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:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plasma levels of antithrombin III, alpha 2-macroglobulin and inter-alpha-trypsin inhibitor, as well as those of various clotting, complement and other plasma factors, were significantly decreased in 18 patients suffering from hyperdynamic septic shock. A similar statistically significant reduction of the concentrations of several plasma factors (prothrombin and antithrombin III, plasminogen and alpha 2-plasmin inhibitor, complement factor C3 and clotting factor XIII) was observed in experimental endotoxaemia. In this model the reduction in the plasma levels of these factors was considerably diminished by the intravenous injection of a granulocytic elastase--cathepsin G inhibitor of lower molecular weight from soybeans. The results of both studies indicate that consumption of plasma factors in the course of Gram-negative sepsis proceeds not only via the classical routes (by activation of the clotting, fibrinolytic and complement cascades by system-specific proteinases such as thrombokinase or the plasminogen activator) but also to an appreciable degree of unspecific degradation of plasma factors by neutral proteinases such as elastase and cathepsin G. The endotoxin-induced release of both sorts of proteinases, the system-specific ones and the unspecific lysosomal proteinases from leucocytes and other cells, is likely to be mainly responsible for the consumption of antithrombin III and alpha-2-macroglobulin via complex formation (followed by elimination of the complexes) and the increased turnover of the inter-alpha-trypsin inhibitor as observed in the clinical study. The therapeutic use of an exogenous elastase--cathepsin G inhibitor in the experimental model was stimulated by the observation that human mucous secretions contain and acid-stable inhibitor of the neutral granulocytic proteinases, called HUSI-I or antileucoproteinase. This inhibitor protects mucous membranes and soluble proteins against proteolytic attack by leucocytic proteinases released in the course of a local inflammatory response. Preliminary results indicate that HUSI-I, which is produced by the epithelial cells of mucous membranes, does not belong to any known structural type of acid-stable proteinase inhibitor. The search for other candidates suitable for medication in humans led to the discovery of a potent elastase--cathepsin G inhibitor, called eglin, in the leech Hirudo medicinalis. This acid-stable inhibitor with a molecular weight close to 8100 has an unusual structural property in that the structure of the molecule is not stabilized by any disulphide bridge.
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PMID:Proteinase inhibitors in severe inflammatory processes (septic shock and experimental endotoxaemia): biochemical, pathophysiological and therapeutic aspects. 39 95

The effect of human neutrophil elastase and cathepsin G on recombinant tissue factor pathway inhibitor (TFPI) was investigated. A weak inhibition by TFPI of both elastase (Ki = 0.4 microM) and cathepsin G (Ki = 0.1 microM) was observed. Neutrophil elastase rapidly cleaved TFPI at the Thr87-Thr88 bond situated at the link between Kunitz domains I and II. Cleavage of TFPI by cathepsin G was also observed, but the reaction was much slower and resulted in a number of fragments. Proteolytic cleavage by both elastase and cathepsin G resulted in destruction of inhibitor function with respect to TFPI's inhibition of factor Xa. Cleavage by neutrophil elastase was capable of restoring factor Xa amidolytic activity after its initial inhibition by TFPI. Inhibition of cathepsin G by TFPI was strongly augmented by stoichiometric amounts of factor Xa. However, the augmentation was temporary, presumably due to concomitant cleavage of TFPI by cathepsin G. These observations may have implications for the putative effect of neutrophil leukocyte stimulation on the regulation of the tissue factor-mediated coagulation pathway. Conversely, formation of a factor Xa/TFPI complex may reduce or modulate the proteolytic potential of stimulated leukocytes by temporary inhibition of cathepsin G.
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PMID:Effect of leukocyte proteinases on tissue factor pathway inhibitor. 151 13

