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

The involvement of specific aspartic acid (D) and glutamic acid (E) residues of the recombinant (r) kringle 2 (K2) domain of tissue-type plasminogen activator (tPA) in stabilizing its interaction with omega-amino acid ligands has been assessed by examination of these binding events subsequent to site-directed mutagenesis of the relevant amino acid residues. We have expressed and purified nonconservative alanine (A) replacement mutants at the following amino acid sequence locations in r-K2tPA:E17 (r-[K2tPA/E17A]), E75 (r-[K2tPA/E75A]), and D78 (r-[K2tPA/D78A]). More conservative E for D replacements were generated at the only other anionic (at neutral pH) amino acids of r-[K2tPA], viz., D57 (r-[K2tPA/D57E]) and D59 (r-[K2tPA/D59E]). Each of these variant polypeptides was then utilized for binding investigations with a series of omega-amino acids. No substantial differences were found in the binding constants (pH 8.0, 25 degrees C) for the ligands, 6-aminohexanoic acid (6-AHxA), 7-aminoheptanoic acid (7-AHpA), L-lysine, and trans-(aminomethyl)cyclohexane-1-carboxylic acid (AMCHA), among wild-type (wt) r-K2tPA, r-[K2tPA/E17A], r-[K2tPA/E75A], and r-[K2tPA/D78A]. On the other hand, dramatic effects on this same binding were observed in recombinant mutants with alterations at D57 and D59. In these cases, even with the most conservative replacements, i.e., r-[K2tPA/D57E] and r-[K2tPA/D59E], the Kd values for these ligands were increased approximately 3-6-fold and 18-85-fold, respectively. NMR analysis of these variants suggested that no substantial gross conformational changes occurred as a result of the mutations made, but some localized alterations in amino acid microenvironments did take place.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Specific anionic residues of the recombinant kringle 2 domain of tissue-type plasminogen activator that are responsible for stabilization of its interaction with omega-amino acid ligands. 838 82

Adjuncts to thrombolytic agents have improved coronary patency and prevented early reocclusion after thrombolysis in acute myocardial infarction. The aim of this study was to compare in a canine thrombolysis model the effects of Ro 43-5054 = N-(N-[N-(p-amidinobenzoyl)-beta-alanyl]-L-alpha-aspartyl)-3- phenyl-L-alanine-trifluor-acetate, a new glycoprotein IIb-IIIa receptor antagonist with aspirin or heparin. Six groups of 10 dogs each were studied. A platelet-rich coronary thrombus was induced in open-chest dogs by electrical stimulation. In addition to recombinant tissue-type plasminogen activator (30 micrograms/kg.min during 60 min), the dogs received 1) saline, 2) heparin 200 U/kg + 50 U/kg.hr i.v., 3) aspirin 10 mg/kg i.v., 4) heparin+aspirin, 5) Ro 43-5054 (3 micrograms/kg.min) and 6) heparin+Ro 43-5054. The overall reperfusion rate was 70% (range, 60-90%) and comparable in all the six groups. During the 120-min observation period, episodes of reocclusion were observed in the absence of antiplatelet therapy (group 1 and 2) irrespective of heparin treatment. Aspirin prevented coronary reocclusion in half of the reperfused dogs (group 3 and 4). However, after reinforcement of the thrombogenic stimulus, 80% of the reperfused dogs treated with aspirin showed reocclusion, whereas none of them reoccluded when treated with Ro 43-5054. Thus, inhibition of platelet activation by the selective, nonpeptidic glycoprotein IIb-IIIa receptor antagonist Ro 43-5054, although without effect on the time to reperfusion, better protected than aspirin against early reocclusion after thrombolytic therapy.
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PMID:Effects of heparin, aspirin and a synthetic platelet glycoprotein IIb-IIIa receptor antagonist (Ro 43-5054) on coronary artery reperfusion and reocclusion after thrombolysis with tissue-type plasminogen activator in the dog. 842 48

