Functional characterization of monoclonal antibodies directed against fibrin binding domains of tissue-type plasminogen activator

Fibrin interacts with tissue-type plasminogen activator (tPA) via the finger and the kringle 2 domains. Three monoclonal antibodies against tPA, designated MPW3VPA, MPW6VPA, and MPW7VPA, which react with epitopes in the tPA molecule involved in fibrin binding, were characterized. The IgM monoclonal antibody MPW6VPA, directed against an epitope close to the finger and epidermal growth factor domains, stimulated plasminogen activation only in the absence of CNBr-fibrinogen fragments by increasing kcat in a dose-dependent fashion, an effect which was not restricted to the intact molecule. These results suggest that MPW6VPA mimics the initial effect of fibrin bound to the tPA molecule, which results in a change of kcat values. The MPW6VPA effect was reversed by another antibody, MPW3VPA, also directed against epidermal growth factor and finger domains. The latter antibody also inhibited plasminogen activation by tPA in the presence of CNBr-fibrinogen fragments in a dose-dependent, apparently noncompetitive way. No effect of MPW3VPA was seen in the absence of CNBr-fibrinogen fragments. MPW7VPA directed against kringle 2 of tPA inhibited plasminogen activation by tPA only when CNBr-fibrinogen fragments were present. This inhibition was apparently competitive and dose-dependent. These data suggest that MPW3VPA interferes with the first phase of fibrin binding to tPA, whereas MPW7VPA interferes with the second phase of fibrin binding to the tPA molecule via kringle 2, resulting in Km changes.     

Characterization of the binding of human tissue-type plasminogen activator to platelets

Cell surface binding sites for the constituent proteins of the fibrinolytic system may play a role in the localization and regulation of fibrinolysis. In the present study, specific binding of recombinant human tissue-type plasminogen activator (rt-PA) to human blood platelets was identified and characterized. 125I-labeled rt-PA was found to bind specifically, saturably, and reversibly to the surface of gel-filtered platelets, reaching equilibrium within 5 min at 22 degrees C. Scatchard analysis revealed a single class of binding sites. Unstimulated platelets bound 120,000 +/- 24,000 (mean +/- S.D.) molecules/platelet with an apparent Kd of 340 +/- 25 nM, whereas thrombin-stimulated platelets bound 290,000 +/- 32,000 molecules/platelet with an apparent Kd of 800 +/- 60 nM. Binding of 0.1 microM 125I-rt-PA was greater than 90% reversible by a 50-fold excess of unlabeled rt-PA. Binding was not inhibited by fibrinogen or single chain urokinase-type plasminogen activator, but plasminogen partially competed for binding of 125I-rt-PA to platelets (up to 40% displacement). These findings indicate that the platelet surface possesses a large number of specific, low affinity binding sites for t-PA and provide further evidence for the role of platelets in localization and regulation of fibrinolysis.     

Heterogeneity of human tissue-type plasminogen activator

Tissue-type plasminogen activator (t-PA) from human melanoma cells (Bowes) was purified by immunosorbent chromatography on affinospecific polyclonal antibodies and gel filtration in the presence of KSCN. The immunosorbent eluate contained three major components of greater than 200, 85 and 65 kDa, respectively. The 65 kDa t-PA component could be separated by gel filtration on Ultrogel AcA44 in the presence of KSCN to a pure preparation yielding a unique N-terminal amino acid sequence. Immunoblot analysis, using affinospecific antibodies against t-PA, was a specific and sensitive method to identify different types of t-PA (I-IV), as well as t-PA-inhibitor complexes and degradation products in unstimulated melanoma cell culture fluids. Furthermore, the t-PA preparations, produced by phorbol ester-treated melanoma cells, were free of type IV and thus differed physiochemically from the constitutively produced t-PA preparations. The composition of t-PA from mammalian cell cultures is thus more complex than hitherto described.     

组织型纤溶酶原激活剂的纯化制备

简述了用于大规模生产组织型纤溶酶原激活剂(tPA)的重组动物细胞及其培养工艺。从重组tPA的大规模、快速纯化的角度考虑,对tPA的纯化制备方法进行了简要评述。     

Structural domains of human tissue-type plasminogen activator that confer stimulation by heparin

Heparin has been shown recently to stimulate the activity of human tissue-type plasminogen activator (t-PA). To investigate this effect further, mutant proteins lacking various domains of t-PA were screened for the ability to be stimulated by heparin. Those mutants harboring either the finger domain or the 2nd kringle were found to have enhanced enzymatic activity in the presence of heparin. Only mutants containing these structures would bind to heparin-agarose beads; monoclonal antibodies directed against these domains blocked binding. The stimulatory effect of heparin was more pronounced in finger-containing mutants than kringle-2 proteins. Earlier results had localized the fibrin-binding domains to the same two structures. Unlike heparin, the 2nd kringle was shown to be more important than the finger for fibrin stimulation. Our results have implications for producing recombinant t-PA variants for use in thrombolytic therapy.     

