Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.68 (tissue plasminogen activator)
 11,311 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We selected six peptide sequences as belonging to potential epitopes of tissue plasminogen activator (tPA) using, as the main criterion for their choice, the location of the peptide sequences on the surface of the protein molecule. The six peptides (corresponding to amino acids 4-8, 11-16, 96-101, 272-277, 371-376 and 514-519) were synthesized, coupled to carrier proteins and injected into rabbits. All of these peptides elicited antibodies and 15-75% binding of the corresponding iodinated peptide was obtained with a 1:100 dilution of antiserum. Only two anti-(peptide) sera [anti-(tPA96-101) and anti-(tPA272-277)] reacted with intact tPA and its heavy chain in Western immunoblotting analysis. These two peptides sequences and fragment tPA11-16 appear to be involved in the structure of native antigenic epitopes of tPA, since they were recognized and antibodies present in antisera raised against native tPA. There was no interaction between anti-(tPA4-8) and anti-(tPA371-376) sera with intact one-chain or two-chain tPA. In the case of anti-(tPA4-8) cleavage of one-chain tPA to two-chain tPA and reduction of disulfide bonds exposed this epitope.
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PMID:Production and characteristics of human tissue plasminogen-activator antibodies with region-oriented synthetic peptides. 137 86

Hybrid molecules containing the catalytic domain of either tissue plasminogen activator (tPA) or single chain urokinase-type plasminogen activator (scuPA), and the fibrin binding domain of a murine antifibrin monoclonal antibody were constructed using either cDNA or genomic DNA encoding the plasminogen activator and genomic DNA encoding antifibrin monoclonal antibody 59D8. In order to optimize expression of these fusion proteins in hybridoma cells, we compared plasminogen activator 3' UT domains (which decrease mRNA stability) with immunoglobulin and beta globin 3' UT domains (which increase mRNA stability). The presence of the plasminogen activator 3' UT domain resulted in approximately tenfold lower steady-state mRNA levels, and 300 to 500-fold lower levels of expressed functional protein. The initial goal of these studies was to increase the fibrinolytic potency and selectivity of tPA or scuPA. Fusion proteins comprising an antifibrin antibody domain and the catalytic domain of either tPA or scuPA were expressed and shown to have very different properties. The fusion protein that comprised the Fab portion of an antifibrin antibody and the catalytic domain of tPA, while displaying antigen binding properties indistinguishable from those of the parent antibody and amidolytic activity similar to that of tPA, was not more efficient than tPA in an in vitro clot lysis assay. In contrast, it had been shown that tPA chemically coupled to the same antibody was four- to sixfold more efficient in fibrinolysis both in vitro and in vivo. A recombinant scuPA-antifibrin antibody hybrid, however, was sixfold more potent than scuPA in vitro and 20-fold more potent in a rabbit thrombolysis model. An explanation for this apparent discrepancy may relate to the requirement for stimulation by fibrin in order for tPA to achieve its maximal catalytic activity, a property that was demonstrated to have been lost in the antifibrin-tPA fusion protein. In contrast, the activity of urokinase is independent of the presence of fibrin. This may explain the greater success achieved in enhancing catalytic activity in the urokinase-antifibrin fusion protein. It is of additional interest that fibrin or soluble fibrin fragments stimulate the catalytic activity of both tPA and the isolated tPA B chain, demonstrating that at least part of the enhanced catalytic activity of tPA observed in the presence of fibrin is independent of fibrin binding either by the tPA kringles or finger domain (or any heavy chain domain). These data indicate that it is possible to construct recombinant hybrid molecules in which both plasminogen activator catalytic function and antibody binding are preserved.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Hybrid molecules: insights into plasminogen activator function. 180 66

The binding of t-PA to fibrin is mediated both by its "finger" (F) and its "kringle 2" (K2) domain. In addition, these domains are involved in the stimulation of t-PA activity by fibrin. We analyzed the kinetic characteristics of Glu-plasminogen activation by t-PA and a set of t-PA deletion mutants in the absence and the presence of desA-fibrin. In the absence of desA-fibrin, the activity of t-PA (variants) is determined by the presence of the protease domain, irrespective of the composition of the amino-terminal heavy chain. In the presence of the cofactor desA-fibrin, the activity of t-PA (variants) is dependent on the domain composition of the heavy chain. The activity of t-PA is stimulated 2,400 fold by desA-fibrin, whereas the activity of the mutant lacking the K1 domain (del. K1) increases 936 fold in the presence of this cofactor. Mutants lacking either the K2 domain (del. K2) or the F domain (del. F) exhibit an enhanced activity upon desA-fibrin addition of 200 and 210 fold, respectively. DesA-fibrin has no stimulatory effect on the activity of the mutant containing only the serine-protease domain (del.FE K1 K2) nor on the activity of the variant containing only the K1 domain and the serine-protease domain (del. FE K2). Furthermore, we determined the relative fibrin affinity of each t-PA variant, which is similarly dictated by the composition of the heavy chain.
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PMID:Kinetic characterization of tissue-type plasminogen activator (t-PA) and t-PA deletion mutants. 190 54

