Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plasminogen activator inhibitor-1 (PAI-1) plays particularly important role in regulation of homeostasis of fibrinolytic system. It neutralizes active molecules of tissue-type and urokinase-type plasminogen activators. PAI-1 is synthesized mainly in endothelial cells but it is present also in other cells. It was found in vitro that elevated expression of PAI-1 gene and increase in PAI-1 concentration released from endothelium is caused by many different biologically active substances. Among them there are thrombin, lipopolysaccharides, cell growth factors, cytokines and also glucocorticoids and phorbol esters. In this work mechanisms of regulation of PAI-1 synthesis and role of this protein in homeostasis of fibrinolytic system are described.
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PMID:[Progress in knowledge about fibrinolysis regulation]. 799 70

Plasminogen activator inhibitor-1 (PAI-1), the main physiological inhibitor of tissue-type plasminogen activator (t-PA), may occur in three interconvertible conformations: active, latent, and substrate. To delineate specific domains in the PAI-1 molecule responsible for its conformational flexibility and associated functional diversity, four mutants of PAI-1 (with the amino acids at positions P12, P10, P8, and P6, respectively, substituted with proline) were expressed in Escherichia coli, purified, and characterized. Wild-type PAI-1 (wtPAI-1) had a specific activity of 21 +/- 10% (mean +/- S.D., n = 3) of the theoretical maximum value. PAI-1-P12 (Ala-->Pro at P12), PAI-1-P10 (Ser-->Pro at P10), and PAI-1-P8 (Thr-->Pro at P8) had specific activities of 0.06 +/- 0.03% (n = 3), 2.6 +/- 1.0% (n = 4), and 2.7 +/- 1.1% (n = 3), respectively (p < 0.03 versus wtPAI-1). PAI-1-P6 (Val-->Pro at P6) has a specific activity of 12 +/- 3.3% (n = 3) of the theoretical maximum value (p = not significant versus wtPAI-1). SDS-polyacrylamide gel electrophoresis of mixtures of wtPAI-1 or PAI-1-P6 with a 2-fold molar excess of t-PA yielded a mixture of a covalent 110-kDa t-PA.PAI-1 complex (15-25%), nonreactive 45-kDa material (44-67%), and a 41-kDa band (18-31%) representing cleaved PAI-1. PAI-1-P12, PAI-1-P10, and PAI-1-P8 behaved as substrates, yielding predominantly the 41-kDa cleavage product (85-91%) and a small amount (9-15%) of non-reactive material. NH2-terminal amino acid sequencing revealed that cleavage occurred at the P1-P1' bond (Arg346-Met347). Incubation of PAI-1-P12, PAI-1-P10, or PAI-1-P8 with a 2-fold molar excess of urokinase-type plasminogen activator, plasmin, or thrombin also primarily generated a 41-kDa cleavage product (62-89%). Incubation of wtPAI-1 and PAI-1-P6 at 37 degrees C resulted in a loss of inhibitory activity, whereas the substrate behavior of PAI-1-P12, PAI-1-P10, and PAI-1-P8 remained unaltered. Treatment of the three substrate-like mutants with guanidinium Cl did not induce inhibitory activity. In conclusion, point mutations at positions P12, P10, and P8 yield PAI-1 variants with stable substrate properties, which may facilitate more detailed structure/function studies.
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PMID:Conversion of plasminogen activator inhibitor-1 from inhibitor to substrate by point mutations in the reactive-site loop. 803 24

