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)

Previous reports have shown that neuronal and glial cells express functionally active thrombin receptors. The thrombin receptor (PAR-1), a member of a growing family of protease activated receptors (PARs), requires cleavage of the extracellular amino-terminus domain by thrombin to induce signal transduction. Studies from our laboratory have shown that PAR-1 activation following the addition of thrombin or a synthetic thrombin receptor activating peptide (TRAP) induces motoneuron cell death both in vitro and in vivo. In addition to increasing motoneuron cell death, PAR- 1 activation leads to decreases in the mean neurite length and side branching in highly enriched motoneuron cultures. It has been suggested that motoneuron survival depends on access to sufficient target-derived neurotrophic factors through axonal branching and synaptic contacts. However, whether the thrombininduced effects on motoneurons can be prevented by neurotrophic factors is still unknown. Using highly enriched avian motoneuron cultures, we show here that alone, soluble chick skeletal muscle extracts (CMX), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and glial cell line-derived neurotrophic factor (GDNF) significantly increased motoneuron survival compared to controls, whereas nerve growth factor (NGF) did not have a significant effect on motoneuron survival. Furthermore, cotreatment with muscle-derived agents (i.e., CMX, BDNF, GDNF) significantly prevented the death of motoneurons induced by alpha-thrombin. Yet, non-muscle-derived agents (CNTF and NGF) had little or no significant effect in reversing thrombin-induced motoneuron death. CMX and CNTF significantly increased the mean length of neurites, whereas NGF, BDNF, and GDNF failed to enhance neurite outgrowth compared to controls. Furthermore, CMX and CNTF significantly prevented thrombin-induced inhibition of neurite outgrowth, whereas BDNF and GDNF only partially reversed thrombin-induced inhibition of neurite outgrowth. These findings show differential effects of neurotrophic factors on thrombin-induced motoneuron degeneration and suggest specific overlaps between the trophic and stress pathways activated by some neurotrophic agents and thrombin, respectively.
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PMID:Prevention of thrombin-induced motoneuron degeneration with different neurotrophic factors in highly enriched cultures. 1008 90

Three members of the family of protease-activated receptors (PARs), PARs-1, -3 and -4, have been identified as thrombin receptors. PAR-1 is expressed by primary myoblast cultures, and expression is repressed once myoblasts fuse to form myotubes. The current study was undertaken to investigate the hypothesis that thrombin inhibits myoblast fusion. Primary rodent myoblast cultures were deprived of serum to promote myoblast fusion and then cultured in the presence or absence of thrombin. Thrombin inhibited myoblast fusion, but another notable effect was observed; 50% of control cells were apoptotic within 24 h of serum deprivation, whereas less than 15% of thrombin-treated cells showed signs of apoptosis. Proteolysis was required for the effect of thrombin, but no other serine protease tested mimicked the action of thrombin. Neither a PAR-1- nor a PAR-4-activating peptide inhibited apoptosis or fusion, and myoblast cultures were negative for PAR-3 expression. Myoblasts exposed to thrombin for 1 h and then changed to medium without thrombin accumulated apoptosis inhibitory activity in their medium over the subsequent 20 h. Thus the protective action of thrombin appears to be effected through cleavage of an unidentified thrombin receptor, leading to secretion of a downstream apoptosis inhibitory factor. These results demonstrate that thrombin functions as a survival factor for myoblasts and is likely to play an important role in muscle development and repair.
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PMID:Thrombin, a survival factor for cultured myoblasts. 1009 88

A second protease-activated receptor (PAR-2) that could be activated by trypsin or more physiologically by mast cell tryptase has been recently cloned. Both the structure and activation mechanism of PAR-2 was similar to the functional thrombin receptor (PAR-1). Although many effects of the coagulation protease thrombin on the vascular endothelium could be attributed to PAR-1 activation, very little is known about the physiological and pathophysiological role of PAR-2. We investigated whether stimulation of PAR-2 on endothelial cells induced two cellular responses that play a central role in primary and secondary haemostasis: the release of high molecular weight von Willebrand factor (hmw-VWF) from Weibel-Palade bodies and the de novo synthesis of tissue factor (TF) mRNA and protein. Human umbilical vein endothelial cells (HUVEC) were incubated with agonists for PAR-2 at 37 degrees C. Both trypsin and SLIGKV increased TF mRNA and activity and induced the release of hmw-VWF due to elevated levels of cytosolic Ca2+. Trypsin (10 nm) induced a 6-fold increase of TF mRNA and reduced time until fibrin clot formation to 37%, indicating trebling of the cell surface located TF activity. Stimulation of HUVEC with the PAR-2 agonist peptide SLIGKV induced a dose-dependent increase of TF mRNA up to 6 times and TF activity up to 3 times. Release of hmw-VWF was achieved both after incubation of HUVEC with trypsin and SLIGKV and was directly depending on intracellular Ca2+ mobilization. To make results comparable to the functional thrombin receptor, homologous experiments were carried out using the PAR-1 agonists thrombin and SFLLRN.
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PMID:Endothelial protease-activated receptor-2 induces tissue factor expression and von Willebrand factor release. 1023 35