The [Arg15,Glu52]aprotinin gene has been constructed from a synthetic [Glu52]-aprotinin gene via an exchange of the appropriate DNA cassette. The gene has been fused to the N-terminal part of the bacteriophage MS-2 polymerase and expressed in a temperature inducible E. coli expression system. The produced fusion protein is deposited as inclusion bodies. Pure and functionally active [Arg15,Glu52]aprotinin has been obtained after cleavage of the purified fusion protein and renaturation of the aprotinin homologue. Recombinant [Arg15,Glu52]aprotinin shows good inhibition of human anionic and cationic trypsin (Ki less than or equal to 10(-11)M) and of human plasma kallikrein (Ki = 3.2 x 10(-10)M). The inhibition constants for human plasmin are Ki = 1.3 x 10(-10)M and for human urinary kallikrein Ki = 10(-11)M. No inhibition was found with the human proteinases thrombin, coagulation factor Xa, urokinase, tissue plasminogen activator, cathepsin G, leukocyte elastase and pancreatic elastase.
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PMID:Expression, isolation and characterization of recombinant [Arg15,Glu52]aprotinin. 246 33

The human intracellular serine proteinase inhibitor, proteinase inhibitor 6 (PI-6), was expressed in the methylotropic yeast Pichia pastoris. The PI-6 cDNA was modified to encode six histidine residues immediately after the initiation codon, and was placed under the control of the P. pastoris alcohol oxidase promoter in the vector pHIL-D2. On the methanol induction, active recombinant PI-6 was produced within the yeast cells, and following cell lysis, was separated from yeast proteins by affinity chromatography using nickel nitrilo-tri-acetic acid (NTA) resin. The interaction of recombinant PI-6 with a range of serine proteinases was studied. Second order association rate constants (ka) were derived for the interaction with trypsin (1.8 x 10(6) M-1 s-1), thrombin (1.2 x 10(5) M-1 s-1), urokinase plasminogen activator (4.0 x 10(4) M-1 s-1), plasmin (1.3 x 10(6) M-1 s-1), and activated protein C (7.5 x 10(3) M-1 s-1). By monitoring complex formation, recombinant PI-6 was also shown to interact with factor Xa. No complex formation was observed with chymotrypsin, human leukocyte elastase, cathepsin G and tissue plasminogen activator, although PI-6 is apparently a substrate for chymotrypsin, leukocyte elastase and cathepsin G.
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PMID:Production and characterization of recombinant human proteinase inhibitor 6 expressed in Pichia pastoris. 754 63

1. In order to characterize the physiological functions of the domain structure of secretory leukoprotease inhibitor (SLPI), the biological capacities of half-length SLPIs, (Ser1-Pro54)SLPI and (Asn55-Ala107)SLPI, were investigated and compared with those of full-length SLPI. 2. The activities of these inhibitors against several serine proteases were determined using synthetic chromogenic substrates. The inhibitory capacity of the C-terminal domain, (Asn55-Ala107)SLPI, was as strong as that of full-length SLPI against human neutrophil elastase (NE), cathepsin G and chymotrypsin. It possessed less trypsin inhibitory activity than intact SLPI. For the N-terminal domain of SLPI, (Ser1-Pro54)SLPI, no inhibitory activity could be detected against the serine proteases tested in this study. 3. The inhibitory activity of (Asn55-Ala107)SLPI against the proteolysis of the natural substrates elastin and collagen by NE was comparable with that of full-SLPI (elastin, IC50 = 907 +/- 31 nM for SLPI, 767 +/- 33 nM for (Asn55-Ala107)SLPI; collagen, IC50 = 862 +/- 36 nM for SLPI, 727 +/- 47 nM for (Asn55-Ala107)SLPI). 4. The binding affinities of full- and half-length SLPIs for heparin were measured by affinity column chromatography. Full-length SLPI showed high affinity for heparin while the binding capacities of both half-length SLPIs were lower. (Concentration of NaCl for elution, 0.45 M for SLPI, 0.24 M for (Ser1-Pro54)SLPI, 0.27 M for (Asn55-Ala107)SLPI). 5. The effects of full-SLPI and (Asn55-Ala107)SLPI on blood coagulation were measured using the activated partial thromboplastin time (APTT). Full-length SLPI prolonged clotting time dose dependently(1.25, 2.5 and 5.0 microM), whereas (Asn55-AlalO7)SLPI had no effect even at the highest concentration.6. In conclusion, the C-terminal domain of SLPI is a promising candidate for the treatment of inflammatory diseases in which participation of neutrophil proteases has been suggested.
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PMID:Pharmacological activity of the C-terminal and N-terminal domains of secretory leukoprotease inhibitor in vitro. 758 15

