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 binding of plasminogen activator inhibitor-1 (PAI-1) to serine proteinases, such as tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), is mediated by the exosite interactions between the surface-exposed variable region-1, or 37-loop, of the proteinase and the distal reactive center loop (RCL) of PAI-1. Although the contribution of such interactions to the inhibitory activity of PAI-1 has been established, the specific mechanistic steps affected by interactions at the distal RCL remain unknown. We have used protein engineering, stopped-flow fluorimetry, and rapid acid quenching techniques to elucidate the role of exosite interactions in the neutralization of tPA, uPA, and beta-trypsin by PAI-1. Alanine substitutions at the distal P4' (Glu-350) and P5' (Glu-351) residues of PAI-1 reduced the rates of Michaelis complex formation (k(a)) and overall inhibition (k(app)) with tPA by 13.4- and 4.7-fold, respectively, whereas the rate of loop insertion or final acyl-enzyme formation (k(lim)) increased by 3.3-fold. The effects of double mutations on k(a), k(lim), and k(app) were small with uPA and nonexistent with beta-trypsin. We provide the first kinetic evidence that the removal of exosite interactions significantly alters the formation of the noncovalent Michaelis complex, facilitating the release of the primed side of the distal loop from the active-site pocket of tPA and the subsequent insertion of the cleaved reactive center loop into beta-sheet A. Moreover, mutational analysis indicates that the P5' residue contributes more to the mechanism of tPA inhibition, notably by promoting the formation of a final Michaelis complex.
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PMID:The contribution of the exosite residues of plasminogen activator inhibitor-1 to proteinase inhibition. 1459 4

We have recently shown that the NH(2)-terminal fragment (PrP23-110) of the human cellular prion protein (PrP(c) ) stimulates t-PA mediated plasminogen activation. PrP23-110 contains an N-terminal lysine cluster (LC1; K(23),K(24), K(27)) and a C-terminal one (LC2; K(101),K(104),K(106),K(110)). To study their biological function we have substituted all lysine residues of each cluster by alanine and generated the recombinant PrP proteins PrP23-110sLC1 and PrP23-110sLC2. The ability of the mutant proteins to stimulate plasminogen activation was assayed. We found that both lysine clusters are essential for t-PA mediated plasminogen activation. We further studied the binding of soluble PrP23-110 to immobilized t-PA or plasminogen using surface plasmon resonance. The recorded binding curves could not be modeled by classical 1:1 binding kinetics suggesting oligomerisation of PrP23-110. Further plasmon resonance studies show that indeed PrP23-110 binds to itself and that glycosaminoglycans modify this interaction. Binding of t-PA or plasminogen to PrP23-110 was no longer influenced by glycosaminoglycans when PrP23-110 was immobilized on the chip surface. Thus a possible role of heparin as a cofactor in the stimulation of plasminogen activation by t-PA could be the generation of a PrP23-110 form with both lysine clusters accessible for binding of t-PA and plasminogen.
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PMID:Both lysine-clusters of the NH2-terminal prion-protein fragment PrP23-110 are essential for t-PA mediated plasminogen activation. 1498 21

In this letter we report the synthesis and biochemical evaluation of selective, irreversible diphenyl phosphonate inhibitors for urokinase plasminogen activator (uPA). A diphenyl phosphonate group was introduced on the substratelike peptide Z-d-Ser-Ala-Arg, and modification of the guanidine side chain was investigated. A guanylated benzyl group appeared the most promising side chain modification. A k(app) value in the 10(3) M(-1) s(-1) range for uPA was obtained, together with a selectivity index higher than 240 toward other trypsin-like proteases such as tPA, thrombin, plasmin, and FXa.
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PMID:Development of irreversible diphenyl phosphonate inhibitors for urokinase plasminogen activator. 1511 82

In order to characterize tissue plasminogen activator (t-PA) binding to gamma-chain residues in fibrinogen, we generated variant fibrinogens substituting alanine for gamma D316, gamma D318, gamma D320, and gamma K321. We measured thrombin-catalyzed polymerization and found normal polymerization with gamma K321A, no polymerization with gamma D316A, and, as reported by Lounes et al. in 2002, impaired polymerization with gamma D318A and gamma D320A. We measured t-PA binding in a solid-phase assay, and t-PA activity by the generation of plasmin. Comparing normal fibrin with fibrinogen, we found a seven-fold increase in binding and a two-fold increase in activity. Binding to all variant fibrinogens was the same as normal. In contrast, t-PA binding to all variant fibrins was weaker than binding to normal fibrin, 2.5-fold for gamma K321A, seven-fold for gamma D320A and 10-fold for gamma D316A and gamma D318A. Plasmin generation in the presence of variant fibrinogens was similar, although not identical, to normal, and plasmin generation in the presence of variant fibrins was impaired for the Asp to Ala variants. As the three variants with the weakest t-PA binding and least activity also showed impaired polymerization, our results support previous findings demonstrating the DD:E complex, found in the normal fibrin polymer, is necessary for the fibrin enhanced binding of t-PA and activation of plasminogen.
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PMID:Investigation of residues in the fibrin(ogen) gamma chain involved in tissue plasminogen activator binding and plasminogen activation. 1531 Nov 53

Monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) can be used directly as sorbents for affinity chromatography after solid phase peptide synthesis. The quality of the synthesized products, the amount of grown peptides on a support and the reproducibility of the process must be considered. A determination of the quantity of the introducing beta-Ala (and, consequently, the total amount of synthesized peptide) was carried out. Three peptides complementary to recombinant tissue plasminogen activator (t-PA) have been synthesized using Fmoc-chemistry on GMA-EDMA disks. The peptidyl ligands were analysed by amino acid analysis, ES-MS and HPLC methods. The affinity binding parameters were obtained from frontal elution data. The results were compared with those established for GMA-EDMA affinity sorbents formed by the immobilization of the same but separately synthesized and purified ligands. The immobilization on GMA-EDMA disks was realized using a one-step reaction between the amino groups of the synthetic ligand and the original epoxy groups of monolithic material. The affinity constants found for two kinds of sorbent did not vary significantly. Finally, the directly obtained affinity sorbents were tested for t-PA separation from a cellular supernatant.
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PMID:Solid phase peptide synthesis on epoxy-bearing methacrylate monoliths. 1563 24

Previous studies have suggested that thrombin interacts with integrins in endothelial cells through its RGD (Arg-187, Gly-188, Asp-189) sequence. All existing crystal structures of thrombin show that most of this sequence is buried under the 220-loop and therefore interaction via RGD implies either partial unfolding of the enzyme or its proteolytic digestion. Here, we demonstrate that surface-absorbed thrombin promotes attachment and migration of endothelial cells through interaction with alpha(v)beta(3) and alpha(5)beta(1) integrins. Using site-directed mutants of thrombin we prove that this effect is mediated by the RGD sequence and does not require catalytic activity. The effect is abrogated when residues of the RGD sequence are mutated to Ala and is not observed with proteases like trypsin and tissue-type plasminogen activator, unless the RGD sequence is introduced at position 187-189. The potent inhibitor hirudin does not abrogate the effect, suggesting that thrombin functions through its RGD sequence in a non-canonical conformation. A 1.9-Angstroms resolution crystal structure of free thrombin grown in the presence of high salt (400 mm KCl) shows two molecules in the asymmetric unit, one of which assumes an unprecedented conformation with the autolysis loop shifted 20 Angstroms away from its canonical position, the 220-loop entirely disordered, and the RGD sequence exposed to the solvent.
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PMID:Thrombin functions through its RGD sequence in a non-canonical conformation. 1599 37

The charge of Lys300(c143) located within a flexible loop(297-313) of sc-uPA has been identified as an important determinant for its high intrinsic activity. Mutations affecting the flexibility of the loop also modulate the intrinsic activity. Glu-plasminogen activation by sc-uPA is strongly promoted by fibrin fragment E but not fibrin fragment D-dimer, whereas plasminogen activation by t-PA is strongly promoted by fragment D-dimer but not fragment E. To further investigate the effect of conformation changes in the flexible loop on catalytic properties of sc-uPA, cassette mutations at Pro309(c152) were made and characterized. It was found that the activation of Pro309(c152) mutants by Lys-plasmin was only moderately affected. In contrast, the intrinsic and two-chain activities of Pro309(c152) mutants against S2444 were both significantly decreased. The two-chain activities of these mutants against Glu-plasminogen were also reduced in a range of 1.1- to 127-fold. The mutations of Pro309(c152) to Trp/Phe and Arg/Asp more significantly affected both intrinsic and two-chain activities, while only a moderate decrease in activity was found with mutations to Ala/Ser/Thr. In contrast to wild-type sc-uPA, plasminogen activation by Pro309(c152) mutants was found to be promoted by both fibrin fragment E and D-dimer. In the presence of 2.0 microM D-dimer, plasminogen activation by mutant Pro309(c152) --> His was promoted by 22-fold, while only 2.0-fold promotion was found with mutant Pro309(c152) --> Gly. In conclusion, these findings demonstrated that conformation changes in the flexible loop of sc-uPA not only affect its intrinsic and two-chain activity, but also extend its promotion of plasminogen activation by fragment E to D-dimer.
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PMID:Mutagenesis at Pro309 of single-chain urokinase-type plasminogen activator alters its catalytic properties. 1623 30