Several studies have shown that both neuronal and glial cells express functional thrombin receptors as well as prothrombin transcripts. Recently, we (and others) have shown that alpha-thrombin induces apoptotic cell death in different neuronal cell types, including motoneurons, in culture. Thrombin-induced effects on different cells are mediated through the cell surface protease-activated thrombin receptor, PAR-1. Furthermore, it has been shown that, in contrast to thrombin, which induces proteolysis of other proteins besides its receptor, the thrombin receptor agonist peptide, serine-phenylalanine-leucine-leucine-arginine-asparagine-proline (SFLLRNP), is only known to activate this receptor. However, whether activation of the thrombin receptor in vivo affects the development of spinal cord motoneurons is not known. Here, we show that treatment with a synthetic SFLLRNP peptide induced a dose-dependent degeneration and death of spinal motoneurons both in highly enriched cultures and in the developing chick embryo in vivo. However, cotreatment with caspase inhibitors completely abolished SFLLRNP-induced motoneuron death both in vitro and in vivo. These results suggest that developing motoneurons express functionally active PAR-1 whose activation leads to cell death through stimulation of the caspase family of proteins. Our findings also suggest a novel and deleterious role for PAR-like receptors in the central nervous system, different from their previously known functions in the vascular and circulatory system.
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PMID:Activation of the protease-activated thrombin receptor (PAR)-1 induces motoneuron degeneration in the developing avian embryo. 1033 38

1. Proteases regulate cells by cleaving proteinase-activated receptors (PARs). Thrombin and trypsin cleave PAR-1 and PAR-2 on neurons and astrocytes of the brain to regulate morphology, growth and survival. We hypothesized that thrombin and mast cell tryptase, which are generated and released during trauma and inflammation, regulate enteric neurons by cleaving PAR-1 and PAR-2. 2. We detected immunoreactive PAR-1 and PAR-2 in > 60 % of neurons from the myenteric plexus of guinea-pig small intestine in primary culture. A large proportion of neurons that expressed substance P, vasoactive intestinal peptide or nitric oxide synthase also expressed PAR-1 and PAR-2. We confirmed expression of PAR-1 and PAR-2 in the myenteric plexus by RT-PCR using primers based on sequences of cloned guinea-pig receptors. 3. Thrombin, trypsin, tryptase, a filtrate from degranulated mast cells, and peptides corresponding to the tethered ligand domains of PAR-1 and PAR-2 increased [Ca2+]i in > 50 % of cultured myenteric neurons. Approximately 60 % of neurons that responded to PAR-1 agonists responded to PAR-2 agonists, and > 90 % of PAR-1 and PAR-2 responsive neurons responded to ATP. 4. These results indicate that a large proportion of myenteric neurons that express excitatory and inhibitory neurotransmitters and purinoceptors also express PAR-1 and PAR-2. Thrombin and tryptase may excite myenteric neurons during trauma and inflammation when prothrombin is activated and mast cells degranulate. This novel action of serine proteases probably contributes to abnormal neurotransmission and motility in the inflamed intestine.
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PMID:Thrombin and mast cell tryptase regulate guinea-pig myenteric neurons through proteinase-activated receptors-1 and -2. 1035 15

The thrombin receptor PAR-1 is activated by alpha-thrombin to stimulate cells, including platelets, through the tethered-ligand sequence SFLLRN. We have discovered a novel series of heterocycle-peptide hybrids comprised of a tripeptide segment, such as Cha-Arg-Phe, and an N-terminal heterocyclic group, many of which behave as full PAR-1 agonists. Certain compounds with an aminotriazole group, such as 4 and 16, are nearly as potent as SFLLRN-NH2 in inducing platelet aggregation. Also, some arylethenoyl "N-capped" compounds, such as 52 and 57, exhibit mixed PAR-1 agonist-antagonist activity.
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PMID:Heterocycle-peptide hybrid compounds. Aminotriazole-containing agonists of the thrombin receptor (PAR-1). 1036 Jul 49

