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)

Possible mechanisms underlying the development of fibrinolysis inhibition following trauma were studied. In order to investigate the role of intravascular coagulation dogs were subjected to infusions of thrombin or endotoxin, which both caused an increase in urokinase inhibitor activity in serum after 24 and 48 hours. The inhibitor increase following thrombin infusion was not, however, prevented by previous defibrinogenation with Defibrase or by induction of thrombocytopenia with antiplatelet serum, suggesting that neither platelets nor fibrinogen are necessary for the post-traumatic occurrence of fibrinolysis inhibition. In all groups subjected to infusion of thrombin an increase in plasma free fatty acids (FFA) was observed. The role of this increase for the development of fibrinolysis inhibition was tested by infusion of norepinephrine alone and in combination with nicotinic acid. Norepinephrine caused an increase of FFA after 2 hours and in urokinase inhibitor activity after 24-48 hours. Both of these were diminished by high doses of nicotinic acid, indicating that the release of FFA rather than intravascular coagulation might be the principal mechanism underlying the occurrence of fibrinolysis inhibition following trauma.
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PMID:Induction of endogenous fibrinolysis inhibition in the dog. Effect of intravascular coagulation and release of free fatty acids. 126 38

Protease nexin I (PNI), a 43,000- to 50,000-dalton glycoprotein, is a potent thrombin and urokinase inhibitor produced by many mammalian cells, including human glia, in tissue culture. PNI is a member of the growing superfamily of serine protease inhibitors now known as serpins, but, unlike many others of this family, it has not yet been detected in normal human plasma. Of interest to neurobiology and neurologic disease, PNI is identical to a glia-derived neurite-promoting factor, glia-derived nexin (GDN). Antibody to PNI stains the periphery of senile amyloid plaques in brain tissue from patients with Alzheimer's disease (AD), along with another serpin, alpha 1-antichymotrypsin (alpha 1-ACT). A soluble form of the beta-amyloid precursor protein (beta APP), containing a Kunitz-type trypsin inhibitor domain, the beta APP751 form, is identical to protease nexin II (PNII), a 100,000-dalton serine protease inhibitor present in a number of tissues besides the brain. PNII/beta APP is also found in normal and AD CSF. We found a 47,000-dalton PNI, a thrombin- and urokinase-inhibiting serpin, in normal human CSF by Western blotting using a monospecific antibody. We also demonstrated biologically active PNI capable of forming complexes with serine proteases 125I-urokinase or 125I-thrombin.
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PMID:Protease nexin I, thrombin- and urokinase-inhibiting serpin, concentrated in normal human cerebrospinal fluid. 162 Mar 46

A protein that has several similarities to protease nexin I, a fibroblast thrombin and urokinase inhibitor, has been detected on platelets (Gronke RS, Bergman BL, and Baker JB: J Biol Chem 262:3030, 1987). On incubation of platelets with 125I-thrombin, this platelet protein forms complexes with 125I-thrombin that are found both in the incubation medium and, as demonstrated here, associated with purified platelet plasma membranes. The present results indicate that interaction with the platelet surface may modulate the conformation and function of this platelet form of protease nexin I (PNIp) because: (a) an antibody against protease nexin I inhibited released PNIp, but not platelet-bound PNIp from complexing 125I-thrombin, and (b) whereas PNIp extracted from platelets bound both thrombin and urokinase, platelet-bound PNIp bound only thrombin. In experiments using several different platelet isolation methods, PNIp accounted for a large fraction of the rapid high affinity binding of 125I-thrombin to platelets. However, platelets isolated and maintained in the presence of metabolic inhibitors failed to take added thrombin into 125I-thrombin-PNIp complexes. This finding suggests that PNIp is released from inside platelets during activation, and thus does not function to transmit the primary activating signal that is generated by thrombin binding to platelets.
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PMID:A form of protease nexin I is expressed on the platelet surface during platelet activation. 291 87

