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)

Coronary artery reocclusion after thrombolysis with human recombinant tissue-type plasminogen activator (rt-PA) is related to the short half-life of this agent in plasma. K2P, a mutant of rt-PA lacking the fibronectin fingerlike, epidermal growth factor-like and first kringle domains (amino acids 6 to 173) and having the glycosylation site Asn184 mutagenized to Gln, has been produced in Chinese hamster ovary cells. In this study we compared the thrombolytic effect of K2P and rt-PA in dogs with electrically induced coronary artery thrombosis. Both agents were given intravenously in equimolar amounts over 20 min after the occlusive thrombus was stable for 30 min; dogs were monitored for 1 h after reperfusion if flow occurred. Coronary blood flow was restored by rt-PA in 6 (60%) of 10 dogs. The restored flow lasted for 49 +/- 12 min and mean flow at 60 min from the start of reperfusion was 7 +/- 3 ml/min. The reocclusion rate was 50% (three of six dogs). Flow was restored in five (100%) of five dogs by K2P. The restored blood flow lasted during the entire 1-h observation period in all but one dog and mean flow at 60 min was 49 +/- 16 ml/min (p less than 0.02 vs. flow in rt-PA-treated dogs). Restored coronary blood flow showed marked cyclic flow variations in rt-PA-treated but not in K2P-treated dogs.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Sustained reflow in dogs with coronary thrombosis with K2P, a novel mutant of tissue-plasminogen activator. 160 30

A modified variant of human tissue-type plasminogen activator (t-PA) lacking the finger domain (F), the growth factor domain (G) and the first kringle domain (K1), has an extended plasma half-life in vivo, compared to that of t-PA. When the variant (denoted K2P) was tested in vitro for its ability to lyse human plasma clots we found that the activity was characterized by a time lag phase and a sigmoidal dose-response curve. However, an attenuation of the lag phase in vitro was observed both when K2P was mixed with t-PA in a w/w ratio of 4:1 and when K2P was allowed to lyse a clot that had been pre-exposed to t-PA i.e. submitted to a limited plasmic digestion. Dosis that in vitro caused 50% lysis within 6 h were calculated from individual dose-response curves and were for K2P, t-PA and K2P/t-PA (4:1 w/w) 540 ng/ml, 360 ng/ml and 310 ng/ml, respectively. These results indicated a synergistic effect between K2P and t-PA. However, the data from individual dose-response curves showed that the effect of the K2P/t-PA mixture never was better than that of t-PA alone, and the synergistic effect in vitro is therefore considered to be of limited use. The thrombolytic activity in vivo was evaluated in a rabbit jugular vein thrombus model. Despite the lag phase observed in vitro, K2P was approximately 3 times as effective as t-PA in vivo (bolus injection). The thrombolytic effect of K2P was further potentiated when it was administered together with a small amount of t-PA (4:1 w/w).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Synergism between tissue-type plasminogen activator and a genetically engineered variant lacking the finger domain, the growth factor domain and the first kringle domain. 171 Aug 37

A gene encoding a variant (lacking amino acids 6-173) of human tissue-type plasminogen activator (t-PA), consisting only of the second kringle domain (K2) and the serine protease domain (P), was fused to a DNA segment coding for the signal peptide of staphylococcal protein A and a synthetic gene coding for a protein with ability to bind immunoglobulin G (IgG). The fusion protein which was synthesized in Escherichia coli and secreted into the growth medium, was found to be fibrinolytically active. Purification of the fusion protein was performed in a single step by affinity chromatography with immobilized IgG. Enzymatically active K2P was liberated from the fusion protein by cleavage at a unique Asn-Gly dipeptide sequence using hydroxylamine. These results demonstrate that a variant of human t-PA can be synthesized and secreted by E. coli as a fibrinolytically active fusion protein, which upon specific cleavage yields an active variant t-PA of the expected size.
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PMID:Synthesis and secretion of a fibrinolytically active tissue-type plasminogen activator variant in Escherichia coli. 190 33

