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)

The proteolytic action of thrombin on its receptor (protease-activated receptor-1 or PAR-1) results in a conformational change in which the new N-terminal sequence auto-activates the receptor. Peptide analogs of this N-terminal sequence (TRAPs) are able to mimic the effect of thrombin and an extensive search has led to the definition of the structural requirement for the agonist and antagonist activity on thrombin receptors in several peripheral systems. Thrombin plays an important role in central and peripheral nervous system development and PAR-1 is present in neurons and astrocytes. We have now characterized thrombin receptors pharmacologically in cultured rat astrocytes by using [3H]thymidine incorporation and reversal of stellation induced by Bt2cAMP as end-points. Thrombin increased [3H]thymidine incorporation into DNA with an EC50 of 1 nM and induced a complete reversion of cell stellation. The effects of thrombin on [3H]thymidine incorporation were mimicked by TRAP-14 (EC50 = 3 microM) and a peptide containing non-natural amino acids Ala-Phe(p-F)-Arg-Cha-HArg-Tyr-NH2 (A6Y; EC50 = 0.8 microM). Similarly, these two peptides reversed Bt2cAMP-induced stellation. The effect of thrombin, TRAP-14 and A6Y on [3H]thymidine incorporation into DNA was significantly prevented by L9R, a 9-amino-acid peptide (Leu-Val-Arg-D-Cys-Gly-Lys-His-Ser-Arg; IC50 = 180 microM against thrombin and TRAP-14 and 800 microM against A6Y) previously described as an antagonist in human platelet aggregation. L9R antagonized also thrombin effects on astrocyte morphology. These results demonstrate that rat astrocytes express PAR-1 receptors which are pharmacologically similar to those previously characterized in human platelets.
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PMID:Pharmacological characterization of protease-activated receptor (PAR-1) in rat astrocytes. 910 86

These studies have examined the effects of thrombin-related agonists in stimulating thromboxane production by human platelets. The results presented show that (1) the maximal response elicited by thrombin receptor agonist peptide (TRAP) stimulation was 40% to 50% of that seen with thrombin or the thrombin mutant Thrombin Quick I; (2) pretreatment of platelets with prostaglandin E1 or genistein resulted in differential inhibition of thromboxane production in response to TRAP compared with either enzyme agonist; (3) an antibody to the thrombin receptor cleavage site that inhibits increases in intracellular [Ca2+] only partially reduced thromboxane production in response to 5 nmol+L thrombin and 15 nmol/L Thrombin Quick I; (4) preincubation with 20 mumol/L TRAP resulted in desensitization to further stimulation by 100 mumol/L TRAP, but not by 100 nmol/L thrombin; and (5) the response to thrombin after TRAP desensitization was completely inhibited by the tyrosine kinase inhibitor genistein and was independent of an intracellular [Ca2+] flux, The cumulative results may be explained by the existence of two proteolytically activated receptors that result in thromboxane production in response to thrombin. One is the thrombin receptor/substrate, PAR-1. Stimulation through the second receptor/substrate depends on a genistein-sensitive step, is independent of an intracellular Ca2+ flux, and is initiated by a thrombin-activated receptor that does not depend on interaction with anion-binding exosite I, as previously indicated by the relative activity of Thrombin Quick I in stimulating platelet aggregation and thromboxane production. The proposed second thrombin receptor on platelets represents an additional member of the class of proteolytically activated receptors.
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PMID:Thrombin-induced thromboxane synthesis by human platelets. Properties of anion binding exosite I-independent receptor. 943 1

Thrombin is a serine protease involved in haemostasis which exerts a number of cellular effects, including stimulating mesenchymal cell migration, proliferation, and has been implicated both in normal wound healing and pathological conditions associated with hyperproliferation of smooth muscle cells such as atherosclerosis and restenosis. We hypothesize that thrombin, in addition to its proliferative effects, may also influence the deposition of matrix proteins at sites of vascular injury by directly stimulating smooth muscle cell procollagen production. 10 nM thrombin significantly stimulated rat aortic smooth muscle cell procollagen production by 34 +/- 3% compared to media control cells over a 48 h incubation period, and increased steady state alpha1(I) procollagen mRNA levels by up to 104 +/- 22%. These effects are mediated via interaction of thrombin with the PAR-1 receptor since TRAP (Thrombin Receptor Activating Peptide) stimulated procollagen production by 23 +/- 0.5%. In addition, conditioned medium from thrombin-treated cells stimulated procollagen production by 30 +/- 3% suggesting that thrombin is acting via the production and/or release of an autocrine mediator. These data suggest a novel role for thrombin in vascular wound healing and the development of pathological conditions associated with increased connective tissue deposition.
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PMID:Thrombin stimulates smooth muscle cell procollagen synthesis and mRNA levels via a PAR-1 mediated mechanism. 949 99

