EC:3.4.21.68 - FACTA Search

Gene/Protein Disease Symptom Drug Enzyme Compound
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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 the strictly conserved tryptophan-25 (W25) residue in the structural stability and omega-amino acid ligand binding properties of the recombinant (r) kringle 2 (K2) domain of tissue-type plasminogen activator (tPA) has been investigated. Two conservative mutants were constructed and expressed that contained W25-->F and W25-->Y substitutions. The binding (dissociation) constants (Kd) for three ligands, viz., 6-aminohexanoic acid (EACA), 7-aminoheptanoic acid (7-AHpA), and L-lysine (Lys), to these polypeptides were determined by intrinsic fluorescence titrations. In the case of r-[K2tPA/W25F], the Kd values for these ligands were found to be 37, 16, and 89 microM for EACA, 7-AHpA, and Lys, respectively. For r-[K2tPA/W25Y], the Kd values for these same ligands were 64, 9, and 115 microM, respectively. The wild-type (wt) kringle domain possessed Kd values of 43, 6, and 85 microM for EACA, 7-AHpA, and Lys, respectively. The effect of these mutations on the stability of the r-[K2tPA] domain has been examined by differential scanning colorimetry. The temperature of maximum heat capacity (Tm) of wt-r-[K2tPA] (75.6 degrees C) was dramatically reduced to 50.8 and 58.0 degrees C for r-[K2tPA/W25F] and r-[K2tPA/W25Y], respectively. In the presence of EACA, the Tm values were increased to 86.1, 61.7, and 68.7 degrees C, respectively, indicating that EACA does interact with the r-[K2tPA] mutants and stabilizes their native conformations, similar to the case with wt-r-[K2tPA].(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Role of the strictly conserved tryptophan-25 residue in the stabilization of the structure and in the ligand binding properties of the kringle 2 domain of tissue-type plasminogen activator. 831 51

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

A series of chimeric urokinase-type plasminogen activator (uPA) genes, which contain combinations of kringle domains of human plasminogen (HPg) in place of the uPA kringle (KuPA), has been constructed and expressed. Some of the resulting recombinant (r) variant uPA chimeras contain modules that potentially mediate the macroscopic binding of HPg to its activation effectors, fibrin(ogen) and 6-aminohexanoic acid (EACA). Such binding sites are not possessed by KuPA, but are present in certain of the HPg kringles, viz., kringle 1 (K1HPg), kringle 4 (K4HPg), and kringle 5 (K5HPg). The recombinant (r) chimeras constructed included molecules with replacements of KuPA with K1HPg (r-[KuPA-->K1HPg]uPA), and with KuPA replaced by double kringle combinations of K1HPgK4HPg (r-[KuPA-->K1HPgK4HPg]uPA), K2HPgK3HPg (r-[KuPA-->K2HPgK3HPg]uPA), and K4HPgK5HPg (r-[KuPA-->K4HPgK5HPg]uPA). All of these variant genes, along with their wild-type (wt) r-uPA counterparts, were expressed in human kidney 293 cells. In cases wherein EACA-binding kringles from HPg have been placed in uPA, this property has been retained in the chimeric molecule and employed as an essential part of the purification procedures for the variants. The steady state amidolytic activity of two-chain (tc) wtr-uPA toward the chromogenic substrate, H-D-pyroglutamyl-Gly-L-Arg-p-nitroanilide (S2444), is characterized by a kcat/KM (pH 7.4, 37 degrees C) of 120 s-1 mM-1. This value ranges from 92 s-1 mM-1 (tcr-[KuPA-->K1HPg]uPA) to 166 s-1 mM-1 (tcr-[KuPA-->K1HPgK4HPg]uPA) for each of the variants, demonstrating that the catalytic efficiency of the active site is altered only in a small way by changes in the noncatalytic domain of uPA. Small differences are also observed in the abilities of these tcr variants to interact with the fast-acting plasma inhibitor of uPA, viz., plasminogen activator inhibitor-1 (PAI-1). The second-order rate constant for the interaction of PAI-1 with tcr-uPA, 0.46 x 10(7) M-1s-1 (pH 7.4, 10 degrees C), ranges from 0.29 x 10(7) M-1s-1 (tcr-[KuPA-->K1HPgK4HPg]uPA) to 1.08 x 10(7) M-1s-1 (tcr-[KuPA-->K4HPgK5HPg]uPA), for the tcr-chimeric variants. Neither wtr-uPA nor any of its chimeric r-variants interacted macroscopically with a fibrin clot under conditions that allowed binding of 74% of single-chain r-tissue-type plasminogen activator. However, the tcr-chimeric uPA variants provided HPg-enriched clot lysis times between 0.2 (r-[KuPA-->K1HPgK4HPg]uPA) and 2.4 (r-[KuPA-->K2HPgK3HPg]uPA) relative to that of wtr-uPA.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The construction and expression of chimeric urokinase-type plasminogen activator genes containing kringle domains of human plasminogen. 851 11

