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

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.
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PMID:High resolution analysis of functional determinants on human tissue-type plasminogen activator. 190 May 16

The steady-state kinetics of the amidolytic activity of single chain tissue-type plasminogen activator (tPA) were analyzed in the presence or absence of different molecular forms of fibrinogen degradation products. Single chain tPA showed a Km value of 1.6 mM and kcat value of 4.9/s toward the chromogenic substrate H-D-Ile-Pro-Arg-p-nitroanilide (S-2288). In the presence of infinite concentrations of fibrinogen, kinetic constant was calculated as about 8-times higher than that in the absence of fibrinogen, mainly caused by the decrease of Km value. The dissociation constant (Ka) for this stimulation by fibrinogen was 2.9 microM. When the same assay was conducted with fragment X or fragment D of fibrinogen, the kinetic constants increased 3.2 and 2.9-times, respectively, whereas no enhancement was obtained by fragment E. Neither lysine analogues nor monoclonal antibody toward domains of finger and epidermal growth factor of tPA quench the enhancement by fibrinogen. This enhancement was not observed in the case of the two chain form of tPA. These results indicate that fibrinogen enhances the amidolytic activity of single chain tPA by binding to kringle 2 domain or light chain through D domain of fibrinogen.
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PMID:Stimulation of the amidolytic activity of single chain tissue-type plasminogen activator by fibrinogen degradation products: possible fibrin binding sites on single chain tissue-type plasminogen activator molecule. 190 67

The effects of 4 monoclonal antibodies against human tissue-type plasminogen activator (t-PA) on binding of t-PA to lysine, fibrin, and heparin, and on fibrin-mediated activation of one-chain t-PA-amidolytic activity were investigated. The association constants of the antibodies were determined in a direct assay to be equal to 0.125 l/nmol, 0.225 l/nmol, 0.4 l/nmol, and 0.5 l/nmol for mAB 5, mAB 16, mAB 25, and mAB 31, respectively. All 4 monoclonal antibodies inhibited binding of intact t-PA to lysine-Sepharose and fibrin, and they suppressed fibrin-mediated activation of one-chain t-PA-amidolytic activity. Binding analysis demonstrated that mAB 25 inhibited t-PA binding to lysine-Sepharose and to fibrin as well as fibrin-mediated enhancement of one-chain t-PA-amidolytic activity in a competitive manner with inhibitor constants of 5 nmol/l, 3 nmol/l and 10 nmol/l, respectively. It was also shown that free lysine counteracts the association of t-PA with the antibodies. Binding of t-PA to heparin is only moderately affected by the 4 antibodies. Since t-PA possesses two homologous kringle domains which contain fibrin (lysine) binding sites, the results underline the importance of a lysine binding site for fibrin binding by intact t-PA and show that the binding of the enzyme to fibrin and lysine is mediated by the same binding site of a kringle domain. The parallel effects of antibodies on fibrin binding and on fibrin-mediated enhancement of one-chain t-PA amidolytic activity proves that the site of fibrin binding is identical with the site of fibrin activation. The binding site of heparin apparently differs from lysine and fibrin binding sites.
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PMID:Effects of monoclonal antibodies on tissue-type plasminogen activator (t-PA) binding to lysine, fibrin and heparin and on fibrin-mediated enhancement of one-chain t-PA amidolytic activity. 190 50

The sequence fibrinogen-A alpha-(148-160) can mimic part of the fibrin-induced rate enhancement of the activation of plasminogen by tissue-type plasminogen activator. Previously we have reported that the lysine residue at position A alpha-157 is crucial. During our further investigations on A alpha-157 we found that lysine at position A alpha-157 may be replaced by glutamic acid. This unexpected finding prompted us to re-investigate the requirements of this position. We prepared analogues of A alpha-(148-160) in which the lysine residue at position A alpha-157 was replaced by lysine derivatives (acetyl-lysine, benzyloxycarbonyl-lysine and methanesulphonylethyloxycarbonyl-lysine), acidic residues (aspartic acid and glutamic acid), basic residues (arginine and ornithine), polar residues (glutamine and methanesulphonylethyloxycarbonylornithine), apolar residues (alanine, valine, norleucine and glutamic acid 4-nitrobenzyl ester) and glycine. These analogues were tested for their stimulatory activity. When aspartic acid, glutamic acid 4-nitrobenzyl ester or norleucine is present at position A alpha-157 in A alpha-(148-160) virtually all stimulatory capacity is lost. With valine at position A alpha-157 the stimulatory activity is marginal. None of the other replacements at position A alpha-157 caused loss of rate-enhancing properties. From these results we conclude that for the rate-enhancing effect of A alpha-(148-160) the side chain of the amino acid residue at position A alpha-157 must fulfill certain requirements: there must be one (as in alanine) or no (as in glycine) carbon atom in the side chain, or at least two carbon atoms and a polar group (charged or uncharged) to which a rather bulky group (such as the benzyloxycarbonyl group) or a polar group (such as the methanesulphonylethyloxycarbonyl group) may be attached. The highest activity [even higher than native A alpha-(148-160)] was obtained with ornithine, methanesulphonylethyloxycarbonylornithine or methanesulphonylethyloxycarbonyl-lysine at position A alpha-157.
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PMID:Structural requirements of position A alpha-157 in fibrinogen for the fibrin-induced rate enhancement of the activation of plasminogen by tissue-type plasminogen activator. 190 25

