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)

Granzymes are a family of serine proteases that are harbored in cytoplasmic granules of activated T lymphocytes and are released upon target cell interaction. Immediate and complete neurite retraction was induced in a mouse neuronal cell line when total extracts of granule proteins were added. This activity was isolated and identified as granzyme A. This protease not only induced neurite retraction at nanomolar concentrations but also reversed the stellation of astrocytes. Both effects were critically dependent on the esterolytic activity of granzyme A. As neurite retraction is known to be induced by thrombin, possible cleavage and activation of the thrombin receptor were investigated. A synthetic peptide spanning the N-terminal thrombin receptor activation sequence was cleaved by granzyme A at the authentic thrombin cleavage site Leu-Asp-Pro-Arg-Ser. Antibodies to the thrombin receptor inhibited both thrombin and granzyme A-mediated neurite retraction. Thus, T-cell-released granzyme A induces cellular responses by activation of the thrombin receptor. As brain-infiltrating CD4+ lymphocytes are the effector cells in experimental allergic encephalomyelitis, granzyme A released in the brain may contribute to the etiology of autoimmune disorders in the nervous system.
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PMID:Granzyme A released upon stimulation of cytotoxic T lymphocytes activates the thrombin receptor on neuronal cells and astrocytes. 805 66

Granzyme A is a serine protease stored in cytoplasmic granules of cytotoxic and helper T lymphocytes. This protease seems to elicit thrombin receptor-mediated responses in neural cells, thereby triggering neurite retraction and reversal of astrocyte stellation. Here we report that granzyme A does not cause platelet aggregation even at concentrations that are more than two orders of magnitude higher than the EC50 for granzyme A in causing morphological changes in neural cells. However, granzyme A blocks thrombin-induced platelet aggregation in a dose-dependent manner without affecting the response to either ADP or to the peptide agonist of the thrombin receptor SFLLRN that corresponds in sequence to the tethered ligand domain. The inability of granzyme A to cause aggregation and its inhibition of thrombin-induced aggregation were seen in platelets from man, rat and mouse. Granzyme A does not affect the catalytic activity of thrombin in cleaving a chromogenic substrate or the macromolecular substrate fibrinogen. However, granzyme A does seem to cleave the thrombin receptor on platelets to produce a weak Ca2+ signal and reduce the response to subsequent challenge with thrombin, but does not induce a signal in thrombin-stimulated platelets. It is proposed that granzyme A interacts with the thrombin receptor found on platelets in a manner that is insufficient to cause aggregation, but sufficient to compete with thrombin for the receptor. These results suggest that granzyme A cleaves the thrombin receptor at a rate that is insufficient to cause platelet aggregation but is sufficient to cause morphological changes in neural cells. Furthermore, these observations demonstrate that granzyme A release occurring during immune responses within blood vessels would not directly cause platelet aggregation.
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PMID:The serine protease granzyme A does not induce platelet aggregation but inhibits responses triggered by thrombin. 864 80

Granzymes, serine proteases located in the granules of cytotoxic T cel ls and NK cells, are essential for induction of target cell apoptosis. However, since cytotoxic cells constitutively secrete a portion of their synthesized granzymes, these proteases could mediate extracellular functions independent of their role in the lytic event. Thrombin, another serine protease, can induce cytokine production in a number of different cell types. In this study, we test the hypothesis that granzymes, like thrombin, can regulate cell-mediated immunity by inducing the production of different cytokines. We show that granzyme A (GA) stimulates IL-6, IL-8, and TNF-alpha production by human PBMC and purified monocytes. In contrast, monocytes exposed to thrombin had enhanced IL-8 production with no induction of IL-6 or TNF-alpha production. However, monocytes exposed to either GA or thrombin had enhanced phagocytic activity. The enzymatic activity of GA and thrombin was required for the induction of cytokine production and for the enhancement of phagocytic activity. The induction of different cytokine profiles by GA vs thrombin suggested that GA activates monocytes via a receptor that was different from the thrombin receptor. This conclusion was strengthened by the fact that GA was incapable of inducing Ca2+ mobilization in insect cells transfected with the thrombin receptor. These results suggest that enzymatically active GA mediates important immunoregulatory functions through signaling pathways that does not involve thrombin receptor activation.
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PMID:Extracellular activities of human granzyme A. Monocyte activation by granzyme A versus alpha-thrombin. 878 23