In a recent report, we showed that alanine can replace glycine at the amino terminus of synthetic B-knobs that bind to human fibrin(ogen). We now report a survey of 13 synthetic peptides with the general sequence XHRPYam, all tested with regard to their ability to delay fibrinolysis in an in vitro system activated by t-PA, the results being used as measures of binding affinity to the betaC hole. Unexpectedly, some large and bulky amino acids, including methionine and arginine, are effective binders. Amino acids that branch at the beta carbon (valine, isoleucine, and threonine) do not bind effectively. Crystal structures were determined for two of the peptides (GHRPYam and MHRPYam) complexed with fibrin fragment D-dimer; the modeling of various other side chains showed clashing in the cases of beta-carbon substituents. The two crystal structures also showed that the enhanced binding observed with pentapeptides with carboxyl-terminal tyrosine, compared with that of their tetrapeptide equivalents, is attributable to an interaction between the tyrosine side chain and a guanidino group of a nearby arginine (beta406). The equivalent position in gamma-chains of human fibrin(ogen) is occupied by a lysine (gamma338), but in chicken and lamprey fibrin(ogen), it is an arginine, just as occurs in beta chains. Accordingly, the peptides GPRPam and GPRPYam, which are surrogate A-knobs, were tested for their influence on fibrin polymerization with fibrinogen from lamprey and humans. In lampreys, GPRPYam is a significantly better inhibitor, but in humans, it is less effective than GPRPam, indicating that in the lamprey system the same tyrosine-arginine interaction can also occur in the gamma-chain setting.
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PMID:Probing the beta-chain hole of fibrinogen with synthetic peptides that differ at their amino termini. 1768 24

Although chronic thromboembolic pulmonary hypertension (CTEPH) is characterised by the persistence of organised thrombus, few pro-thrombotic risk factors have been identified in subjects with the disease. The aim of the present study was to compare the prevalence of eight functionally relevant haemostatic polymorphisms between CTEPH subjects and healthy controls. Genomic DNA was isolated from 214 CTEPH subjects and 200 healthy controls, and analysed for Factor V Leiden, prothrombin guanine (G) to adenine (A) substitution at nucleotide 20210 (20210G>A), plasminogen activator inhibitor-1 4G/5G, tissue plasminogen activator 7351 cytosine (C)>thymidine (T), Factor XIII 100G>T, fibrinogen Aalpha substitution of threonine with alanine at position 312 (Thr312Ala), fibrinogen Bbeta substitution of arginine with lysine at position 448 (Arg448Lys) and fibrinogen Bbeta 455G>A polymorphisms. A significant difference was demonstrated in fibrinogen Aalpha Thr312Ala genotype and allele frequencies between CTEPH subjects and controls. The presence of the alanine allele significantly increased the risk of CTEPH. The fibrinogen Aalpha alanine 312 allele alters fibrinogen alpha-alpha chain cross-linkage and has previously been associated with both increased risk of embolisation and increased resistance to thrombolysis. An association between this polymorphism and chronic thromboembolic pulmonary hypertension, therefore, supports an embolic aetiology for this disease, and may provide a mechanism by which thrombus persists following an acute event.
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PMID:Fibrinogen Aalpha Thr312Ala polymorphism is associated with chronic thromboembolic pulmonary hypertension. 1805 60

Nutrient utilization and byproduct accumulation were monitored in Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Five) cell lines during growth and following viral infection in suspension cultures in order to develop a better understanding of cell metabolism and to acquire information relevant to large scale fed-batch bioreactors. The utilization of glucose, dissolved oxygen, and amino acids were monitored in Sf-9 cell cultures grown in Sf-900 II serum-free medium (SFM) and in High Five cell cultures grown in both Sf-900 II and Express Five SFM. Using the optimal medium for each cell line, i.e., Sf-900 II SFM for Sf-9 cells and Express Five SFM for High Five cells, the cell growth rate, maximum cell density, specific glucose and glutamine utilization rates, and specific alanine production rate were comparable during cell growth. In addition, the expression level of recombinant human tissue plasminogen activator was comparable in the two cell lines on a per cell basis. It was found, however, that lactate and ammonia accumulated in High Five cell cultures, but not in Sf-9 cell cultures. In addition, High Five cells utilized asparagine more rapidly than glutamine, whereas Sf-9 cells consumed only minimal asparagine, and the oxygen utilization rate was significantly higher in High Five cell cultures. It was also found that the medium had a significant effect on High Five cell metabolism, e.g., the specific glucose utilization rate and the specific lactate and alanine production rates were significantly higher in Sf-900 II SFM than in Express Five SFM. In addition, the maximum cell density and specific asparagine utilization rate were significantly higher in Express Five SFM.
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PMID:Comparison of Trichoplusia ni BTI-Tn-5B1-4 (high five) and Spodoptera frugiperda Sf-9 insect cell line metabolism in suspension cultures. 1863


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