High resolution analysis of functional determinants on human tissue-type plasminogen activator

Sixty-four variants of human tissue-type plasminogen activator (tPA) were produced using recombinant DNA techniques. Charged residues were converted to alanine in clusters of from one to four changes per variant; these clusters spanned all the domains of the molecule. The variants were expressed by mammalian cells and were analyzed for a variety of properties. Variants of tPA were found that had reduced activity with respect to each tested property; in a few cases increased activity was observed. Analysis of these effects prompted the following conclusions: 1) charged residues in the nonprotease domains are less involved in fibrin stimulation of tPA activity than those in the protease domain, and it is possible to increase the fibrin specificity (i.e. the stimulation of tPA activity by fibrin compared to fibrinogen) by mutations at several sites in the protease domain; 2) the difference in enzymatic activity between the one- and two-chain forms of tPA can be increased by mutations at several sites on the protease domain; 3) binding of tPA to lysine-Sepharose was affected only by mutations to kringle-2, whereas binding to fibrin was affected most by mutations in the other domains; 4) clot lysis was influenced by mutations in all domains except kringle-2; 5) sensitivity to plasminogen activator inhibitor-1 seems to reside exclusively in the region surrounding residue 300. A model of the tPA protease domain has been used to map some of the critical residues and regions.     

Binding of tissue-type plasminogen activator with human endothelial cell monolayers. Characterization of the high affinity interaction with plasminogen activator inhibitor-1

The formation and release of covalent complexes between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) limits the application of equilibrium radioligand binding analysis to characterize the interaction between t-PA and human umbilical vein endothelial cell (HUVEC) monolayers. To avoid this difficulty, we used a recombinant mutant of t-PA, S478A rt-PA, in which alanine has been substituted for the active-site serine. Although the mutant is incapable of covalently reacting with PAI-1, 125I-labeled S478A rt-PA binding to HUVEC monolayers is specific and reversible and is characterized by a high affinity (Kd of 1.5 nM) and a large number of sites (1.5 x 10(6)/cell). This binding was shown to occur through noncovalent interaction with PAI-1 in the HUVEC monolayer by the fact that a monoclonal anti-PAI-1 antibody (MA-7D4) completely blocked S478A rt-PA binding. Two solution-phase assays with recombinant PAI-1 (rPAI-1) confirmed this noncovalent interaction: complexes between 125I-S478A rt-PA and rPAI-1 could be isolated by immunoprecipitation with anti-PAI-1 antibodies, and S478A rt-PA competed with rt-PA for inactivation by rPAI-1. In contrast diisopropylphosphate rt-PA (in which the active site serine is chemically modified) showed minimal binding to HUVEC monolayers, as a result of impaired interaction with PAI-1, in the two assays. Thus, both wild-type rt-PA and S478A rt-PA interact with the HUVEC monolayer through PAI-1. With rt-PA this results in the formation of covalent rt-PA.PAI-1 complexes that are released from the monolayer into the supernatant. With S478A rt-PA this results in the formation of noncovalent complexes that remain associated with the HUVEC monolayer, thereby identifying a large pool of reactive PAI-1 molecules in the monolayer.     

Characterization of interaction of active-site serine mutants of tissue-type plasminogen activator with plasminogen activator inhibitor-1

To define determinants of interactions of tissue-type plasminogen activator (t-PA) with plasminogen activator inhibitor type-1 (PAI-1), we utilized site-directed mutagenesis to substitute either threonine or glycine for the active-site serine of tissue-type plasminogen activator. Assays of conditioned media of transfected cells demonstrated that the threonine substitution markedly decreased but did not entirely abolish plasminogen activating activity. In contrast, the glycine substitution yielded a mutant with absolutely no detectable plasminogen activating activity. Wild-type t-PA formed stable complexes with PAI-1. However, even when exogenous inhibitor was present in the medium or purified mutant was added to plasma that had been rendered PAI-1-rich in vivo, the mutants were present in the free form exclusively judging from results of fibrin autography and Western blot analysis. Thus, despite maintenance of some residual plasminogen-activating activity associated with preservation of the hydroxyl group at the active site, the threonine mutant did not form stable complexes with inhibitor. The glycine mutant, developed so that steric hindrance or other unfavorable interactions at the modified active site would be minimal, was similarly incapable of forming complexes with PAI-1. These results show that the presence of an active site serine residue is necessary for formation of stable complexes between t-PA and PAI-1.     