Thrombin, the ultimate enzyme in the blood coagulation cascade, has prominent actions on various cells, including neurons. As in platelets, thrombin increases [Ca2+]i mobilization in neurons, and also retracts neurites. Both these effects are mediated through a G protein-coupled, proteolytically activated receptor for thrombin (PAR-1). Prolonged exposure to thrombin kills neurons via apoptosis, that may also involve PAR-1 activation. Increased [Ca2+]i has been a unifying mechanism proposed for cell death in several neurodegenerative diseases. Thrombin-elevated calcium levels may activate intracellular cascades in neurons leading to cell death. Since thrombin mediates its diverse effects on cells through both heterotrimeric and monomeric G proteins, we also explored what effect altering differential G protein coupling would have on the neuronal response to thrombin. We studied calcium mobilization by thrombin in a model motor neuronal cell line, NSC19, using fluorescence image analysis. Confirming effects in other neuronal types, thrombin caused dramatic increases in [Ca2+]i levels, both transiently and after prolonged exposure, which involved activation and cleavage of the PAR-1 receptor. Using enzyme linked immunosorbent assay (ELISA) and dot-blot analysis, we found that the N-terminal fragment of PAR-1 was released into the medium after exposure to thrombin. We confirmed that PAR-1 protein and mRNA expression occurred in motor neurons. We found that cholera toxin inhibited thrombin-mediated Ca2+ influx, pertussis toxin did not significantly alter thrombin action, and lovastatin, a small 21-kDa Ras GTPase (Rho) modulator, showed a tendency to reduce the thrombin effect. These data indicate that thrombin-increased [Ca2+]i, sufficient to trigger cell death in motor neurons, might be approached in vivo by modulating thrombin signaling through PAR-1.
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PMID:Calcium mobilization and protease-activated receptor cleavage after thrombin stimulation in motor neurons. 958 68

The protease-activated family of G protein-coupled receptors includes PAR-1 and PAR-3, which are activated by thrombin, and PAR-2, which is activated by trypsin and tryptase. PAR-2 has recently been shown to be expressed in human endothelial cells. In the present studies, we have examined the expression of PAR-2 in other cells, particularly vascular smooth muscle, and tested whether the receptors are functional. The results show that PAR-2 is present in human aorta and coronary artery smooth muscle cells, as well as in arteries traversing the walls of the small intestine. It was also detected in human keratinocytes, sweat glands, intestinal smooth muscle, and intestinal epithelium, but not at all in myocardial smooth muscle and only inconsistently in intestinal veins and venules. Activation of aortic smooth muscle cells in culture with PAR-2 peptide agonists caused a transient increase in the cytosolic Ca2+ concentration. In contrast, PAR-2 mRNA could not be detected in saphenous vein smooth muscle cells, and the same cells placed in culture showed little, if any, response to the PAR-2 agonist peptides. These observations show that PAR-2 is widely distributed in human vascular smooth muscle, particularly in arteries. However, this is not a universal finding and at least some venous smooth muscle cells, including those in saphenous veins, apparently do not express the receptor in detectable amounts.
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PMID:Differential expression of functional protease-activated receptor-2 (PAR-2) in human vascular smooth muscle cells. 959 43