The heparin-binding p30 protein amphoterin is proposed to mediate adhesive interactions of the advancing plasma membrane in migrating and differentiating cells. Since the NH2-terminal part of amphoterin is exceptionally rich in lysine residues, we have studied its interactions with plasminogen and tissue plasminogen activator (t-PA). On immunostaining of N18 neuroblastoma cells, amphoterin and t-PA showed a close co-localization in the filopodia of the leading membrane and in the substrate-attached material. In purified systems, both t-PA and plasminogen bound to immobilized amphoterin, and their binding was inhibited by the lysine analogue epsilon-aminocaproic acid. Plasminogen bound to immobilized amphoterin was activated by t-PA, and this resulted in effective degradation of the immobilized amphoterin. Correspondingly, amphoterin-bound t-PA activated plasminogen. In solution amphoterin accelerated t-PA-catalyzed plasminogen activation maximally 46-fold. The results indicate that t-PA and plasminogen form through their lysine-binding sites a complex with amphoterin, which results in acceleration of plasminogen activation and effective degradation of amphoterin. We suggest that local acceleration of t-PA-catalyzed plasminogen activation by amphoterin at the leading membrane enhances the penetration of growing cytoplasmic processes through extracellular materials during cell migration, differentiation and regeneration. The amphoterin-mediated adhesion at the leading membrane may be transient in nature, because the protein also enhances its own breakdown by accelerating t-PA-catalyzed plasminogen activation.
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PMID:Interactions of plasminogen and tissue plasminogen activator (t-PA) with amphoterin. Enhancement of t-PA-catalyzed plasminogen activation by amphoterin. 190 31

Fibrinogen-NDSK complex is a model of protofibril having some features of the fibrin polymer structure. This complex has been studied for its ability to stimulate the plasminogen activation by t-PA. The fibrinogen-NDSK complex have increased the rate of plasminogen activation by t-PA as compared to fibrinogen or NDSK taken separately. This acceleration had slow and fast phases. Lys-plasminogen was activated more effectively as compared to glu-plasminogen. The kinetic parameters of glu- and lys-plasminogen activation at fast phase were: Km--0.18 and 0.015 mu/M, Kkat--0.27 and 0.06 s-1, respectively. Fibrinogen X2--fragments, deprived of alpha C-domains and NH2-end peptides of bB-chains, formed complexes with NDSK, which however did not stimulate the plasminogen activation by t-PA. These findings have shown that the fibrinogen-NDSK complex is an effective stimulator of the plasminogen activation by t-PA. The activating ability of the complex may be due to structures formed in the course of fibrinogen and NDSK polymerization as a result of alpha C-domain interaction.
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PMID:[Plasminogen activation by a tissue activator and effector properties of fibrinogen-N-terminal disulfide (N-DSK) fibrin complex]. 192 82

Monocytes and monocytoid cell lines previously have been shown to express receptors for plasminogen and urokinase (u-PA). In the present study, the monocytoid cell lines, U937 and THP-1, are shown to bind tissue plasminogen activator (t-PA) in a specific, saturable, and reversible manner. These cells bound t-PA with low affinity (kd = 0.67 to 0.97 mumol/L) and high capacity (0.71 to 3.3 x 10(6) receptors/cell). Human peripheral blood monocytes bound t-PA with a kd (0.9 mumol/L) similar to that of the monocytoid cells but with a lower capacity (0.17 x 10(6) sites/cell). These binding parameters also were similar to the low-affinity interaction of t-PA with endothelial cells as measured with the cells in suspension (kd = 0.73 mumol/L and 1.1 x 10(6) sites/cell). Lysine analogues and active or diisopropylfluorophosphate-inactivated u-PA inhibited t-PA binding to monocytes, monocytoid cells, and endothelial cells with similar IC50 (concentration producing 50% inhibition) values, suggesting that the same recognition specificity mediates t-PA binding to all of these cell types. The existence of a high-affinity binding site for t-PA on monocytoid cells was also explored in detail. Unlike endothelial cells where plasminogen activator inhibitor-1 has been implicated in mediating a high-affinity interaction of t-PA with the cells, no evidence for a role of this inhibitor in ligand binding to the monocytoid cells was found. Furthermore, using both high and low 125I-t-PA concentrations, competition analyses with lysine analogues or u-PA, or treatment of the cells with carboxypeptidase B, failed to indicate the presence of distinguishable classes of t-PA binding sites. In sum, low-affinity receptors for t-PA are expressed at high density on monocytes and monocytoid cells, identifying a new element in the fibrinolytic arsenal of these cells.
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PMID:Binding of tissue plasminogen activator to human monocytes and monocytoid cells. 193 47