Urokinase-related proteins in human urine occur mainly as a 1:1 complex of urokinase with an inhibitor (Stump, D. C., Thienpont, M., and Collen, D. (1986) J. Biol. Chem. 261, 1267-1273). BALB/c mice were immunized with this urokinase-urokinase inhibitor complex and spleen cells fused with mouse myeloma cells, resulting in hybridomas producing monoclonal antibodies. Three antibodies reacting with the complex but not with urokinase were utilized to develop a sensitive (0.5 ng/ml) enzyme-linked immunosorbent assay for the urokinase inhibitor, which was used for monitoring its purification by chromatography on zinc chelate-Sepharose, concanavalin A-Sepharose, SP-Sephadex C-50, and Sephadex G-100. A homogenous glycoprotein of apparent Mr 50,000 was obtained with a yield of 40 micrograms/liter urine and a purification factor of 320. One mg of the purified protein inhibited 35,000 IU of urokinase within 30 min at 37 degrees C. This protein was immunologically related to both the purified urokinase-urokinase inhibitor complex and to the inhibitor portion dissociated from it by nucleophilic dissociation. It was immunologically distinct from all known protease inhibitors, including the endothelial cell-derived fast-acting inhibitor of tissue-type plasminogen activator, the placental inhibitor of urokinase and protease nexin. In electrophoresis the protein migrated with beta-mobility. Inhibition of urokinase occurred with a second order rate constant (k) of 8 X 10(3) M-1 s-1 in the absence and of 9 X 10(4) M-1 s-1 in the presence of 50 IU of heparin/ml. The urokinase inhibitor was inactive towards single-chain urokinase-type plasminogen activator and plasmin, but it inhibited two-chain tissue-type plasminogen activator with a k below 10(3) M-1 s-1 and thrombin with a k of 4 X 10(4) M-1 s-1 in the absence and 2 X 10(5) M-1 s-1 in the presence of heparin. The concentration of this urokinase inhibitor in plasma from normal subjects determined by immunoassay was 2 +/- 0.7 micrograms/ml (mean +/- S.D., n = 25). The protein purified from plasma by immunoabsorption had the same Mr, amino acid composition, and immunoreactivity as the urinary protein. Furthermore, when urokinase was added to plasma, time-dependent urokinase-urokinase inhibitor complex formation was observed at a rate similar to that observed for the inhibition of urokinase by the purified inhibitor from urine. This urokinase inhibitor, purified from human urine, most probably represents a new plasma protease inhibitor.
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PMID:Purification and characterization of a novel inhibitor of urokinase from human urine. Quantitation and preliminary characterization in plasma. 309 4

Serum-free culture medium collected from primary monolayer cultures of human articular chondrocytes was found to inhibit human urokinase [EC 3.4.21.31] activity. Although chondrocyte culture medium contained a small amount of endothelial-type plasminogen activator inhibitor which could be demonstrated by reverse fibrin autography, most of the urokinase inhibitory activity of chondrocyte culture medium was shown to be due to a different molecule from endothelial-type inhibitor, since it did not react with a specific antibody to this type of inhibitor. The dominant urokinase inhibitor in chondrocyte culture medium was partially purified by concanavalin A-Sepharose affinity chromatography. The partially purified inhibitor inhibited high-Mr urokinase more effectively than low-Mr urokinase, but no obvious inhibition was detected against tissue-type plasminogen activator, plasmin, trypsin, and thrombin. The inhibitor had an apparent Mr of 43,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and it was unstable to sodium dodecyl sulfate, acid, and heat treatments. Inhibition of urokinase by the inhibitor was accompanied with the formation of a sodium dodecyl sulfate-stable high-Mr complex between them. Inhibition and complex formation required the active site of urokinase. The partially purified inhibitor was thought to be immunologically different from the known classes of plasminogen activator inhibitors, including endothelial-type inhibitor, macrophage/monocyte inhibitor, and protease nexin, since it did not react with specific antibodies to these inhibitors.
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PMID:Detection and partial characterization of a specific plasminogen activator inhibitor in human chondrocyte cultures. 314 40