The kinetic parameters were determined for the hydrolysis of a peptide based on the activation site of the thrombin receptor (residues 38-60) by thrombin and 12 other proteases. The kcat and Km values for the cleavage of this peptide (TR39-40) by thrombin were 107 s-1 and 1.3 microM; the kcat/Km of TR39-40 is among the highest observed for thrombin. A model is presented that reconciles the parameters for cleavage of the peptide with the concentration dependence of cellular responses to thrombin. Cleavage of TR39-40 was not specific for thrombin. The pancreatic proteases trypsin and chymotrypsin hydrolysed TR39-40 efficiently (kcat/Km > 10(6) M-1.s-1). Whereas trypsin cleaved TR39-40 at the thrombin activation site (Arg41-Ser42), chymotrypsin hydrolysed the peptide after Phe43. This chymotryptic cleavage would result in inactivation of the receptor. The efficient cleavage of TR39-40 by chymotrypsin (kcat/Km approximately 10(6) M-1.s-1) was predominantly due to a low Km value (2.8 microM). The proteases factor Xa, plasmin, plasma kallikrein, activated protein C and granzyme A also hydrolysed TR39-40 at the Arg41-Ser43 bond, but exhibited kcat/Km values that were at least 10(3)-fold lower than that observed with thrombin. Both tissue and urokinase plasminogen activators as well as granzyme B and neutrophil elastase were unable to cleave TR39-60 at appreciable rates. However, neutrophil cathepsin G hydrolysed the receptor peptide after Phe55. Like the chymotryptic cleavage, this cleavage would lead to inactivation of the receptor, but the cathepsin G reaction was markedly less efficient; the kcat/K(m) value was almost four orders of magnitude lower than that for thrombin. In addition to the above cleavage sites, a secondary site for thrombin and other arginine-specific proteases was identified at Arg46, but the cleavage at this site only occurred at very low rates and is unlikely to be significant in vivo.
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PMID:Cleavage of the thrombin receptor: identification of potential activators and inactivators. 894 6

Novel aryl derivatives of benzamidine were synthesized and tested for their inhibitory potency against bovine trypsin, rat skin tryptase, human recombinant granzyme A, human thrombin, and human plasma kallikrein. All compounds show competitive inhibition against these proteases with Ki values in the micromolar range. X-ray structures were determined to 1.8 A resolution for trypsin complexed with two of the para-substituted benzamidine derivatives, 1-(4-amidinophenyl)-3-(4-chlorophenyl)urea (ACPU) and 1-(4-amidinophenyl)-3-(4-phenoxyphenyl)urea (APPU). Although the inhibitors do not engage in direct and specific interactions outside the S1 pocket, they do form intimate indirect contacts with the active site of trypsin. The inhibitors are linked to the enzyme by a sulfate ion that forms an intricate network of three-centered hydrogen bonds. Comparison of these structures with other serine protease structures with noncovalently bound oxyanions reveals a pair of highly conserved oxyanion-binding sites in the active site. The positions of noncovalently bound oxyanions, such as the oxygen atoms of sulfate, are distinct from the positions of covalent oxyanions of tetrahedral intermediates. Noncovalent oxyanion positions are outside the "oxyanion hole." Kinetics data suggest that protonation stabilizes the ternary inhibitor/oxyanion/protease complex. In sum, both cations and anions can mediate Ki. Cation mediation of potency of competitive inhibitors of serine proteases was previously reported by Stroud and co-workers [Katz, B. A., Clark, J. M., Finer-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Moore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612].
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PMID:Oxyanion-mediated inhibition of serine proteases. 983 2

MNEI (monocyte/neutrophil elastase inhibitor) is a 42 kDa serpin superfamily protein characterized initially as a fast-acting inhibitor of neutrophil elastase. Here we show that MNEI has a broader specificity, efficiently inhibiting proteases with elastase- and chymotrypsin-like specificities. Reaction of MNEI with neutrophil proteinase-3, an elastase-like protease, and porcine pancreatic elastase demonstrated rapid inhibition rate constants >10(7) M(-1) s(-1), similar to that observed for neutrophil elastase. Reactions of MNEI with chymotrypsin-like proteases were also rapid: cathepsin G from neutrophils (>10(6) M(-1) s(-1)), mast cell chymase (>10(5) M(-1) s(-1)), chymotrypsin (>10(6) M(-1) s(-1)), and prostate-specific antigen (PSA), which had the slowest rate constant at approximately 10(4) M(-1) s(-1). Inhibition of trypsin-like (plasmin, granzyme A, and thrombin) and caspase-like (granzyme B) serine proteases was not observed or highly inefficient (trypsin), nor was inhibition of proteases from the cysteine (caspase-1 and caspase-3) and metalloprotease (macrophage elastase, MMP-12) families. The stoichiometry of inhibition for all inhibitory reactions was near 1, and inhibitory complexes were resistant to dissociation by SDS, further indicating the specificity of MNEI for elastase- and chymotrypsin-like proteases. Determination of the reactive site of MNEI by N-terminal sequencing and mass analysis of reaction products identified two reactive sites, each with a different specificity. Cys(344), which corresponds to Met(358), the P(1) site of alpha1-antitrypsin, was the inhibitory site for elastase-like proteases and PSA, while the preceding residue, Phe(343), was the inhibitory site for chymotrypsin-like proteases. This study demonstrates that MNEI has two functional reactive sites corresponding to the predicted P(1) and P(2) positions of the reactive center loop. The data suggest that MNEI plays a regulatory role at extravascular sites to limit inflammatory damage due to proteases of cellular origin.
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PMID:The serpin MNEI inhibits elastase-like and chymotrypsin-like serine proteases through efficient reactions at two active sites. 1174 53