To investigate structure-function relationships in tissue-type plasminogen activator (t-PA) we deleted the following domains in the heavy chain: a) The epidermal growth factor domain (t-PA del. G), b) the finger domain, and the epidermal growth factor domain (t-PA del. FG), and c) the finger, the epidermal growth factor and Kringle 1 (t-PA del. FGK1). To study specifically the function of the growth factor domain we made two substitutions of d) 8 amino acids (consensus sequence) in the growth factor domain (t-PA G-CS) and e) the whole domain with factor IX growth factor domain (t-PA G-IX). Finally, f) an analogue with substitution in the finger domain (fibronectin consensus sequence) was constructed (t-PA F-CS). A reduced fibrin binding of all the analogues was found. The fibrin stimulated activity of all analogues was also reduced and correlated to the fibrin binding. In contrast, the activity of the analogues in the clot lysis assay and the plate assay were only slightly reduced as compared to authentic t-PA. This suggested that at high fibrin concentrations the decreased fibrin affinity was less critical for obtaining a high fibrinolytic activity. All analogues had a prolonged half-life in vivo as compared to authentic t-PA. The assumption of clearance mechanism involving mainly the growth factor region (or Kringle 1) was not challenged by the observation of a prolonged half-life for the substitution analogue t-PA F-CS.2+off
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PMID:Fibrin affinity and clearance of t-PA deletion and substitution analogues. 211 Oct 48

A peptide fragment of tissue plasminogen activator (tPA) corresponding to amino acid residues 4-8 (tPA4-8) was synthesized, coupled to thyroglobulin and injected into rabbits. Antibodies specific to the peptide tPA4-8 were purified immunochemically on the pentapeptide coupled to CNBr-Sepha rose 4B. Anti-tPA4-8 antibodies, reacted with iodinated peptide tPA4-8, showing a relatively high binding affinity (KD = 2.3 x 10(-8) M). There was no interaction between anti-tPA4-8 antibodies and native one- or two-chain tPA. However, reduction of disulfide bonds unmasked the epitope on the heavy chain of tPA which became accessible to anti-tPA4-8 antibodies. Similarly, complexing of tPA with alpha 1-antitrypsin inhibitor resulted in unmasking of the epitope formed by amino acid residues in the positions 4-8. Presented data suggest that complexing of tPA with inhibitors results in conformational changes occurring in the "finger" domain of tPA molecule and such conformational transition can be detected by antipeptide antibodies. Therefore, anti-tPA4-8 antibodies may be employed as sequence-specific reporter molecules to monitor local conformational changes in tPA molecule.
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PMID:The conformation changes of the finger domain of tissue type plasminogen activator during the activator-inhibitor reaction. 211 44

An antigen assay based on a monoclonal antibody directed against the light chain of tissue-type plasminogen activator (t-PA) was developed to quantify seven recombinant (r) t-PA deletion mutant proteins. These recombinant proteins were then employed to map different epitopes on t-PA which interact with a panel of twenty-three monoclonal anti-t-PA antibodies. Twenty were directed against domains on the heavy chain, two against the "finger" domain, three against the "epidermal growth factor-like" domain, five against the kringle 1 domain, and ten against the kringle 2 domain. Only three monoclonal anti-t-PA antibodies interact with the light chain. The finding that the epitopes of each of the monoclonals could be determined with the deletion mutant proteins supports the hypothesis of autonomous folding of structural domains and emphasizes the validity of the use of the recombinant t-PA-deletion mutant proteins for structure-function studies.
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PMID:Mapping of epitopes on human tissue-type plasminogen activator with recombinant deletion mutant proteins. 243 98

Six monoclonal antibodies (mIgG) and a polyclonal antibody (pIgG) directed against human tissue-type plasminogen activator (t-PA) were tested for their species specificity towards human or murine t-PA. Whereas pIgG as well as several mIgGs discriminated poorly between these two t-PA species, one mIgG (clone E3) was highly specific for human t-PA. Inhibition and binding studies of human t-PA by mIgGs revealed high affinity-high inhibitory (E3) as well as high affinity-poor inhibitory (B1) mIgGs. The relative affinity of two mIgGs for human t-PA was found to be equal or even superior to that of pIgG. Immunoblotting of reduced two-chain t-PA and of an isolated heavy chain of t-PA prepared by recombinant DNA technology, showed that the E3 antibody was directed against the heavy chain of t-PA.
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PMID:Monoclonal antibodies directed against human tissue-type plasminogen activator: a characterization of their species specificity, affinity and heavy-chain binding. 243 3