Inactivation by slow acting inhibitors in plasma is of little consequence for thrombolysis with wild type t-PA, since it is rapidly cleared from the blood stream and constantly replenished through infusion. However, it becomes increasingly important as the clearance rate of t-PA is reduced, through mutagenesis, to enable the molecule to be long acting and administered by a single bolus injection. The substitution of serine for alanine at position 473 substantially reduced the slow inactivation that occurs at pharmacological levels of t-PA in plasma. Approximately 70% of the activity of A473S remained after 4 h incubation in human plasma compared to approximately 25% for wild type t-PA. Wild type t-PA and A473S showed the same stability in alpha-2-antiplasmin depleted plasma, indicating that the resistance of A473S to inactivation is a result of reduced reactivity towards alpha-2-antiplasmin, the primary slow acting inhibitor of t-PA. The second order rate constant for the inactivation of A473S by purified alpha-2-antiplasmin was approximately 4 fold less than that of wild type t-PA, which is consistent with the results obtained in plasma. Substitution of threonine at position 473 also produced inhibitor resistance, but glycine did not. The substitution of charged or bulky residues at position 473 destroyed enzymatic activity. The mechanism of inhibitor resistance for A473S and A473T appears to be a reduced reactivity towards substrates with arginine at the P1 position. The A473S mutation adds well to T103N, a mutation that causes an approximate 9 fold reduction in the clearance rate of t-PA. The double mutation variant, T103N, A473S had normal plasma clot lysis activity, and was stable in plasma over a 4 h incubation period at 37 degrees C in vitro.
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PMID:Substitution of serine or threonine at position 473 of tissue-type plasminogen activator increases its stability in plasma. 871 88

Variants of tissue-type plasminogen activator (t-PA) were constructed with selected cysteines replaced by alanine to evaluate the role of an unpaired cysteine, which has been presumed to be in the growth factor module. C75A, C83A, C84A and CC83-84AA variants of t-PA were expressed transiently in human embryonic kidney cells. The biochemical properties of these variants provided experimental evidence to identify the unpaired cysteine in t-PA. Assays of amidolytic activity, plasminogen activation (in the presence or absence of fibrinogen or fibrin), plasma clot lysis, fibrin binding, clearance in mice, and interaction with a panel of monoclonal antibodies were performed as the basis for comparing these variants with wild-type t-PA. In all assays, C83A t-PA was biochemically equivalent to wild-type t-PA. C75A t-PA, C84A t-PA and CC83-84AA t-PA variants exhibited reduced activities in a variety of functional assays. These variants displayed two-to threefold lower activity in fibrinogen or fibrin stimulated plasminogen activation, and fivefold reduced plasma clot lysis activity compared with that of wild-type t-PA. The affinity of C75A t-PA and C84A t-PA for fibrin was decreased more than two orders of magnitude compared with C83A t-PA or wild-type t-PA. Plasma clearance of C75A t-PA and C84A t-PA was reduced 2-fold in mice. The C75A, C84A and CC83-84AA variants displayed significantly decreased reactivity with anti-tPA monoclonal antibodies specific for finger/growth factor domain epitopes. These data are consistent with a disulfide linkage of Cys75 with Cys84 and that Cys83 exists as an unpaired sulfhydryl. The significance of the unpaired cysteine is as yet undetermined since C83A t-PA and wild-type t-PA are functionally equivalent.
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PMID:Locating the unpaired cysteine of tissue-type plasminogen activator. 873 95

Surface-associated plasmin(ogen) may contribute to the invasive properties of various cells. Analysis of plasmin(ogen)-binding surface proteins is therefore of interest. The N-terminal variable regions of M-like (ML) proteins from five different group A streptococcal serotypes (33, 41, 52, 53 and 56) exhibiting the plasminogen-binding phenotype were cloned and expressed in Escherichia coli. The recombinant proteins all bound plasminogen with high affinity. The binding involved the kringle domains of plasminogen and was blocked by a lysine analogue, 6-aminohexanoic acid, indicating that lysine residues in the M-like proteins participate in the interaction. Sequence analysis revealed that the proteins contain common 13-16-amino-acid tandem repeats, each with a single central lysine residue. Experiments with fusion proteins and a 30-amino-acid synthetic peptide demonstrated that these repeats harbour the major plasminogen-binding site in the ML53 protein, as well as a binding site for the tissue-type plasminogen activator. Replacement of the lysine in the first repeat with alanine reduced the plasminogen-binding capacity of the ML53 protein by 80%. The results precisely localize the binding domain in a plasminogen surface receptor, thereby providing a unique ligand for the analysis of interactions between kringles and proteins with internal kringle-binding determinants.
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PMID:Identification of a plasminogen-binding motif in PAM, a bacterial surface protein. 874 39