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

To describe the role of the lysyl binding site in the interaction of tissue-type plasminogen activator (t-PA, FGK1K2P) with a forming fibrin clot, we performed binding experiments with domain deletion mutants GK1K2P, K2P, and the corresponding point mutants lacking the lysyl binding site in the absence and the presence of epsilon-amino caproic acid (EACA). Occupation of the lysyl binding site in the K2 domain with EACA has a pronounced effect on the binding of FGK1K2P to a fibrin clot (C50 = 77 +/- 11 nM versus 376 +/- 45 nM with EACA). Deleting the lysyl binding site in the K2 domain (substitution D236N) also impairs fibrin binding but to a lesser extent (C50 = 169 +/- 20 nM). Although the binding of K2P to a fibrin clot is weak (C50 = 1163 +/- 490 nM), it still is 2 orders of magnitude stronger than the binding of EACA to K2P. Therefore it was surprising to find that deletion of the lysyl binding site in K2P completely abolishes fibrin binding. Even when both the F domain and the lysyl binding site were deleted, considerable fibrin binding is still observed (C50 = 557 +/- 126 nM), suggesting other than F and K2-mediated interactions. The binding of FGK1K2P, FGK1K2P (D236N), GK1K2P, and GK1K2P (D236N) to fibrin could be competitively inhibited by FGK1K2P and K2P, indicating that all molecules recognize the same interaction sites on a fibrin clot. Based on these results, a new model for the interaction of t-PA with a forming fibrin clot is proposed. The fibrin binding sites in t-PA are not confined to the F and K2 domain. The main role of the lysyl binding site in the K2 domain of t-PA appears indirect rather than direct, most likely stabilizing a conformation favorable for fibrin binding.
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PMID:The role of the lysyl binding site of tissue-type plasminogen activator in the interaction with a forming fibrin clot. 775 76

We have created a novel thrombolytic agent by the combination of mutation with partial deletion of tissue-type plasminogen activator (t-PA). We constructed Escherichia coli expression vectors for (i) native t-PA (nt-PA) and its derivatives; (ii) K1K2P, consisting of kringle 1 (K1), kringle 2 (K2), and protease (P) domains; (iii) K2P, consisting of K2 and P domains; (iv) D-nt-PA; (v) D-K1K2P; and (vi) D-K2P. The latter three are point mutants of nt-PA, K1K2P, and K2P, respectively, in which Arg275 (number corresponds to that of nt-PA) has been mutated to Asp. The production of nt-PA and its derivatives was remarkably improved by (i) removal of the 3' noncoding region of nt-PA cDNA from expression vectors and (ii) expression in mutant E. coli derived from E. coli HB101, which is insensitive to heat-shock inductions. The proteins produced were precipitated as insoluble aggregates in the cells and were renatured to active forms by extraction with 8 M urea followed by dialysis against a redox buffer containing GSH and GSSG. The renaturation yield depended on the pH of the buffer and the number of disulfide bonds of the proteins (nt-PA << K1K2P < K2P). The mutation of Arg275 (the plasmin cleavage site) caused an increase in the catalytic enhancement by fibrin and a decrease of the interaction with plasminogen activator inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Production and characterization of a novel tissue-type plasminogen activator derivative in Escherichia coli. 776 73

Two different techniques have been used to study the complex formation of recombinant human plasminogen activator inhibitor type-1, PAI-1, with either recombinant human two-chain tissue plasminogen activator, tc tPA (EC 3.4.21.68), or the tPA deletion variants tc K2P, containing the kringle 2 domain and the proteinase domain, and P, containing only the proteinase domain. The same value for Kon, 2.10(7) M-1s-1 for binding of PAI-1 was found for the three tPA forms by direct detection of the complex formation in real time by surface plasmon resonance, BIAcore, or indirectly by monitoring the time course of the inhibition of tPA using the chromogenic substrate N-methylsulfonyl-D-Phe-Gly-Arg-4-pNA-acetate. Apparently, no conformational change is involved in the rate-limiting step, since the kon value was found to be independent of the temperature from 20 to 35 degrees C. By the BIAcore technique, it was found that the complex between PAI-1 and tPA covalently coupled to the surface, was stable at 25 degrees C, since no dissociation was seen in buffer. However, extended treatment with 1 M NH4OH destroyed the complex with t 1/2 = 5 h. The same kon values and complex composition were found by measuring either the binding of tPA to PAI-1 captured on the monoclonal antibody MAI-11 or the binding of PAI-1 to tPA captured on the monoclonal antibody 2:2 B10. Quantification of the complex composition between PAI-1 captured on the monoclonal antibody MAI-11 with either tPA, K2P or P gave a one-to-one ratio with the fraction of active PAI-1, consistent with the results from SDS-PAGE and the specific activity of PAI-1. The complexes of the three tPA forms with PAI-1 captured on a large surface of MAI-11 dissociated more rapidly from MAI-11, with the same apparent koff, kdis, = 2.10(-3) s-1, compared with 0.7-10(-3) s-1 for the dissociation of PAI-1 alone. In consistance, the Kd, calculated from the direct determination of the kon and koff for the association of different form of PAI-1 to a small surface of MAI-11, was found to be higher for PAI-1 in complex with tPA than for free active PAI-1. Apparently, upon complex formation, a change is induced in PAI-1 at the binding epitope for MAI-11.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Plasminogen activator inhibitor type-1 interacts exclusively with the proteinase domain of tissue plasminogen activator. 781 90