We have designed and constructed a DNA sequence encoding human tissue plasminogen activator (tPA) with convenient restriction sites that flank each of the domains of the heavy chain. To accomplish this, the first 1095 bases of the gene coding for the mature protein were synthesized with unique restriction sites engineered into the interdomainal regions. This synthetic construction was then ligated to a cDNA fragment of the tPA gene that encoded the active site, thus generating a full-length tPA gene. The gene products produced by Chinese hamster ovary (CHO) cells transfected with either the tPA cassette gene or the tPA cDNA gene were then compared with the tPA produced by Bowes melanoma cells to determine whether or not synthetic interdomainal amino acid changes had an effect on the biochemical characteristics of the molecule. Specifically, molecular weight, specific activity, enhancement by fibrinogen fragments and kinetic constants were analysed. None of the properties examined were significantly different from those of the native melanoma tPA. Therefore, the cassette gene described herein should provide considerable versatility and precision in the construction of tPA mutants by facilitating the manipulation of the finger, growth factor and kringle domains, and likewise should be useful in assessing the function of these domains within the tPA molecule. We present this cassette gene system as a model for the analysis of protein domain function applicable to other multi-domain proteins.
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PMID:Construction of a tissue plasminogen activator gene having unique restriction sites to facilitate domain manipulation: a model for the analysis of multi-domain proteins. 249 53

Our method for constructing an antifibrin antibody-t-PA chimeric protein can be adapted to form other bifunctional, antibody-targeted proteins. Once an appropriate targeting antibody is obtained, the investigator can derive the heavy chain loss variant cell lines and clone the functional heavy chain rearrangement transcribed by the hybridoma. Other useful reagents include antisera directed against mouse Fab and antisera against whatever effector component is to be combined with the antibody. These are helpful during the screening of transfectants and the characterization of the secreted fusion protein, and they allow for protein purification by affinity chromatography. An assay of the functional activity of the effector domain is also desirable. The apparent retention of enzymatic activity and substrate specificity in our antibody-targeted plasminogen activator hybrid demonstrates that even complex molecules with strict folding requirements and multiple intrachain disulfide bonds can be used to form hybrid recombinant proteins. We have documented by electrophoretic transfer blotting that the heavy chain-t-PA fusion protein is secreted in association with light chain in the form of a 180-kDa dimer. The heavy chains appear to be attached by disulfide bonds at the hinge region, as is the case with the heavy chains of natural immunoglobulins. Our method can be adapted to various uses. More or less of the antibody constant region could be employed, depending on the desired geometry and the immunologic interactions mediated by the Fc domain. We have made a recombinant fusion peptide containing an additional 100 constant region amino acids but found that its targeting and catalytic abilities did not differ from those of the smaller molecule. Recent reports indicate that it is possible to express an antibody Fv that has full antigen recognition and binding properties; such small immunoglobulins could minimize potential immunogenicity while affording full targeting capability. The use of a human constant region sequence may also provide a less immunogenic molecule, and, by transferring the complementarity-determining regions of the monoclonal antibody into human variable region sequence, it may be possible to completely "humanize" an antibody-directed chimeric protein. The application of these and other innovative approaches should soon make antibodies an attractive means of targeting a wide range of molecules, both in scientific investigation and in medical therapy.
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PMID:Recombinant antibodies possessing novel effector functions. 251 68

The interactions between tPA domains that are important for catalysis are poorly understood. We have probed the function of interdomain interactions by generating tPA variants in which domains are duplicated or rearranged. The proteins were expressed in a transient mammalian expression system and tested in vitro for their ability to activate plasminogen, induce fibrinolysis and bind to a forming fibrin clot. Duplication of the heavy chain domains of tPA produced enzymatically active tPA variants, many of which demonstrated similar in vitro amidolytic and fibrinolytic activity and similar fibrin affinity to the parent molecule. Zymographic analysis of the domain duplication tPA variants showed one major active species for each variant. Selection of the residues duplicated and the interdomain spacing were found to be critical considerations in the design of tPA variants with duplicated domains. We also rearranged the domains of tPA such that kringle 1 replaced the second kringle domain and vice versa. An analysis of these variants indicates that the first kringle domain can confer fibrin affinity to a tPA variant and function in place of kringle 2. Therefore, in wild-type tPA, the functions of kringle 1 and kringle 2 must be dependent partially on their orientation within the heavy chain of the protein. The functional autonomy of the heavy and light chains of tPA is demonstrated by the activity of a tPA variant in which the order of the heavy and light chains was reversed.
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PMID:Tissue-type plasminogen activator variants with domain duplications and rearrangements. 255 97


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