Proteolytically activated receptors (PARs) represent an emerging subset of seven transmembrane G protein-coupled receptors that mediate cell activation events by receptor cleavage at distinct scissile bonds located within receptor amino termini. Differential genomic blotting using a yeast artificial chromosome known to contain the PAR-1 and PAR-2 genes identified the PAR-3 gene within a PAR gene cluster spanning approximately 100 kilobases at 5q13. The PAR-3 gene is relatively small (approximately 12 kilobases); and, like the PAR-1 and PAR-2 genes, it displays a two-exon structure, with the majority of the coding sequence and the proteolytic cleavage site contained within the larger second exon. Sequence analysis of the 5'-flanking region demonstrates that the promoter is TATA-less, similar to that seen with PAR-1, with the identification of nucleic acid motifs potentially involved in transcriptional gene regulation, including AP-1, GATA, and octameric sequences. PAR-3 transcripts were apparent in human vascular endothelial cells, although at considerably lower levels than those of PAR-1 and not significantly modulated by the endothelial cell stimulus tumor necrosis factor-alpha. Likewise, although PAR-3 mRNA was evident in human platelets, receptor cell surface expression was modest (approximately 10%) compared with that of PAR-1. Thus, although PAR-3 is postulated to represent a second thrombin receptor, its modest endothelial cell and platelet expression suggest that PAR-3 activation by alpha-thrombin is less relevant for physiological responses in these mature cells. Rather, given its disparately greater expression in megakaryocytes (and megakaryocyte-like human erythroleukemia cells), a regulatory role in cellular development (by protease activation) could be postulated.
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PMID:The human proteinase-activated receptor-3 (PAR-3) gene. Identification within a Par gene cluster and characterization in vascular endothelial cells and platelets. 961 15

The serine protease, thrombin, is both a potent agonist for platelet aggregation and a mitogen inducing the proliferation of other cell types. Many cellular responses to thrombin are mediated by a G-protein-coupled thrombin receptor (protease-activated receptor-1, PAR-1). This represents the prototype of a new family of proteolytically cleaved receptors that includes PAR-2 and the recently identified PAR-3. Like PAR-1, PAR-3 is a potential thrombin receptor. Their similar gene structure, mechanism of activation, and colocalization to 5q13 raises the question of a common evolutionary origin and of their belonging to a clustered gene family. Construction of a physical map of the 5q13 region by pulsed-field gel electrophoresis (PFGE) has allowed us to identify six potential CpG islands and to establish a linkage of the PAR genes. Southern blot analysis showed that they were in a cluster on a 560-kb Asc I fragment, in the order PAR-2, PAR-1, and PAR-3. PAR-1 and PAR-2 genes were contained within the identical 240-kb Not I fragment, thus confirming a tight linkage between them. The localization of other CpG islands suggested that more PAR-family genes may be present.
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PMID:Protease-activated receptor genes are clustered on 5q13. 963 95

Protease-activated receptors (PARs) are a family of G protein-coupled receptors activated by a tethered ligand sequence within the amino terminal that are revealed by site-specific proteolysis. The thrombin-sensitive PAR-1 and trypsin-activated PAR-2 mediate endothelium-dependent vascular relaxation in a number of species. Because both thrombin and trypsin-like enzymes have been implicated in coronary artery disease, the purpose of this study was to investigate whether similar receptors are present in human coronary arteries. Thrombin (0.001 to 0.1 U/mL) and trypsin (0.001 to 1 U/mL) caused concentration- and endothelium-dependent relaxations of human coronary artery ring segments suspended in organ chambers for isometric tension recording and contracted with the thromboxane A2 mimetic U46619. These relaxations were dependent on the catalytic activity of each enzyme and were inhibited by the NO synthase inhibitor NG-nitro-L-arginine (100 micromol/L) and the NO scavenger oxyhemoglobin (20 micromol/L). The synthetic PAR-1 tethered ligand sequence SFLLRN-NH2 (0.01 to 10 micromol/L) also caused endothelium-dependent relaxation of U46619-contracted human coronary arteries; however, the equivalent PAR-2 ligand SLIGKV-NH2 caused almost no relaxation. In addition, desensitization to either thrombin or trypsin resulted in cross-desensitization to the other enzyme but had only a minimal affect on the response to SFLLRN-NH2. Therefore, we conclude that human coronary artery endothelial cells possess a PAR-1-like receptor that is potently activated by thrombin, trypsin, and SFLLRN-NH2 to cause NO-mediated vascular relaxation. Once cleaved, this receptor is recycled in a truncated form, able to respond to exogenous application of only its tethered ligand sequence, suggesting the presence of another endogenous activator possibly acting independently of receptor cleavage.
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PMID:Atypical protease-activated receptor mediates endothelium-dependent relaxation of human coronary arteries. 964 27