We have recently described a new serine proteinase inhibitor, proteinase inhibitor 6 (PI-6). This serpin has features that suggest it may function intracellularly, but its close resemblance to ovalbumin serpins like plasminogen activator inhibitor 2 (PAI-2) raises the possibility that it is secreted to regulate an extracellular proteinase. To determine whether PI-6 is secreted, we have examined its cellular distribution by immunohistochemistry and have attempted to induce its release from platelets and from cultured cells. We find that PI-6 is present in endothelial and epithelial cells, but it is apparently cytoplasmic and it is not released from cells in response to phorbol ester, dibutyryl cAMP or tumor necrosis factor alpha treatment. It is also not released from activated platelets. The addition of a conventional signal peptide to the amino terminus of PI-6 directed its translocation into the endoplasmic reticulum (ER), resulting in glycosylation but not secretion of the molecule. By contrast, the addition of the same signal peptide to PAI-2 markedly enhanced its translocation and secretion. Glycosylated PI-6 was sequestered in the ER and was incapable of interacting with thrombin. The failure of PI-6 to move along the secretory pathway, and the loss of inhibitory function of ER-localized PI-6, demonstrates that unlike PAI-2, PI-6 is not naturally secreted. Taken together, these results suggest that PI-6 has evolved to fulfil an intracellular role and that it represents a new type of cellular serpin.
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PMID:Proteinase inhibitor 6 cannot be secreted, which suggests it is a new type of cellular serpin. 857 59

We purified a novel serine proteinase inhibitor (serpin)-like protein from the bovine brain and named it B-43 from its molecular mass, 43 kDa. A cleaved peptide from B-43 was copurified with the native B-43. Partial amino acid sequencing of the purified B-43 showed that this protein was homologous to glia-derived nexin/protease nexin-1 (GDN/PN-1), plasminogen activator inhibitor 2, leukocyte elastase inhibitor (LEI) and placental thrombin inhibitor (PTI) among the serpins. Although B-43 had a similar amino acid composition to these serpins, the biochemical features of B-43 were different from them. B-43 did not form sodium dodecyl sulfate (SDS)-resistant serpin-proteinase complexes with thrombin, urokinase, pancreatic elastase and plasmin, suggesting that these proteinases were not the targets of B-43. In contrast to GDN/PN-1, B-43 did not have an affinity for heparin. B-43, having different biochemical properties from GDN/PN-1, appears to be an additional serpin expressed in the brain.
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PMID:Purification of a novel serpin-like protein from bovine brain. 884 89