Fucoidan [sulfated poly (L-fucopyranose)] was compared with 6-aminohexanoic acid (6-AH) or CNBr-cleaved fibrinogen (CNBr-Fbg) alone or in combination in enhancing the activation of glutamic plasminogen (Glu-Plg) or lysine plasminogen (Lys-Plg) by two-chain tissue plasminogen activator (t-PA) or LMwt-urokinase or by streptokinase. Fucoidan enhanced the t-PA activation of Glu-Plg or Lys-Plg at Plg concentrations greater than 75nM, while stimulation by CNBr-Fbg of t-PA activation followed saturation kinetics of Michaelis-Menton. During t-PA activation of Glu-Plg, a high degree of synergism was observed between 6-AH and fucoidan while the enhancement by CNBr-Fbg was not influenced by fucoidan and was reversed by 6-AH. Fucoidan alone at higher concentrations was effective in enhancing the activation of Glu-Plg by urokinase while the combination of fucoidan and 6-AH showed additive effect in enhancing the activation of Lys-Plg. The activation of Glu-Plg by streptokinase was reversed by fucoidan in a manner similar to that reported for 6-AH. The results are interpreted to suggest that CNBr-Fbg and 6-AH compete with each other for the same lysine binding sites (LBS) on the Plg molecule while fucoidan acted synergistically with 6-AH in enhancing the t-PA activation of Glu-Plg by a different mechanism. The double reciprocal plot for the interaction of Glu-Plg and urokinase also showed a significantly higher affinity between the two in presence of fucoidan.
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PMID:Effect of fucoidan during activation of human plasminogen. 853 20

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

The interactions of fucoidan with glutamic plasminogen (Glu-Plg), two-chain tissue plasminogen activator (t-PA), LMwt-urokinase, thrombin, and antithrombin III (AT-III) were investigated using fucoidan-sepharose affinity chromatography. The results showed 1) a high degree of affinity between fucoidan-sepharose and Glu-Plg; Lmwt-urokinase and thrombin while t-Pa and AT-III did not bind with fucoidan-sepharose. 2) The double reciprocal plot for the LMwt-urokinase activation of Glu-Plg showed that plasminogen activator inhibitor (PAI-1) inhibited this reaction in a noncompetitive manner and that the presence of fucoidan decreased Km for this interaction by 50% and increased Kcat by 30-fold, 3) The double reciprocal plot for the t-PA activation of Glu-Plg showed that PAI-1 inhibited this reaction in a competitive manner and that fucoidan in conjunction with 6-aminohexanoic acid (6-AH) increased Kcat for this interaction by 5-fold without affecting Km. 4) Fucoidan enhanced the interaction of thrombin with both AT-III and heparin cofactor II (HC-II) and it was more effective than unfractionated heparin of LMwt-heparin in enhancing the interaction of HC-II with thrombin.
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PMID:Interaction of fucoidan with proteases and inhibitors of coagulation and fibrinolysis. 930 16

We have previously demonstrated a low-affinity (0.8 microM, non-covalent complex formation between high-molecular-mass kininogen (HK) and plasminogen (Plg) which prevented Plg interaction with glioma and endothelial cells. We have now extended our previous observations by exploring the potential complex formation between Plg and low-molecular-mass kininogen (LK) and between LK and HK with Plg cleaved with human neutrophil elastase (HNE). Plg cleavage by HNE (PlgHNE) yielded kringles 1-3, kringle 4 and mini-plasminogen. PlgHNE was subjected to SDS/PAGE under non-reducing conditions, followed by western blotting, and incubated with either 125I-HK or 125I-LK. Autoradiograms revealed that 125I-HK bound to miniplasminogen and to kringles 1-3 but not to kringle 4 and the presence of 10 mM 6-aminohexanoic acid (Ahx) disrupted only the interaction with kringles 1-3. In contrast, 125I-LK bound to miniplasminogen but not to kringles 1-3 or 4 and Ahx had no effect at all. The complex formation of either HK (0.67 microM) or LK (3 microM) with Plg (1.5 microM) did not affect its conversion to plasmin by tissue plasminogen activator (t-PA) (10 U/ml) in the presence of a tissue plasminogen stimulator (0.14 microM). However, the rate of conversion of plasminogen to plasmin by t-PA was affected when platelets were added to the reaction mixture. Since HK (0.83 microM) has been shown to inhibit plasmin-induced platelet aggregation, we investigated whether this inhibitory property is found within the heavy chain shared by HK and LK. We found that LK inhibited plasmin-induced platelet aggregation, but a 4-fold molar excess was required when compared to HK. Compared to plasmin, 3-5-fold molar excess of miniplasmin is required to induce platelet aggregation, indicating the important role of kringles 1-3 for plasmin interactions with these cells. These results indicate that HK and LK-mediated inhibition of plasmin-induced platelet aggregation is likely due to complex formation with kringle 5 without interfering with plasmin's active site. We found an additional interaction between HK and kringles 1-3 enhancing the inhibitory effect, presumably by interfering with plasmin's interaction with platelets. This HK and LK-associated modulation of plasmin-induced platelet aggregation may serve as a template to develop synthetic peptides as novel therapeutic agents to prevent some of the plasmin-associated thrombocytopenia seen during thrombolytic therapy.
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PMID:High-molecular-mass and low-molecular-mass kininogens block plasmin-induced platelet aggregation by forming a complex with kringle 5 of plasminogen/plasmin. 942 7