Characterization of the interaction in vivo of tissue-type plasminogen activator with liver cells

The interaction in vivo of 125I-labeled tissue-type plasminogen activator (t-PA) with the rat liver and the various liver cell types was characterized. Intravenously injected 125I-t-PA was rapidly cleared from the plasma (t1/2 = 1 min), and 80% of the injected dose associated with the liver. After uptake, t-PA was rapidly degraded in the lysosomes. The interaction of 125I-t-PA with the liver could be inhibited by preinjection of the rats with ovalbumin or unlabeled t-PA. The intrahepatic recognition site(s) for t-PA were determined by subfractionation of the liver in parenchymal, endothelial, and Kupffer cells. It can be calculated that parenchymal cells are responsible for 54.5% of the interaction of t-PA with the liver, endothelial cells for 39.5%, and Kupffer cells for only 6%. The association of t-PA with parenchymal cells was not mediated by a carbohydrate-specific receptor and could only be inhibited by an excess of unlabeled t-PA, indicating involvement of a specific t-PA recognition site. The association of t-PA with endothelial cells could be inhibited 80% by the mannose-terminated glycoprotein ovalbumin, suggesting that the mannose receptor plays a major role in the recognition of t-PA by endothelial liver cells. An excess of unlabeled t-PA inhibited the association of 125I-t-PA to endothelial liver cells 95%, indicating that an additional specific t-PA recognition site may be responsible for 15% of the high affinity interaction of t-PA with this liver cell type. It is concluded that the uptake of t-PA by the liver is mainly mediated by two recognition systems: a specific t-PA site on parenchymal cells and the mannose receptor on endothelial liver cells. It is suggested that for the development of strategies to prolong the half-life of t-PA in the blood, the presence of both types of recognition systems has to be taken into account.     

Characterization of a tissue-type plasminogen activator from porcine urine

Porcine urine, unlike human urine, does not contain detectable amounts of urokinase-type plasminogen activator (u-PA). The plasminogen activator present in porcine urine is of tissue-type (t-PA) as identified by the following criteria. (1) Porcine urine PA exhibits an Mr of 65,000 similar to the Mr of human t-PA (64-70,000) but distinct from the Mr of human u-PA (55,000). (2) Antibodies against human t-PA bind and inhibit crude and purified porcine urine PA, while human u-PA-specific antibodies do not react with porcine urine PA. (3) Plasminogen activation by porcine urine PA is markedly stimulated in the presence of fibrinogen fragments. (4) Porcine urine PA activity is not affected by concentration of amiloride substantially suppressing human u-PA activity.     

Interactions between the finger and kringle-2 domains of tissue-type plasminogen activator and plasminogen activator inhibitor-1.

We have shown that plasminogen activator inhibitor-1 (PAI-1) inhibits the fibrin binding of both the single chain and two chain forms of tissue-type plasminogen activator (tPA) through two different mechanisms. PAI-1 inhibits the finger domain-dependent fibrin binding of diisopropylfluorophosphate-inactivated single chain tPA and the kringle-2 domain-dependent fibrin binding of diisopropylfluorophosphate-inactivated two chain tPA. In accordance with the data, preformed complexes of single chain tPA/PAI-1 and of two chain tPA/PAI-1 lost the fibrin binding abilities mediated by the finger and kringle-2 domains, respectively. These effects of PAI-1 appear to be mediated by steric hindrance of the fibrin binding sites after PAI-1 binding to adjacent regions in the functional domains of tPA. We thus propose a model in which a PAI-1 binding site resides in the finger domain of a single chain, and plays a role in the reversible association of single chain tPA and PAI-1. Conformational changes may take place during the conversion of single chain tPA to two chain tPA, resulting in burying of the original PAI-1 binding site and exposure of an alternate PAI-1 binding site on the surface of the kringle-2 domain.     