Serine proteinase inhibitors (serpins) are classically regulators of extracellular proteolysis, however, recent evidence suggests that some function intracellularly. Such "ovalbumin" serpins include the human proteinase inhibitors 6 (PI-6), 8 (PI-8), and 9 (PI-9), plasminogen activator inhibitor 2, and the monocyte/neutrophil elastase inhibitor. PI-9 is a potent granzyme B (graB) inhibitor that has an unusual P1 Glu and is present primarily in lymphocytes. In a search for the murine equivalent of PI-9 we screened cDNA libraries, and performed reverse transcriptase-polymerase chain reaction on RNA isolated from leukocyte cell lines and from lymph nodes and spleens of allo-immunized mice. We identified 10 new ovalbumin serpin sequences: two resemble PI-8, two resemble PI-9, and the remaining six have no obvious human counterparts. By RNA analysis only one of the two sequences resembling PI-9 (designated SPI6) is present in mouse lymphocytes while the other (a partial clone designated mBM2A) is predominantly in testis. SPI6 comprises a 1.8-kilobase cDNA encoding a 374-amino acid polypeptide that is 68% identical to PI-9. mBM2A is 65% identical to PI-9 and over 80% identical to SPI6. Although the reactive loops of SPI6 and mBM2A differ from PI-9, both contain a Glu in a region likely to contain the P1-P1' bond. SPI6 produced in vitro using a coupled transcription/translation system formed an SDS-stable complex with human graB and did not interact with trypsin, chymotrypsin, leukocyte elastase, pancreatic elastase, thrombin, or cathepsin G. Recombinant SPI6 produced in a yeast expression system was used to examine the interaction with human graB in more detail. The second-order rate constant for the interaction was estimated as 8 x 10(4) M-1 s-1, and inhibition depended on the Glu in the SPI6 reactive center. The SPI6 gene was mapped to the same region on mouse chromosome 13 as Spi3, which encodes the murine homolog of PI-6. We conclude that even though their reactive centers are not highly conserved, SPI6 is a functional homolog of PI-9, and that the regulation of graB in the mouse may involve a second serpin encoded by mBM2A. Our identification of multiple sequence homologs of PI-8 and PI-9, and six new ovalbumin serpins, is consonant with the idea that the larger set of granule and other proteinases known to exist in the mouse (compared with human) is balanced by a larger array of serpins.
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PMID:A new family of 10 murine ovalbumin serpins includes two homologs of proteinase inhibitor 8 and two homologs of the granzyme B inhibitor (proteinase inhibitor 9). 918 75

Plasminogen activator inhibitor 2 (PAI-2) is produced by activated monocytes in two forms, intracellular and secreted. We have studied the distribution of these two forms in unstimulated human peripheral blood monocytes and after stimulation by thrombin. Fetal calf serum (FCS) in the culture medium was absolutely necessary for accumulation of intracellular PAI-2; but not for synthesis and secretion. Even at a concentration as low as 0.1%, FCS restored accumulation of intracellular PAI-2. Increasing concentrations of FCS resulted in an increase in the ratio of intracellular to secreted PAI-2. The factor that promoted accumulation of intracellular PAI-2 was not a platelet product. Failure of monocytes to accumulate PAI-2 did not reflect leakage due to cell death, as assessed by LDH in culture supernatants. We propose that accumulation of intracellular PAI-2 is not simply due to poor secretion, but is an active process that is modulated by factor(s) found in serum.
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PMID:The distribution of the secreted and intracellular forms of plasminogen activator inhibitor 2 (PAI-2) in human peripheral blood monocytes is modulated by serum. 956 98

Plasminogen activator inhibitor 2 (PAI-2) has been shown to inhibit apoptosis in transfected cells. We have investigated this phenomenon in activated human monocytes, which are a physiological source of intracellular PAI-2. Apoptosis of monocytes was rapidly induced by removal of serum, addition of hydrogen peroxide, or binding of a monoclonal antibody to Fas. Treatment of monocytes with thrombin or lipopolysaccharide (LPS) inhibited apoptosis of monocytes and also up-regulated intracellular PAI-2. Increased apoptosis was accompanied with increased activity of caspases 3 and 8. Thrombin or LPS treatment of monocytes decreased the activity of both caspases, which correlated with protection from apoptosis. The role for PAI-2 in protection of monocytes from apoptosis was studied. Monocytes were transfected with antisense oligonucleotides that blocked PAI-2 antigen, and antisense for PAI-2 had no effect on apoptosis of monocytes. No interaction was evident between PAI-2 and recombinant caspases 3 and 8 in vitro. PAI-2 was not a substrate for caspases during apoptosis of monocytes, although some cleavage of recombinant PAI-2 by caspase 3 was evident in vitro. This study shows that thrombin or LPS protected monocytes from apoptosis and that PAI-2 did not mediate this inhibitory effect.
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PMID:Thrombin inhibits apoptosis of monocytes and plasminogen activator inhibitor 2 (PAI-2) is not responsible for this inhibition. 1101 Aug 7


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