigen determinants were identified. Thus the data suggest that tPA binds to HUVEC by two principally different mechanisms. One is mediated by PAI-1, which binds and inactivates tPA with a functional active site. The other binding is achieved by components which react with sites on the activator molecule other than structures of the A-chain or the active site.

scholarly journals Secretion of Active Recombinant Human Tissue Plasminogen Activator Derivatives in Escherichia coli

2001 ◽  
Vol 67 (6) ◽  
pp. 2657-2664 ◽  
Author(s):  
Jiradej Manosroi ◽  
Chatchai Tayapiwatana ◽  
Friedrich Götz ◽  
Rolf G. Werner ◽  
Aranya Manosroi
Keyword(s):  
Escherichia Coli ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Specific Activity ◽  
Stop Codon ◽  
Active Form ◽  
Site Directed Mutagenesis ◽  
Amidolytic Activity ◽  
Tissue Plasminogen ◽  
Kringle 2

ABSTRACT The DNA fragment coding for kringle 2 plus serine protease domains (K2S) of tissue plasminogen activator (tPA) was inserted into a phagemid vector, pComb3HSS. In the recombinant vector, pComb3H-K2S, the K2S gene was fused togpIII of ΦM13 and linked to the OmpA signal sequence. The resulting gene, rK2S-gpIII, was inducibly expressed in Escherichia coli XL-1 Blue. The protein was presented on the phage particle. To stop the expression of gpIII,a stop codon between K2S and the gpIIIgene was inserted by site-directed mutagenesis. This mutated vector, MpComb3H-K2S, was transformed in XL-1 Blue. After induction with IPTG (isopropyl-β-d-thiogalactopyranoside), rK2S was found both in the periplasm as an inactive form of approximately 32% and in the culture supernatant as an active form of approximately 68%. The secreted form of rK2S was partially purified by ammonium sulfate (55%) precipitation. The periplasmic form was isolated from whole cells by chloroform extraction. The fibrin binding site of kringle 2 was demonstrated in all expressed versions (phage-bound, periplasmic, and secreted forms) using the monoclonal anti-kringle 2 antibody (16/B). Only the secreted form of rK2S revealed a fibrinogen-dependent amidolytic activity with the specific activity of 236 IU/μg. No amidolytic activity of rK2S was observed in either the periplasmic or the phage-bound form. The secretion of rK2S as an active enzyme offers a novel approach for the production of the active-domain deletion mutant tPA, rK2S, without any requirements for bacterial compartment preparation and in vitro refolding processes. This finding is an important technological advance in the development of large-scale, bacterium-based tPA production systems.

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