Protease activated receptors (PARs) compose a family of G protein signal transduction receptors activated by proteolysis. In this study, the susceptibility of PARs expressed on human keratinocytes and dermal fibroblasts to the human mast cell proteases tryptase and chymase was evaluated. PAR activation was measured by monitoring cytosolic [Ca2+] in cells loaded with the fluorescent Ca2+ probe Fura-2. Tryptase produced transient cytosolic Ca2+ mobilization in keratinocytes, but not in fibroblasts. Ca2+ mobilization in keratinocytes required enzymatically active tryptase, demonstrated desensitization, and was blocked by pretreatment of cells with the PAR-2 peptide agonist SLIGKV, trypsin, or the phospholipase inhibitor U73122. Heparin, a GAG that binds to tryptase, stabilizing its functional form, also inhibited tryptase-induced Ca2+ mobilization. The maximal response elicited by tryptase was smaller than that observed upon treatment of keratinocytes with trypsin, a known activator of PAR-2, and keratinocytes made refractory to tryptase by pretreatment with the protease remained responsive to trypsin. Pretreatment of keratinocytes with thrombin, an activator of PAR-1 and -3 (thrombin receptors), had no detectable effect on the tryptase or trypsin responses. These data suggest that in keratinocytes tryptase may be activating a subpopulation of PAR-2 receptors. Treatment of keratinocytes or fibroblasts with human chymase did not produce Ca2+ mobilization, nor did it affect Ca2+ mobilization produced by trypsin. However, chymase pretreatment of fibroblasts rapidly inhibited the ability of these cells to respond to thrombin. Inhibition was dependent on chymase enzymatic activity and was not significantly affected by the presence of heparin. This finding is consistent with studies indicating that PAR-1 may be susceptible to proteases with chymotrypsin-like specificity. These results suggest that the proteases tryptase and chymase secreted from mast cells in skin may affect the behavior of surrounding cells by the hydrolysis of PARs expressed by these cells.
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PMID:Reaction of mast cell proteases tryptase and chymase with protease activated receptors (PARs) on keratinocytes and fibroblasts. 964 24

Apoptosis, often also termed "programmed cell death", occurs in normal development in the brain and spinal cord. Important to concepts of disease and potential intervention is the exciting finding that apoptosis is also found after neurotrauma and in a number of neurodegenerative diseases. Although the precise mechanism of neuronal cell loss remains unknown, much emphasis has been placed recently on the activation of cell death protease cascades within the cell. How these cascades may be activated, especially from extracellular influences, is currently poorly understood. Thrombin, the multifunctional coagulation protease, is an early phase modulator at sites of tissue injury and has been shown to induce cell death in neurons by an apoptotic mechanism by activating its receptor, PAR-1. Using a model motor neuronal cell line, NSC19, which we have shown undergoes apoptosis after treatment with classic apoptosis inducers such as the topoisomerase inhibitors camptothecin and etoposide, we unambiguously found that nanomolar thrombin induced characteristic signs of apoptosis. Strikingly, endonucleolysis was accompanied by an increase in caspase-3-like activity in cellular extracts, which correlated with both detection of caspase-induced signature cleavage of the cortical cytoskeleton component nonerythroid spectrin (alpha-fodrin) and identification of increased accessibility of a caspase cleavage domain, using an antibody (Ab127) made against a synthetic peptide KGDEVD. Demonstrating that thrombin activation of death proteases was linked to cell death, we were able to inhibit thrombin-induced apoptosis by using a caspase family inhibitor, benzyloxycarbonyl-Asp-(oMe)-fluoromethyl ketone (Boc-D-FMK). These novel results demonstrate that thrombin serves as an extracellular "death signal" to activate intracellular protease pathways. These pathways lead to apoptotic cell death and can be modulated by inhibiting caspase activity downstream to PAR-1.
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PMID:Thrombin is an extracellular signal that activates intracellular death protease pathways inducing apoptosis in model motor neurons. 965 39


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