Plasminogen activator inhibitor-1 (PAI-1) is a member of the serpin superfamily of proteins and is the fast acting inhibitor of both urinary plasminogen activator and tissue-type plasminogen activator. We have assessed the functional significance of reactive center residues on the carboxy-terminal side of the cleavage site of recombinant human PAI-1. Using site-directed mutagenesis, the P1'-P5' residues (P1' is the first residue on the carboxy-terminal side of the protease cleavage site) of the wild-type PAI-1 reactive center sequence were replaced with the corresponding sequences of plasminogen activator inhibitor-2, antithrombin, alpha 2-antiplasmin and protease nexin I. Rate constants of inhibition of the serine proteases urinary plasminogen activator, tissue-type plasminogen activator, plasmin and thrombin by the variants were determined. The results suggest a crucial role for both reactive center length and sequence in the inhibition of plasminogen activators by PAI-1. Analysis of substitutions at positions P4' and P5' both confirms and extends our previous work demonstrating a favorable electrostatic interaction between these residues and tissue-type plasminogen activator. None of the variants show dramatic increases in the rate constants of inhibition of other serine proteases, suggesting that these residues alone are not sufficient to confer protease specificity on PAI-1. Apparently, the determinants of the rapid inhibitory specificity of PAI-1 are localized to the P1'-P5' region of the reactive center and these residues act synergistically to produce the exquisite specificity of PAI-1 for plasminogen activators.
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PMID:Sequence requirements in the reactive-center loop of plasminogen-activator inhibitor-1 for recognition of plasminogen activators. 862 Aug 72

Heparin-coated systems for extracorporeal lung-assist (ECLA) were developed to reduce hemorrhagic risk by lowering the systemic heparinization, monitored by global tests, e.g. activated coagulation time (ACT) and activated partial thromboplastin time (APTT). Since this strategy gives no insight into procoagulant states, five ARDS patients receiving ECLA with heparin-coated systems were investivated for changes in coagulation using both global and extended tests. During ECLA onset the APTT and ACT were within or near normal ranges, platelets decreased 76.5% within 48 h, fibrinogen decreased 28.7%, thrombin-antithrombin-III complexes were elevated before ECLA (53 micrograms/L), but demonstrated an additional peak (238 micrograms/L), plasminogen-activator-inhibitor-1 increased 12-fold, and the C1-inhibitor dropped 14.1%. In conclusion, after the onset of ECLA from a previous prethrombotic state, the precoagulant, anticoagulant, fibrinolytic and complement systems were activated in a similar way to that reported for non-heparinized systems with high-dose heparin. This was however only monitored by an extended test panel which was unable to predict thromboembolic events during ECLA.
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PMID:Global and extended coagulation monitoring during extracorporeal lung assist with heparin-coated systems in ARDS patients. 906 29

The initial step in atherosclerosis is the rapid targeting of monocytes to the sites of inflammation and endothelial injury. Serum levels of intercellular adhesion molecule-1 were found to be increased in ischaemic heart disease patients and polymorphisms in the E-selectin gene were associated with accelerated atherosclerosis in young (age < 40 years) patients, further suggesting a role of inflammation in atherosclerosis. Cholesterol loading in macrophages was found to induce interleukin-8 expression, suggesting an association between foam cell formation and beta 2-integrin-dependent adhesion of leukocytes. Enhanced endothelium-platelet interaction induced by hypercholesterolaemia is mediated by von Willebrand factor, whereas platelet adhesion to subendothelial matrix is mediated by fibulin-fibrinogen complexes. Activated platelets mediate the homing of leukocytes by interaction with the subendothelial matrix under shear stresses that do not allow neutrophil adhesion. They may also contribute to the oxidative modification of LDL, provide a source of lipids for foam cell generation and contribute to smooth muscle cell proliferation. Oxidized LDL induces tissue factor in macrophages that also provide sites for fibrin polymerization and decreases the anticoagulant activity of endothelium by interfering with thrombomodulin expression and inactivating tissue factor pathway inhibitor. Intravascular fibrinolysis induced by tissue-type plasminogen activator or urokinase may contribute to the initiation of atherosclerosis by inducing P-selectin and platelet activating factor as well as to plaque rupture, either directly or indirectly, by activating metalloproteinases. Plasminogen activator inhibitor-1 inhibits smooth muscle cell migration and, in the presence of vitronectin, promotes the clearance of thrombin by LDL receptor-related protein at sites of endothelial injury.
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PMID:Thrombosis and atherosclerosis. 933 57