The way in which three thrombolytic agents, tissue-type plasminogen activator (t-PA), streptokinase (SK) and a genetically engineered variant of t-PA composed of the second kringle and the protease domain (K2P), cause the dissolution of haemostatic plugs of differing ages was investigated in a novel rabbit model. Standardized incisions were made on the rabbit ear and the wounds were left to heal for 0.5 h or 24 h, before the thrombolytic agents were infused. In the absence of heparin, t-PA showed little discrimination between clots of different ages (36% and 28% lysis of the 0.5 h and 24 h wounds, respectively). In contrast, K2P and SK showed a pronounced fresh clot selectivity since they were significantly more effective in lysing fresh clots than old ones (68% and 4% lysis for K2P and 72% and 36% for SK, respectively). In the presence of heparin the potency of t-PA on fresh clots was considerably increased whilst the effect on old clots was not affected, a fresh clot selectivity for t-PA (64% lysis of fresh clots, 24% lysis of old clots) was thus effected. Heparin did not significantly affect the fresh clot selectivity of K2P or SK, although lysis of old clots was increased (from 4% to 36%) when it was given together with K2P. Furthermore, heparin did not affect the time to onset of bleeding nor was the bleeding time prolonged by its addition. The bleeding time observed for t-PA (20-25 min) was markedly shorter than that found for K2P or SK (40-50 min).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A bleeding model in rabbits demonstrate fresh clot selectivity for a genetically engineered variant of tissue-type plasminogen activator and for streptokinase. 839 27

Despite the high overall similarity in primary structure between kringle one (K1) and kringle two (K2) of tissue-type plasminogen activator (t-PA) there exists an enormous functional difference. It is thought that, in contrast to K1, K2 mediates lysine binding and fibrin binding and is involved in stimulation of plasminogen activation by fibrin or derivatives as CNBr fragments of fibrinogen. Hypothesizing that sequence differences are responsible for differences in function, we compared the amino acid sequences of K1 and K2 with a consensus kringle sequence. Six consecutive amino acids unique to K2 of t-PA were found, i.e. from Asn248 to Trp253. To test whether these residues are involved in lysine binding, fibrin binding, and fibrin-dependent plasminogen activation, we constructed a set of t-PA mutant proteins containing only a kringle and the protease (P) domain: K2P, K1P, and k1P. In the latter molecule the original amino acid residues Ala160-Ser165 from K1 were substituted by Asn248-Trp253 from K2. As expected, K2P showed enhanced plasminogen activation in the presence of CNBr fragments of fibrinogen, bound to lysine-Sepharose and to a forming fibrin clot. K1P did not show any of these features. In contrast, k1P could be stimulated by CNBr fragments of fibrinogen and bound to lysine-Sepharose and a forming fibrin clot. These results indicate that at least a part of the functional differences between K1 and K2 of t-PA can be localized to a stretch of 6 amino acid residues from Asn248 to Trp253 present in K2.
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PMID:Introduction of lysine and clot binding properties in the kringle one domain of tissue-type plasminogen activator. 839 5

A number of cell types have previously been shown to bind tissue plasminogen activator (tPA), which in some cases can remain active on the cell surface resulting in enhanced plasminogen activation kinetics. We have investigated several cultured cell lines, U937, THP1, K562, Molt4, and Nalm6 and shown that they bind both tPA and plasminogen and are able to act as promoters of plasminogen activation in kinetic assays. To understand what structural features of tPA are involved in cell surface interactions, we performed kinetic assays with a range of tPA domain deletion mutants consisting of full-length glycosylated and nonglycosylated tPA (F-G-K1-K2-P), DeltaFtPA (G-K1-K2-P), K2-P tPA (BM 06.022 or Reteplase), and protease domain (P). Deletion variants were made in Escherichia coli and were nonglycosylated. Plasminogen activation rates were compared with and without cells, over a range of cell densities at physiological tPA concentrations, and produced maximum levels of stimulation up to 80-fold with full-length, glycosylated tPA. Stimulation for nonglycosylated full-length tPA dropped to 45-60% of this value. Loss of N-terminal domains as in DeltaFtPA and K2P resulted in a further loss of stimulation to 15-30% of the full-length glycosylated value. The protease domain alone was stimulated at very low levels of up to 2-fold. Thus, a number of different sites are involved in cell interactions especially within finger and kringle domains, which is similar to the regulation of tPA activity by fibrin. A model was developed to explain the mechanism of stimulation and compared with actual data collected with varying cell, plasminogen, or tPA concentrations and different tPA variants. Experimental data and model predictions were generally in good agreement and suggest that stimulation is well explained by the concentration of reactants by cells.
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PMID:Regulation of tissue plasminogen activator activity by cells. Domains responsible for binding and mechanism of stimulation. 1021 15


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