Single-chain urokinase-type plasminogen activator or pro-urokinase is a zymogen with an intrinsic catalytic activity which is greater than that of most other zymogens. To study the structural basis for this activity, a three-dimensional homology model was calculated using the crystallographic structure of chymotrypsinogen, and the structure-function relationship was studied using site-directed mutagenesis and kinetic analysis. This model revealed a unique Lys300 in pro-urokinase which could form a weak interaction with Asp355, adjacent to the active site Ser356. It was postulated that this lysine, by its epsilon-amino group, may serve to pull Ser356 close to the active position, thereby inducing the higher intrinsic activity of pro-urokinase. This was consistent with the published finding that a homologous lysine (Lys416) in single chain tissue plasminogen activator when mutated to serine induced some reduction in activity. To test this hypothesis, a site-directed mutant with a neutral residue (Lys300-->Ala) was produced and characterized. The Ala300-pro-urokinase had a 40-fold lower amidolytic activity than that of pro-urokinase. It was also stable in plasma at much higher concentrations than pro-urokinase, reflecting much attenuated plasminogen activation. Plasmin activatability was comparable to that of pro-urokinase, but the resultant two-chain derivative (Ala300-urokinase) had a lower enzymatic activity (approximately 33% that of urokinase) due to a reduction of kcat. Interestingly, the KM of two-chain Ala300-urokinase against plasminogen was 5.8-fold lower than that of urokinase, being similar to that of pro-urokinase which has a KM about 5-fold lower than urokinase. In conclusion, the hypothesis that Lys300 is a key structural determinant of the high intrinsic activity of pro-urokinase was confirmed by these studies. This residue also appears to be important for the full expression of the enzymatic activity of urokinase.
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PMID:A site-directed mutagenesis of pro-urokinase which substantially reduces its intrinsic activity. 891 92

Human neutrophil elastase cleaves angiogenin at the Ile-29/Met-30 peptide bond to produce two major disulfide-linked fragments with apparent molecular weights of 10,000 and 4000, respectively. Elastase-cleaved angiogenin has slightly increased ribonucleolytic activity, but has lost its ability to undergo nuclear translocation in endothelial cells, a process essential for angiogenic activity. Cleavage appears to alter the cell-binding properties of angiogenin, despite the fact that it occurs some distance from the putative receptor-binding site, since the elastase-cleaved protein fails to compete with its native counterpart for nuclear translocation in endothelial cells. Plasminogen specifically accelerates elastase proteolysis of angiogenin. It does not enhance elastase activity toward ribonuclease A or the synthetic peptide substrate MeOSuc-Ala-Ala-Pro-Val-pNA. Plasminogen-accelerated inactivation of angiogenin by elastase might be a significant event in the process of angiogenin-induced angiogenesis since (i) angiogenin and plasminogen circulate in plasma at high concentrations, (ii) angiogenin, especially when bound to actin, activates tissue plasminogen activator to generate plasmin from plasminogen, and (iii) elastase cleaves plasminogen to produce angiostatin, a potent inhibitor of angiogenesis and metastasis. Interrelationships among angiogenin, plasminogen, plasminogen activators, elastase, and angiostatin may provide a sensitive regulatory system to balance angiogenesis and antiangiogenesis.
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PMID:Limited proteolysis of angiogenin by elastase is regulated by plasminogen. 933 Feb 25