Because platelets interact with fibrinolysis in a complex manner, it can be expected that with abnormal platelet numbers and quality this interference can be even more profound. The aim of this work was to study the lysis-resistance of platelet-rich clots in diseases with high platelet counts: polycythemia vera (PV), essential thrombocythemia (ET) and to make comparison with polyglobulia (PG). Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were analyzed by an in vitro clot lysis test. Plasminogen activator inhibitor-1 (PAI-1) activity was measured in plasma and in the supernatants of the washed and gel-filtered platelets after activation by thrombin. The lysis showed decreased speed of PPP-clots in PV and ET. This phenomenon was even more marked in PRP-clots from PV and ET, but further increased lysis resistance after retraction was not observed in PV and ET, most likely due to abnormal platelet functions. Our results suggest that the fibrinolytic activity is reduced in PV and ET, and may play a role both in the increased aptitude for venous thrombosis and in the arterial complications. These are partly caused by higher plasmatic PAI-1 activity as well as by more active platelet PAI-1. The PAI-1 activity was significantly higher in the supernatants of the washed and gel-filtered platelets of PV after activation by thrombin compared with controls. Other factors might have influenced the reduced fibrinolysis.
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PMID:Reduced in vitro clot lysis and release of more active platelet PAI-1 in polycythemia vera and essential thrombocythemia. 968 57

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor of the serpin superfamily which rapidly inactivates tissue plasminogen activator (tPA), but reacts with thrombin at a much slower rate. Based on the previous mutagenesis studies and the X-ray crystal structure of the thrombin E192Q-bovine pancreatic trypsin inhibitor (BPTI) complex, the structural basis for the slow reactivity of thrombin with PAI-1 is investigated in this study. In the crystal structure of the thrombin E192Q-BPTI complex, the reactive site loop of BPTI is stabilized in a canonical conformation by several productive interactions (e.g., Glu39 of thrombin is ion-paired to the P5' Arg, and Gln192 is hydrogen-bonded to the P2 and P4 backbone carbonyls of BPTI). PAI-1 contains Glu residues at both the P4' and P5' positions, and previous mutagenesis studies suggest that these residues make productive interactions with Arg39 of tPA as well as with two other positively charged residues present on the 39-loop of the protease (chymotrypsin numbering). Glu39 and Glu192 of thrombin would be unable to make such productive interactions with PAI-1. Instead, their repulsive interactions with the similarly charged residues and/or the backbone carbonyls of the PAI-1 reactive site loop could restrict the reaction. To test this, the rate constants (k2) for the PAI-1 inactivation of wild-type, E39K, E39Q, E192Q, E192M, and E39K/E192Q thrombins were determined. The inactivation rates of E39K [k2 = (4.3 +/- 0.2) x 10(4) M-1 s-1] and E39Q [k2 = (1.0 +/- 0.1) x 10(4) M-1 s-1] were 50- and 12-fold faster than the inactivation of wild-type thrombin [k2 = (8.6 +/- 0. 5) x 10(2) M-1 s-1], respectively. Relative to thrombin, the PAI-1 inactivation rates were improved 31-fold for E192Q [k2 = (2.7 +/- 0. 5) x 10(4) M-1 s-1] and 5-fold for E192M [k2 = (4.3 +/- 0.8) x 10(3) M-1 s-1] thrombins. With the double mutant E39K/E192Q, the inactivation rate [k2 = (5.4 +/- 0.4) x 10(5) M-1 s-1] was improved 628-fold over wild-type thrombin. These results suggest that repulsive interactions and/or lack of productive electrostatic interactions between PAI-1 and Glu39 and Glu192 of thrombin are responsible for the slow reaction of thrombin with this serpin.
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PMID:Elucidation of the structural basis for the slow reactivity of thrombin with plasminogen activator inhibitor-1. 974 20