A cDNA coding for the E isoform of alpha-1-antiproteinase (also called alpha-1-antitrypsin or alpha-1-proteinase inhibitor) was isolated by oligonucleotide hybridization following immunochemical screening of the rabbit liver cDNA library. The deduced amino acid sequence of the E isoform showed 96.4% identity in 413 residues of the F and S-1 isoforms of rabbit alpha-1-antiproteinase. The N-terminal half of the amino acid residues of the three isoforms was almost identical, but the putative reactive-site loop structure (P8-P'8) was significantly different in the various forms, the P1 site of the E form being glutamic acid. Interaction of the recombinant E form with the various proteinases was investigated by SDS/PAGE, followed by immunoblot analysis. The recombinant protein and trypsin formed a 62 kDa equimolar complex, which gradually became graded to the 37 kDa fragment through several intermediates. The E form also formed a complex of a similar size with elastase and became degraded to the 31 kDa fragment. Several proteinases which cleaved the E form without forming a detectable complex on SDS/PAGE are chymotrypsin, protease V8, pancreas kallikrein, thermolysin, papain and ficin. Other proteinases, with a stringent substrate specificity, such as thrombin, factor Xa, plasmin, plasma kallikrein and cathepsin G, did not attack the E form. Unlike the F and S-1 forms of rabbit plasma alpha-1-antiproteinase, the recombinant E form did not inhibit the amidolytic and proteolytic activities of trypsin. Neither elastase nor protease V8 was inhibited by the E form. Thus the change in the amino acid residues in the reactive-site loop, probably in the P1 site, is responsible for the loss of inhibitory activity of rabbit alpha-1-antiproteinase E. The novel character of the E form could provide a new insight into the interaction of serpin and proteinases.
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PMID:Rabbit alpha-1-antiproteinase E: a novel recombinant serpin which does not inhibit proteinases. 773 71

Z-D-Phe-Pro-boroMpg-OPin (9a)1,2 has been shown previously to be a highly specific inhibitor of thrombin in spite of lacking an arginine-like guanidino group at the P1 site. A range of compounds have been synthesized based upon this lead compound, varying the neutral side chain at the P1 site. Of the 20 examples based upon the structures at P2 and P3 of Z-D-X-Pro (X being Phe or beta,beta-diphenylalanine), all were found to be effective inhibitors of thrombin (Ki's between 10 and 100 nM). Furthermore all exhibited a high specificity toward thrombin having values for a Ki(trypsin)/Ki(thrombin) ratio of between 10- and 100-fold. High ratio values were found for a number of the compounds tested against a range of serine proteinases (plasmin, factor Xa, kallikrein, urokinase, protein Ca, chymotrypsin, elastase, and cathepsin G). As far as potency toward thrombin, compounds containing the methoxypropyl group at P1 were favored over those with a methoxy grouping on a shorter alkyl chain (8) or without the methoxy group (1-5). The compounds display potent anticoagulant activity with values for 18 in thrombin time of 0.63 microM and in activated partial thromboplastin time of 2.0 microM. 11B NMR has been used to confirm interaction of the boron atom with the active site. From the high specificity shown with all the compounds we propose that the compounds, constitute a new class of thrombin inhibitors.
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PMID:Characterization of a class of peptide boronates with neutral P1 side chains as highly selective inhibitors of thrombin. 773 10