Platelet activation is involved in the pathogenesis of cerebrovascular ischemia, but the major agonist involved has yet to be identified. To investigate the role of thrombin in platelet activation in patients with acute ischemic stroke, and while thrombin is the most likely candidate for activation of the thrombin receptor PAR-1 in vivo, we assessed its cleavage and internalization using the antibodies SPAN12, binding to uncleaved PAR-1, and WEDE15, recognizing cleaved and uncleaved, but not internalized PAR-1. In contrast to healthy age-matched controls, platelets from stroke patients exhibited significant cleavage and internalization of PAR-1 (P<0.001) and failed to respond to thrombin in vitro. Enhanced surface expression of CD62P, CD63, TSP-1 and less mepacrine uptake showed platelet degranulation during stroke. Platelets from patients with acute cerebral ischemia are exhausted and desensitized to thrombin through cleavage of PAR-1, indicating that high concentrations of thrombin occur with acute cerebrovascular ischemic events in vivo.
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PMID:Platelets in patients with acute ischemic stroke are exhausted and refractory to thrombin, due to cleavage of the seven-transmembrane thrombin receptor (PAR-1). 1496 Nov 62

Some of extracellular serine proteases with trypsin-like specificity of cleavage have been known to increase the release of inflammatory mediators from various cell types. For instance, two well-known trypsin-like serine proteases circulating in blood, granzyme A (GrA) and thrombin, have been found to promote interleukin (IL)-8 release from an alveolar epithelial A549 cell line. However, the mechanisms by which the proteases promote IL-8 release from the cells are not fully understood. In the present study, using A549 cells we found that (1) thrombin promoted IL-8 release from the cells via a mechanism partially involving activation of protease-activated receptor-1, a G-protein coupled receptor, whereas a recombinant form of GrA (rGrA) did it via a mechanism that does not involve the receptor activation; that (2) unlike rGrA, thrombin did not cause detachment and microtubule disruption of the cells; and that (3) the release of IL-8 induced by rGrA was inhibited in the presence of taxol, a microtubule-stabilizing reagent, whereas that induced by thrombin was not. These findings suggest that rGrA and thrombin promote the release of IL-8 from A549 cells through distinct mechanisms.
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PMID:Granzyme A and thrombin differentially promote the release of interleukin-8 from alveolar epithelial A549 cells. 2042 14

Spontaneous intracerebral hemorrhage (ICH) is one of the most severe types of stroke. Thrombin has been reported to participate in brain repair following ICH and play an important role in angiogenesis. Our previous studies have shown that ICH induces angiogenesis in damaged rat brain, accompanied by upregulation of expression of thrombospondin (TSP)-1 and TSP-2. The aim of the present study was to investigate whether the expression of TSP-1 and TSP-2 was regulated by thrombin in rat brain following ICH. A rat model of ICH was induced by injection of autologous blood into the right globus pallidus (GP). Hirudin, a thrombin specific inhibitor, or thrombin was injected into the GP. Immunohistochemistry, quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) and western blot assays were applied. Results showed that ICH induced an increase in the expression of TSP-1 mRNA and TSP-2 mRNA after ICH, whereas hirudin significantly inhibited the expression of TSPs mRNA after ICH (P<0.05). In contrast, sole thrombin treatment in normal rats induced strong expression of TSP-1 or TSP-2 in the blood vessels around the damaged brain region when compared with those without thrombin treatment. Western blot analysis data confirmed that the protein levels of TSPs were significantly increased when compared with those in the sham control group (P<0.01). These findings support that thrombin positively regulates the expression of TSP-1 and TSP-2 after ICH, which may be involved in modulating angiogenesis in injured brains following ICH.
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PMID:Thrombin promotes the expression of thrombospondin-1 and -2 in a rat model of intracerebral hemorrhage. 2304 6

We have created models to predict cleavage sites for several human proteases including caspase-1, caspase-3, caspase-6, caspase-7, cathepsin B, cathepsin D, cathepsin G, cathepsin K, cathepsin L, elastase-2, granzyme A, granzyme B, matrix metallopeptidase-2 (MMP2), MMP7, MMP9, thrombin, and trypsin-1. Rather than representing the sequence pattern around the potential cleavage site through a series of flags with each flag representing one of the 20 standard amino acids, we first represent each amino acid by its calculated properties. For these calculated properties, we use validated cheminformatic descriptors, such as molecular weight, logP, and polar surface area, of the individual amino acids. Finally, the cleavage site-specific descriptors are calculated through various combinations of the individual amino acid descriptors for the residues surrounding the cleavage site. Some of these combinations do not take into account the location of the residue, as long as it is in a prescribed neighborhood of the potential cleavage site, whereas others are sensitive to the precise order of the residues in the sequence. The key advantage of this approach is that it allows one to perform meaningful calculations with nonstandard amino acids for which little or no data exists. Finally, using both docking and molecular dynamics simulations, we examine the potential for and limitations of protease crystal structures to impact the design of proteolytically stable peptides.
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PMID:A combined cheminformatic and bioinformatic approach to address the proteolytic stability challenge in peptide-based drug discovery. 2627 Mar 98


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