Glomerular fibrin deposition is thought to be one of the factors causing progressive glomerular injury and may be related to defective intraglomerular fibrinolysis. Recently, it was shown that tissue plasminogen activator (t-PA) is produced by mesangial cells and is associated with degradation of the extracellular matrix. This study was designed to clarify the factors regulating t-PA production in human mesangial cells. The levels of t-PA activity, t-PA antigen and t-PA inhibitor-1 (PAI-1) antigen were estimated in the supernatants of cultured human fetal mesangial cells incubated for 72 h with thrombin, IL-Ibeta, IL-6, IL-10, and transforming growth factor-beta (TGF-beta). The t-PA activity was measured by an amidolytic assay, and the levels of t-PA antigen and PAI-1 antigen were also measured by enzyme-linked immunosorbent assay. Thrombin increased t-PA activity and TGF-beta decreased it in parallel with t-PA antigen level, although these agents did not affect the synthesis of PAI-1. Incubation with IL-1beta, IL-6 and IL-10 did not change the t-PA activity. It was concluded that the release of t-PA from human fetal mesangial cells was stimulated by thrombin and inhibited by TGF-beta in parallel with that of t-PA antigen. These factors may participate in the glomerular fibrin deposition and the accumulation of extracellular matrix.
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PMID:Regulation of tissue plasminogen activator production in cultured human fetal mesangial cells. 980 22

Vitronectin is a multifunctional glycoprotein present in blood and in the extracellular matrix. It binds glycosaminoglycans, collagen, plasminogen and the urokinase-receptor, and also stabilizes the inhibitory conformation of plasminogen activation inhibitor-1. By its localization in the extracellular matrix and its binding to plasminogen activation inhibitor-1, vitronectin can potentially regulate the proteolytic degradation of this matrix. In addition, vitronectin binds to complement, to heparin and to thrombin-antithrombin III complexes, implicating its participation in the immune response and in the regulation of clot formation. The biological functions of vitronectin can be modulated by proteolytic enzymes, and by exo- and ecto-protein kinases present in blood. Vitronectin contains an RGD sequence, through which it binds to the integrin receptor alpha v beta 3, and is involved in the cell attachment, spreading and migration. Antibodies against alpha v beta 3 or synthetic peptides containing an RGD sequence are now being tested as therapeutic agents in the treatment of human cancers, bone diseases (e.g. osteoporosis) and in pathological disorders which involve angiogenesis.
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PMID:Vitronectin. 1039 14

Reptilase, defibrase and ancrod are thrombin-like venom enzymes that cleave fibrinogen to release fibrinopeptide-A and generate fibrin monomers. Although these enzymes decrease fibrinogen levels in vivo, presumably by enhancing fibrinolytic activity, the mechanism has not been identified. In the present study, we analyzed their effects on the inhibitors of fibrinolysis. Plasminogen activator inhibitor-1 (PAI-1) was cleaved at its C-terminus by reptilase and lost its specific activity. Alpha2-antiplasmin (alpha2-AP) was cleaved both at the Pro19-Leu20 peptide bond and at its C-terminus by reptilase, and also lost its specific activity. The apparent second-order rate constants (mol/l per min per Batroxobin unit) were 0.22 for the cleavage of PAI-1 (3.2 micromol/l) and 0.19 for that of alpha2AP (6.4 micromol/l), which were approximately 200-fold lower than that (47.0) for the cleavage of fibrinogen (1.1 micromol/l). Neither defibrase nor ancrod cleaved and inactivated these inhibitors. Only reptilase enhanced euglobulin clot lysis in vitro at high concentration, due probably to PAI-1 inactivation. Since all these three enzymes enhance fibrinolysis similarly during defibrination therapy, the neutralization or inactivation of the inhibitors of fibrinolysis appeared not to represent the main mechanism for the enhancement.
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PMID:The cleavage and inactivation of plasminogen activator inhibitor type 1 and alpha2-antiplasmin by reptilase, a thrombin-like venom enzyme. 1075 7


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