Biochemical and functional characterization of human tissue-type plasminogen activator variants obtained by deletion and/or duplication of structural/functional domains

Five cDNA encoding human tissue-type plasminogen activator (t-PA) variants with deletion and/or duplication of structural/functional domains were cloned and expressed in Chinese hamster ovary cells. The mutants included: rt-PA-delta FE (where r represents recombinant), with deletion of the finger (F) and growth factor (E) domains; rt-PA-delta K1 delta K2, with replacement of kringle 1 (K1) by a second copy of kringle 2 (K2); and rt-PA-delta FK1 delta K2, rt-PA-delta EK1 delta K2, and rt-PA-delta FEK1 delta K2, with deletions in rt-PA-delta K1 delta K2 of the finger or growth factor domain or both, respectively. The variant rt-PAs, purified to homogeneity, were obtained essentially as single-chain molecules. CNBr-digested fibrinogen enhanced plasminogen activation between 110-fold with rt-PA-delta EK1 delta K2 and 150-fold with rt-PA-delta FEK1 delta K2 as compared to 140-fold with rt-PA. All rt-PA moieties showed a comparable concentration-dependent binding to fibrin, except rt-PA-delta FE, which had significantly reduced binding that was, however, partially restored by additional replacement of K1 with K2. All the rt-PA variants with two copies of K2 showed increased binding to lysine-Sepharose as compared to rt-PA, whereas rt-PA-delta FE had reduced binding. All rt-PA moieties induced a similar time- and concentration-dependent lysis of a 125I-fibrin-labeled plasma clot immersed in human plasma. Equally effective concentrations (causing 50% clot lysis in 2 h) ranged between 1.0 microgram/ml for rt-PA-delta K1 delta K2 and 1.6 micrograms/ml for rt-PA-delta FE as compared to 0.5 microgram/ml for rt-PA. Thus, replacement in rt-PA of K1 by a second copy of K2, which is known to contain a lysine-binding site, significantly enhances its affinity for lysine, with maintenance of its affinity for intact fibrin. Deletion of the finger and growth factor domains results in decreased fibrin affinity and fibrinolytic potency in a plasma milieu, which are partially restored by replacement of K1 by K2.     

On the reversible interaction of plasminogen activator inhibitor-1 with tissue-type plasminogen activator and with urokinase-type plasminogen activator

The reaction between plasminogen activators and plasminogen activator inhibitor-1 is characterized by an initial rapid formation of an inactive reversible complex. The second-order association rate constant (k1) of complex formation of recombinant two-chain tissue-type plasminogen activator (rt-PA) or recombinant two-chain urokinase-type plasminogen activator (rtcu-PA) by recombinant plasminogen activator inhibitor-1 (rPAI-1) is 2.9 +/- 0.4 x 10(7) M-1 s-1 (mean +/- S.D., n = 30) and 2.0 +/- 0.6 x 10(7) M-1 s-1 (n = 12), respectively. Different molecular forms of tissue- or urokinase-type plasminogen activator which do not form covalent complexes with rPAI-1, including rt-PA-Ala478 (rt-PA with the active-site Ser478 mutagenized to Ala) and anhydro-urokinase (rtcu-PA with the active-site Ser356 converted to dehydroalanine) reduced k1 in a concentration-dependent manner, compatible with 1:1 stoichiometric complex formation between rPAI-1 and these ligands. The apparent dissociation constant (KD) of the complex between rPAI-1 and rt-PA-Ala478, determined as the concentration of rt-PA-Ala478 which reduced k1 to 50% of its control value, was 3-5 nM. Corresponding concentrations of active-site-blocked two-chain rt-PA were 150-250-fold higher. The concentration of anhydro-urokinase which reduced k1 to 50% was 4-6 nM, whereas that of active-site-blocked rtcu-PA was 100-250-fold higher. Recombinant single-chain urokinase-type plasminogen activator had an apparent KD of about 2 microM. These results suggest that inhibition of rt-PA or rtcu-PA by rPAI-1 proceeds via a reversible high affinity interaction which does not require a functional active site but which is markedly reduced following inactivation of the enzymes with active-site titrants.     

Variants of human tissue-type plasminogen activator that lack specific structural domains of the heavy chain.

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.     