Because histidine-rich glycoprotein binds to the kringle 1-3 domain of plasminogen, it may affect fibrinolysis by reducing fibrin-dependent plasmin production, and in this way it could be mechanistically analogous to 6-aminohexanoic acid. We tested this hypothesis by comparing the effects of histidine-rich glycoprotein and 6-aminohexanoic acid in an in vitro assay of fibrin-dependent plasmin production mediated by tissue plasminogen activator. Whereas 1 mM of 6-aminohexanoic acid increased the K(m) of the reaction from approximately 0.22 microM to approximately 1.7 microM, 2 microM of histidine-rich glycoprotein had no discernible effect. Similar results were obtained in an assay based upon fibrin clot lysis. Therefore, we could not document an effect of histidine-rich glycoprotein on the rate of fibrin-dependent plasmin production.
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PMID:Comparison of the effect of histidine-rich glycoprotein and 6-aminohexanoic acid on plasmin production and fibrinolysis in vitro. 1094 92

The interactions of fucoidan with human glutamic type plasminogen (Glu-Plg), porcine pancreatic elastase digested plasminogen fractions and two chain tissue plasminogen activator t-PA) were investigated using fucoidan-Sepharose affinity chromatography. The results showed a high degree of affinity between fucoidan-Sepharose and Glu-Plg or PlgK(1-3) but not with PlgK4 or mini-Plg. Fucoidan-Sepharose also showed a high affinity for t-PA, which was largely reversed by 0.002 M 6-aminohexanoic acid (6-AH). The addition of fucoidan and CNBr-fibrinogen digest (CNBr-Fbg) gave the highest enhancement of the in vitro activation of Glu-Plg by t-PA in the presence of 0.002 M 6-AH. The results of affinity chromatography and enhancement studies suggested a template mechanism, since increasing the concentrations of any one of the two cofactors reversed the enhancement. Enzyme kinetic studies, using double reciprocal plots, showed that the addition of fucoidan-6-AH increased Kcat by 7-fold without affecting Km and addition of CNBr-Fbg lowered Km by 5-fold without significantly affecting Kcat while addition of the two cofactors lowered Km by 16-fold without significantly affecting Kcat. The enhancement by fucoidan-6-AH or by CNBr-Fbg of the in vitro activation of Glu-Plg by t-PA was reversed by plasminogen activator inhibitor 1 (PAI-1). Fucoidan-Sepharose affinity chromatography revealed that the binding of PAI-1 with fucoidan may be responsible for the reversal of the enhancement by fucoidan-6-AH.
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PMID:Mechanism of enhancement by fucoidan and CNBr-fibrinogen digest of the activation of glu-plasminogen by tissue plasminogen activator. 1111 95

Earlier studies on the stimulatory effect of fucoidan, heparin, and cyanogen bromide (CNBr)-fibrinogen digest on the in-vitro activation of glutamic type plasminogen by tissue plasminogen activator, which were performed using subphysiologic ionic strengths of buffers, gave inconsistent results because of the variation in the ionic strengths of the buffers used. Studies were therefore conducted on the effect of these cofactors using 0.05 mol/l Tris buffer containing a physiologic concentration of sodium chloride. The double reciprocal plots of the activation of glutamic type plasminogen by tissue plasminogen activator in the presence of fucoidan and 6-aminohexanoic acid (6-AH) or heparin and 6-AH showed a four- to six-fold increase in K(cat), while the K(m) remained unchanged. On the other hand, there was greater than six-fold lowering of K(m) from 0.213 to 0.035 micromol/l in the presence of CNBr-fibrinogen, while K(cat) was only slightly increased. The ratios of the initial rate of plasmin generation in the presence or absence of the cofactors were plotted against the inverse of the volume fraction of glutamic type plasminogen or of tissue plasminogen activator after serial dilution. The results suggested that the enhancements by fucoidan and 6-AH or CNBr-fibrinogen were due to their interactions directed towards glutamic type plasminogen, while for heparin and 6-AH, the interaction was directed towards tissue plasminogen activator. Circular dichroism studies in the near ultraviolet range (250-308 nm) showed that 6-AH enhanced the circular dichroism spectra of glutamic type plasminogen around certain chromophores, while fucoidan and heparin had no effect, suggesting that the enhancement by the cofactors may be related to the favorable conformational changes of glutamic type plasminogen by 6-AH.
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PMID:The effect of fucoidan, heparin and cyanogen bromide-fibrinogen on the activation of human glutamic-plasminogen by tissue plasminogen activator. 1269 44

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