Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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)
 ...
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.
 ...
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
 ...
PMID:Fibrin affinity and clearance of t-PA deletion and substitution analogues. 211 Oct 48

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.
 ...
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.
 ...
PMID:Monoclonal antibodies directed against human tissue-type plasminogen activator: a characterization of their species specificity, affinity and heavy-chain binding. 243 3

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.
 ...
PMID:Recombinant antibodies possessing novel effector functions. 251 68

The heavy chain of tissue plasminogen activator (t-PA) consists of four domains [finger, epidermal-growth-factor (EGF)-like, kringle 1 and kringle 2] that are homologous to similar domains present in other proteins. To assess the contribution of each of the domains to the biological properties of the enzyme, site-directed mutagenesis was used to generate a set of mutants lacking sequences corresponding to the axons encoding the individual structural domains. The mutant proteins were assayed for their ability to hydrolyze artificial and natural substrates in the presence and absence of fibrin, to bind to lysine-Sepharose and to be inhibited by plasminogen activator inhibitor-1. All the deletion mutants exhibit levels of basal enzymatic activity very similar to that of wild-type t-PA assayed in the absence of fibrin. A mutant protein lacking the finger domain has a 2-fold higher affinity for plasminogen than wild-type t-PA, while the mutant that lacks both finger and EGF-like domains is less active at low concentrations of plasminogen. Mutants lacking both kringles neither bind to lysine-Sepharose nor are stimulated by fibrin. However, mutants containing only one kringle (either kringle 1 or kringle 2) behave indistinguishably from one another and from the wild-type protein. We conclude that kringle 1 and kringle 2 are equivalent in their ability to mediate stimulation of catalytic activity by fibrin.
 ...
PMID:Variants of human tissue-type plasminogen activator that lack specific structural domains of the heavy chain. 284 82

Two-chain tissue-type plasminogen activator (t-PA), which consists of a heavy chain (Mr congruent to 38,000) and a light chain (Mr congruent to 31,000) connected by a disulfide bridge, was reduced with 2-mercaptoethanol and then air-reoxidized at a low protein concentration and carboxamidomethylated. The two chains were separated by means of zinc chelate-agarose, which was found to bind the light chain selectively. The light chain was fully active on the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine p-nitroanilide (S-2288) and partially active on plasminogen. The plasminogen activator activity of the light chain was, in contrast to that of two-chain t-PA, not stimulated by fibrin or fibrinogen fragments. Fibrin-agarose chromatography of radiolabeled chains showed that only the heavy chain bound to fibrin. These results indicate that the active site-containing light chain in t-PA needs the heavy chain for fibrin stimulation of its plasminogen activator activity.
 ...
PMID:Isolation and functional characterization of the heavy and light chains of human tissue-type plasminogen activator. 308 1

Transfected mouse Ltk- cells were employed for transient expression of recombinant human tissue-type plasminogen activator (t-PA; EC 3.4.21.31) or of recombinant-t-PA deletion proteins, encoded by SV40-pBR322-derived t-PA cDNA plasmids. The t-PA cDNA deletion mutants have two features in common, i.e., cDNA programming the signal peptide and the coding region for the light chain. Consequently, recombinant t-PA mutant proteins are efficiently secreted and display plasminogen activator activity. The gene encoding the amino-terminal heavy chain [an array of structural domains homologous to other plasma proteins (finger, epidermal growth factor, and kringle domains)] was mutated using restriction endonucleases to delete one or more structural domains. The stimulatory effect of fibrinogen fragments on the plasminogen activator activity of t-PA was demonstrated to be mediated by the kringle K2 domain and to a lesser extent by the finger/epidermal growth factor region but not by the kringle K1 domain. These data correlate well with the fibrin-binding properties of the recombinant t-PA deletion proteins, indicating that the stimulation of the activity by fibrinogen fragments is based on aligning the substrate plasminogen and t-PA on the fibrin matrix. Our results support the evolutionary concept of exon shuffling, arranging structural domains that constitute autonomous functions of the protein.
 ...
PMID:Autonomous functions of structural domains on human tissue-type plasminogen activator. 308 64

In order to assess which part of the tissue-type plasminogen activator (t-PA) molecule should be (genetically) modified to obtain more-slowly-clearing mutants, two-chain t-PA and its isolated heavy and light chains were radiolabelled and injected into rats. The vast majority of t-PA and the heavy chain disappeared from the blood circulation with half-lives of 2.3 and 1.0 min respectively. The clearance of the light chain was biphasic, owing to complex-formation with plasma proteinase inhibitors. The disappearance of di-isopropylphospho-light chain, which has a blocked active site, was nearly monophasic, with a half-life of 5.7 min. Organ distribution studies showed that hepatic clearance constituted the major pathway in all cases. These results strongly suggest that t-PA is recognized by the liver primarily through the heavy chain.
 ...
PMID:Clearance of the heavy and light polypeptide chains of human tissue-type plasminogen activator in rats. 309 71


1 2 3 4 Next >>