Mapping the functional domains of human transcobalamin using monoclonal antibodies

Recombinant human transcobalamin (TC) was probed with 17 monoclonal antibodies (mAbs), using surface plasmon resonance measurements. These experiments identified five distinct epitope clusters on the surface of holo-TC. Western blot analysis of the CNBr cleavage fragments of TC allowed us to distribute the epitopes between two regions, which spanned either the second quarter of the TC sequence GQLA...TAAM(103-198) or the C-terminal peptide LEPA...LVSW(316-427). Proteolytic fragments of TC and the synthetic peptides were used to further specify the epitope map and define the functional domains of TC. Only one antibody showed some interference with cobalamin (Cbl) binding to TC, and the corresponding epitope was situated at the C-terminal stretch TQAS...QLLR(372-399). We explored the receptor-blocking effect of several mAbs and heparin to identify TC domains essential for the interaction between holo-TC and the receptor. The receptor-related epitopes were located within the TC sequence GQLA...HHSV(103-159). The putative heparin-binding site corresponded to a positively charged segment KRSN...RTVR(207-227), which also seemed to be necessary for receptor binding. We conclude that conformational changes in TC upon Cbl binding are accompanied by the convergence of multiple domains, and only the assembled conformation of the protein (i.e. holo-TC) has high affinity for the receptor.     

A monoclonal antibody against human urokinase: the epitope structure and sequence homology with a human tissue-type plasminogen activator

Our previous study showed that an epitope defined by a monoclonal antibody against human urokinase is located on the 33-Kdalton catalytic domain of the enzyme (Nakamura, M. et al., Cell Struct Funct., 9, 167-179, 1984). The epitope structure was further determined and characterized on one-dimensional SDS-polyacrylamide slab gel maps of CNBr-cleaved polypeptide fragments as well as on their Western blots. A single homogeneous polypeptide with an approximate molecular weight of 3.4-Kdaltons was found to be antigenic. The monoclonal antibody exhibited a stronger inhibition of the enzyme activity than the polyclonal antibodies tested, and cross-reacted with a 65-Kdalton tissue-type plasminogen activator present in Detroit 562 cells. From these results and data made up with the help of a computer comparison of known sequences of urokinase and a tissue-type plasminogen activator, we concluded that the epitope is Cys-Gln-Gly-Asp-Ser-Gly-Gly-Pro-Leu-Val-Cys and contains a catalytically active residue, serine.     

Isolation and functional characterization of the heavy and light chains of human tissue-type plasminogen activator

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.     

A protease-hypersensitive deletion derivative of human tissue-type plasminogen activator

We have constructed amplified Chinese hamster ovary cell lines constitutively synthesizing human tissue-type plasminogen activator (t-PA) or a derivative in which the domains homologous to epidermal growth factor and kringle 1 have been removed [delta(G + K1)]. The properties of the secreted proteins were investigated when synthesized in the presence or absence of the serine protease inhibitor aprotinin in the medium. t-PA in the culture supernatants was either single-chain or two-chain protein. The protease activity of both forms was stimulated by fibrin. The biochemical properties of delta(G + K1) were significantly different when harvested from cells grown under different culturing conditions. Protease activity of delta(G + K1) was stimulated ten- to 20-fold by fibrin when harvested from medium with aprotinin, but was stimulated only two- to three-fold when aprotinin was absent from the serum. Characterization of the secreted proteins revealed that the heavy-chain equivalent of delta(G + K1) is degraded when serine protease inhibitor is absent in the culture medium. These results indicate that the functional and biochemical properties of restructured versions of t-PA may depend on the presence of protease(s) in the culture supernatants.     

Differentiation-dependent localisation of tissue-type plasminogen activator in human bladder urothelium

Human bladder urothelium is able to secrete tissue-type plasminogen activator (tPA). The aim of our study was to analyse localisation of tPA antigen in comparison to differentiation state of cells in samples of histologically normal urothelium and non-invasive tumours of the human urinary bladder. Twenty-five samples of normal urothelium and 31 non-invasive papillary tumours from 36 patients were examined. The presence of tPA antigen was evaluated immunohistochemically. Differentiation of superficial cells was assessed by the presence of urothelial cell differentiation markers, uroplakins (UPs; immunohistochemistry) and cell's apical surface architecture (scanning electron microscopy). All tissue samples stained anti-tPA positive. In normal urothelium, the intensity of anti-tPA staining was the strongest in superficial cells, which were well-differentiated. In tumours, all cell layers stained anti-tPA positive. The intensity of anti-tPA positive reaction in the upper cell layer correlated with the percentage of anti-UP positive superficial cells. Superficial cells showed various differentiation states. The localisation of tPA antigen in human in vivo tissue is not confined to the well-differentiated superficial cells. Our results suggest a positive correlation between tPA secretion and cell differentiation.