The complex of the type-1 plasminogen activator inhibitor (PAI-1) and its target proteinases, the urokinase and tissue-type plasminogen activators (uPA and tPA), but not the free components, bind with high affinity to the endocytosis receptors alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha2MR/LRP) and very-low-density lipoprotein receptor (VLDLR). To characterize the molecular interaction between the complexes and the receptors, alanine codons were introduced into the human PAI-1 cDNA to replace the four basic residues, Arg-78, Lys-82, Arg-120 and Lys-124, as double mutations. The purified recombinant mutant proteins, rPAI-1/R78A-K124A and rPAI-1/K82A-R120A, produced by the yeast Pichia pastoris, were indistinghuisable from wild-type recombinant and natural human PAI-1 with respect to inhibitory activity against uPA, stability of SDS-resistant complexes with uPA, and vitronectin binding. Radiolabelled mutant uPA.PAI-1 complexes bound with a 10- to 20-fold, and 3- to 7-fold reduced affinity to purified alpha2MR/LRP and VLDLR respectively. alpha2MR/LRP-mediated endocytosis of the mutant complexes by COS-1 cells was reduced to 48 and 38% of the level of endocytosis of wild-type PAI-1. Binding of the mutant complexes to the uPA receptor was not affected. These findings suggest that the binding mode of the uPA.PAI-1 complex to both alpha2MR/LRP and VLDLR is similar. The four residues are surface exposed in the region defined by alpha-helix D and beta-strand 1A in the serine protease inhibitor (serpin) structure. Our study represents the first identification of residues in a surface region implicated in molecular recognition of protease.serpin complexes by endocytosis receptors of the low-density lipoprotein receptor family.
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PMID:Binding of urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex to the endocytosis receptors alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein and very-low-density lipoprotein receptor involves basic residues in the inhibitor. 940 75

Erythrina trypsin/tPA inhibitor (ETI) from the seeds of Erythrina caffra retains its native structure and inhibitory function after reducing its two disulfide bonds. In order to elucidate the specific role of these crosslinks, alanine residues were substituted for cysteines after cloning the gene in Escherichia coli. Expression of the recombinant inhibitor and the substitution mutants, C83A, CC39, 83AA, and CC132, 139AA, led to inclusion bodies. After solubilization in guanidinium-chloride (GdmCl)/dithiothreitol and oxidation in glutathione buffer, activity could be recovered at yields up to 80%. The mutant proteins exhibit full inhibitory function without detectable alterations of their native structure. However, their stability is reduced: at acid pH, where the oxidized natural inhibitor retains its native structure, the reduced wildtype protein and the mutants undergo at least partial denaturation, reflected by decreased pH ranges of stability: pH 5-7 for the reduced inhibitor, pH 2.5-8.5 for CC132, 139AA, and pH 3.5-8.5 for C83A and CC39, 83AA. Urea and GdmCl denaturation at pH 7 show hysteresis for both the oxidized inhibitor and the double mutant CC132, 139AA. In contrast, the reduced protein and the other mutants exhibit true equilibrium transitions at pH 7, with urea half-concentrations of 0.9 M and 1.9 M and GdmCl half-concentrations of 0.5 M and 1.0 M, respectively. The stability of Erythrina trypsin/tPA inhibitor follows the sequence: oxidized ETI > CC132, 139AA > CC39, 83AA and C83A > reduced ETI.
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PMID:Effect of disulfide bonds on the structure, function, and stability of the trypsin/tPA inhibitor from Erythrina caffra: site-directed mutagenesis, expression, and physiochemical characterization. 963 Sep 15

SERP-1 is a myxoma virus-encoded serpin, secreted from infected cells, that is required for virulence and has anti-inflammatory activity. We report that purified recombinant SERP-1 forms SDS-stable complexes with urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasmin, thrombin, and factor Xa. N-terminal sequencing confirmed Arg319-Asn320 as the site of reaction. Mutation of these residues to Ala-Ala abolished inhibitory activity but had no effect on the specific cleavage at Thr315-Leu316 seen with elastase and with cathepsin G. Kinetic analysis of the reactions with uPA, tPA, plasmin, thrombin, Xa, and C1s showed second-order rate constants to vary over 3 logs, from kinh = 3 x 10(5) M-1 s-1 with thrombin to approximately 600 M-1 s-1 with C1s, while steady-state inhibition constants ranged from KI = 10 pM with thrombin to approximately 100 nM with C1s. Stoichiometries of inhibition varied between SI = 1.4 +/- 0.1 for uPA to SI = 13 +/- 3 for thrombin. Analysis of the variations in inhibition kinetics shows that when serpins act at low concentrations, comparable with the target protease or with KI (as appears likely for SERP-1 in vivo), inhibitory specificity becomes less dominated by kinh and is increasingly dependent on partitioning within the branched reaction mechanism and on the lifetime of the inhibited complex.
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PMID:Inhibitory specificity of the anti-inflammatory myxoma virus serpin, SERP-1. 969 48


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