The contribution of His64 to the function and stability of tissue plasminogen activator (t-PA) kringle-2 domain (His244 in t-PA numbering) has been studied by using microcalorimetric methods to compare the ligand binding and thermal denaturation behavior of wild-type kringle-2 and mutants having His64 replaced with Tyr or Phe. This site was examined because modeling studies suggested that the His64 side chain could play an important role in ligand binding by forming an ion-pair with the carboxylate of the ligand, L-lysine. Kringle-2 domains were expressed by secretion of the 174-263 portion of t-PA in E. coli and purified as previously described for the wild-type domain. Both mutant proteins retain affinity for L-lysine, although reduced three- to four-fold relative to wild-type, demonstrating that His64 does not interact with the ligand carboxylate through an ion-pair interaction or by hydrogen bonding. The H64Y substitution does result in an altered specificity of the lysine binding site with the mutant domain having greatest affinity for a ligand of 6.8 A chain length, whereas the wild-type domain prefers an 8.8 A long ligand. For both wild-type and mutant, the binding of the optimal chain length ligand is dominated by enthalpic effects (delta H = -6,000 to -7,000 cal/mol) and T delta S accounts for less than 15% of delta G. In addition, the H64Y mutant differs from wild-type in the effect of ligand alpha-amino group modification on binding affinity. Based on examination of the x-ray structure recently determined for wild-type kringle-2, the specificity changes accompanying the H64Y substitution probably result from changes in side chain interactions in the lysine binding site. Thermal denaturation experiments show that the H64Y mutant is also more stable than the wild-type protein with the difference in stabilization free energy (delta delta G) equal to 2.7 kcal/mol at 25 degrees C and pH 3. The increased stability of the mutant appears to be related to the difference in hydrophobicity between His and Tyr.
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PMID:Thermodynamics of ligand binding and denaturation for His64 mutants of tissue plasminogen activator kringle-2 domain. 196

Modification of glutamic and aspartic acid residues of tissue-type plasminogen activator (t-PA) with 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide leads to a decrease in affinity for lysine and fibrin, to a decrease of plasminogen activation activity in the presence of a fibrin mimic, but leaves amidolytic activity and plasminogen activation without fibrin mimic unaffected. Experiments with kringle-2 ligands and a deletion mutant of t-PA (K2P) suggests that glutamic or aspartic acid residues in K2 of t-PA are involved in stimulation of activity, lysine binding and fibrin binding. Mutant t-PA molecules were constructed by site-directed mutagenesis in which one or two of the five aspartic or glutamic acid residues in K2 were changed to asparagine or glutamine respectively. Mutation of Asp236 and/or Asp238 leads to t-PA molecules with 3- to 4-fold lower specific activity in the presence of fibrin mimic and having no detectable affinity for lysine analogs. However, fibrin binding was not influenced. Mutation of Glu254 also leads to a 3- to 4-fold lower activity, but to a much smaller reduction of lysine or fibrin binding. Residues Asp236 and Asp238 are both essential for binding to lysine derivatives, while Glu254 might be involved but is not essential. Residues Asp236, Asp238 and Glu254 are all three involved in stimulation of activity. Remarkably, mutation of residues Asp236 and/or Asp238 appears not to influence fibrin binding of t-PA whereas that of Glu254 does.
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PMID:Involvement of aspartic and glutamic residues in kringle-2 of tissue-type plasminogen activator in lysine binding, fibrin binding and stimulation of activity as revealed by chemical modification and oligonucleotide-directed mutagenesis. 196 88

Thy-1 antigen is expressed at high levels in the thymus and in adult brain of rodents however its function remains undetermined. We report that immobilised Thy-1 binds laminin, fibronectin and the less active precursor form of the tissue type plasminogen activator (t-PA) yet it does not bind urokinase. The incorporation of serine protease inhibitors within the experimental procedures suggested that Thy-1 bound to the lysine-containing, protein-binding domain of t-PA thus leaving the active site available to interact with other proteins. By using an immunocytochemical approach designed to maximally preserve Thy-1 antigenicity, we were able to demonstrate that in the adult rat peripheral nervous system (PNS) Thy-1 was seen to co-localise with laminin on the Schwann cell membranes and accumulated at the nodes of Ranvier within sciatic nerve. The only neuronal structures to express Thy-1 within the PNS were the unmyelinated nerve fibres. In the adult rat central nervous system (CNS), the most distinct and novel association of Thy-1 was its presence along the myelin forming glial cells and their fibres.
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PMID:Thy-1 is a neuronal and glial surface antigen which interacts with matrix proteins and plasminogen activator. 198 Oct 41


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