The ability of intact peripheral blood monocytes to modulate factor V procoagulant activity was studied using electrophoretic and autoradiographic techniques coupled to functional assessment of cofactor activity. Incubation of plasma concentrations of factor V with monocytes (5 x 10(6)/ml) resulted in the time-dependent cleavage of the 330-kDa protein. Activation occurred via several high molecular mass intermediates (> or = 200 kDa) to yield peptides of 150, 140, 120, 94, 91, 82, and 80 kDa, which paralleled the expression of cofactor activity. The cleavage pattern observed differed from that obtained with either thrombin or factor Xa as an activator. The incubation time required to achieve full cofactor activity was dependent on the monocyte donor and ranged from 10 min to 1 h and was consistently slightly lower than that obtained with thrombin-activated factor Va. Cofactor activity was not diminished by additional incubation. The cofactor activity generated bound to the monocyte such that a competent prothrombinase complex was formed at the monocyte membrane surface. Furthermore, within 5 min of factor V addition to monocytes, near maximal cofactor activity (approximately 70%) was bound and expressed on the monocyte membrane. The proteolytic activity toward factor V was associated primarily with the monocyte membrane, as little proteolytic activity was released into the cell-free supernatant. Proteolytic activity was inhibited by diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride. However, the inhibitor profile obtained with alpha 1-antiproteinase inhibitor, alpha 1-antichymotrypsin, and alpha 2-macroglobulin suggested membrane-bound forms of elastase and cathepsin G were mediating, in large part, the proteolysis observed. These data were confirmed using purified preparations of both proteases and a specific anti-human leukocyte elastase antibody. Thus, expression of these proteases at the monocyte surface may contribute to thrombin generation at extravascular tissue sites by catalyzing the activation of the essential cofactor, factor Va, which binds to the monocyte surface and supports the factor Xa-catalyzed activation of prothrombin.
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PMID:Human coagulation factor V is activated to the functional cofactor by elastase and cathepsin G expressed at the monocyte surface. 783 8

Antistasin, a potent inhibitor of the blood coagulation factor Xa, is the prototype of a novel family of serine-proteinase inhibitors. We have now isolated, sequenced and characterized an antistasin-type inhibitor from the medical leech Hirudo medicinalis. Hirustasin (Hirudo antistasin) was purified to apparent homogeneity by cation-exchange and affinity chromatography. Amino acid sequencing of the 55 amino acid protein (M(r) 5866) revealed that hirustasin is the only antistasin-type protein known to consist of one domain only; 27% and 32% sequence identity was found to the first and second domains of antistasin, respectively, and a nearly exact conservation of the spacing of the ten cysteine residues. Hirustasin is the first inhibitor of tissue kallikrein identified in leeches, and is also a tight-binding inhibitor of trypsin, chymotrypsin and neutrophil cathepsin G. However, despite the high similarity to antistasin, particularly in the vicinity of the putative reactive-site peptide bond, hirustasin neither inhibits blood coagulation in vitro nor amidolytic activity of isolated factor Xa. Thus, structural elements other than the reactive site sequence significantly influence the specificity of antistasin-type proteinase inhibitors.
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PMID:Isolation and characterization of hirustasin, an antistasin-type serine-proteinase inhibitor from the medical leech Hirudo medicinalis. 811 45

The myxoma and malignant rabbit fibroma poxviruses are lethal tumorigenic viruses of rabbits whose virulence is modulated by the production of a virus-encoded secreted serine proteinase inhibitor, SERP-1. This viral protein was detected in medium harvested from myxoma and malignant rabbit fibroma virus-infected cells, and its inhibitory profile has been characterized by gel and kinetic analysis. SERP-1 forms complexes with and inhibits the human fibrinolytic enzymes plasmin, urokinase, and two-chain tissue-type plasminogen activator (association rate constants 3.4 x 10(4), 4.3 x 10(4), and 3.6 x 10(4) M-1 s-1 respectively). It is also able to inhibit C1S, the first enzyme in the complement cascade with an association rate constant which was unaffected by the addition of heparin (1.3 x 10(3) M-1 s-1). SERP-1 acts as a substrate for and is cleaved by thrombin, porcine trypsin, human neutrophil elastase, porcine pancreatic elastase, thermolysin, subtilisin, bovine alpha-chymotrypsin, and factor Xa. Incubation with kallikrein and cathepsin G had no effect. The structure of SERP-1 has been modeled on other members of the serpin family which revealed the characteristic serpin architecture apart from the absence of the D-helix. Structural analysis and kinetic assays demonstrate that the absence of this region does not prevent inhibitory activity and furthermore allow the identification of cysteine residues involved in internal and intermolecular disulfide bonding.
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PMID:Inhibition of plasmin, urokinase, tissue plasminogen activator, and C1S by a myxoma virus serine proteinase inhibitor. 841 56


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