1. This study investigates, whether in addition to the thrombin receptor (PAR-1), the proteinase-activated receptor-2 (PAR-2) is present in vascular smooth muscle cells (SMC) and mediates mitogenesis. PAR-2 is activated by low concentrations of trypsin and the synthetic peptide SLIGRL. 2. Stimulation of bovine coronary artery SMC by trypsin (2 nM) caused a 3 fold increase in DNLA-synthesis. A similar effect was observed with 10 nM thrombin. Trypsin-induced mitogenesis was inhibited by soybean trypsin inhibitor, indicating that the proteolytic activity of the enzyme was required for its mitogenic effect. 3. The specific PAR-2-activating peptide SLIGRL or the PAR1-activating peptide SFFLRN did not elicit mitogenesis. 4. When the SMC were exposed to SLIGRL (40 nM), a homologous desensitization of cytosolic Ca2+ mobilization was found after subsequent stimulation with trypsin (40 nM) but not thrombin (15 nM). 5. Trypsin (2 nM) as well as SLIGRL (100 microm) activated the nuclear factor KB (NFkappaB) with a maximum response 2 h after stimulation of the SMC. This suggests that both agonists acted via a common receptor, PAR-2. Maximum activation of NFkappaB by thrombin (10 nM) was detected after 4-5 h. 6. These data suggest that PAR-2 is present in coronary SMC and mediates a mitogenic response. Activation of NFkappaB via either PAR-1 or PAR-2 does not predict mitogenesis.
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PMID:Evidence for proteinase-activated receptor-2 (PAR-2)-mediated mitogenesis in coronary artery smooth muscle cells. 1037 15

1. Protease-activated receptors (PARs) are activated by an irreversible proteolytic mechanism which renders cleaved receptors unresponsive to subsequent challenges with activating enzymes. Non-specific proteolysis of PARs downstream of the activation site also prevents subsequent enzymic activation. Therefore, we investigated the effects of non-activating amino-terminal proteolysis with the bacterial protease thermolysin on PAR-mediated relaxation of porcine coronary artery ring preparations contracted with the thromboxane A2 mimetic U46619 (1-10 nM). 2. Treatment of contracted artery ring segments with thermolysin (0.01-1 u ml-1, 20 min) caused no response, but abolished endothelium-dependent relaxations induced by the enzymic activators of PAR-1, and PAR-2, thrombin (0.01-0.3 u ml-1) and trypsin (0.003-0.1 u ml-1) respectively. The same treatment, however, did not affect similar responses to the proteolysis-independent PAR-1 and PAR-2 activating peptides, SFLLRN-NH2 and SLIGRL-NH2 respectively (0.1-10 microM). 3. The inhibition of responsiveness to trypsin after thermolysin treatment recovered in a time-dependent manner, with maximal recovery (77.3 +/- 8.0% of time controls) occurring 150 min after thermolysin treatment. No recovery of responsiveness to thrombin after thermolysin treatment was observed within this time, however, the thrombin response returned to control levels after 20 h. 4. The recovery of responsiveness to trypsin was inhibited by the translation inhibitor cycloheximide (100 microM; 17.3 +/- 4.7%) and the protein trafficking inhibitor brefeldin A (10 microM; 12.1 +/- 4.8%) but was unaffected by the transcription inhibitor actinomycin D (2 microM; 65.1 +/- 3.6%), which did, however, abolish upregulation of B1-kinin receptors in this preparation. 5. In conclusion, our findings indicate that activation-independent amino-terminal proteolysis of PARs stimulates selective recovery of endothelial cell PAR-2 responsiveness, which appears to be regulated by translation. Such a novel mechanism for the maintenance of responsiveness to enzymic PAR-2 activators may imply that these receptors play important roles in vascular homeostasis.
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PMID:Protease-activated receptor-2 turnover stimulated independently of receptor activation in porcine coronary endothelial cells. 1040 51

Rat glioma C6 cells have been demonstrated to be a suitable model in the investigation of PAR-1-type thrombin receptors in brain. However, anti-PAR-1 antibodies, which should be very helpful tools in studying PAR-1 in rat cells, have not been available up until now. Therefore, we prepared a monoclonal anti-thrombin receptor antibody (Mab COR7-6H9) directed against the peptide sequence GRAVYLNKSRFPPMPPPPFISEDASG in the N-terminus below the thrombin cleavage site of the rat PAR-1-type thrombin receptor. Using this antibody, we demonstrated the presence of PAR-1 binding sites on the plasma membrane of rat glioma C6 cells both with confocal laser fluorescence and with scanning electron microscopy. In addition, Mab COR7-6H9 was shown to block PAR-1-mediated transmembranal signaling as demonstrated by measurement of free intracellular calcium and cyclic AMP. This novel anti-PAR-1 antibody is therefore likely to be a very helpful tool in studying PAR-1-type thrombin receptors in rat brain.
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PMID:Investigation of PAR-1-type thrombin receptors in rat glioma C6 cells with a novel monoclonal anti-PAR-1 antibody (Mab COR7-6H9). 1044 40

Proteinase-activated receptors (PARs), ubiquitous surface molecules participating on many biological processes have been recently discovered. Specific receptors for thrombin (PAR-1 and PAR-3) and trypsin (PAR-2) are described in this review. They belong to a family of G protein-coupled receptors activated by amino acid sequence of N-terminal part of bound ligand revealed by site-specific proteolysis. PARs participate in tissue growth and differentiation, regeneration and reparation, inflammatory response regulation, malignant transformation, but even in vascular tonus and blood pressure regulation. (Fig. 5, Ref. 35.)
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PMID:Thrombin and trypsin receptors: the same mechanism of signalling on cellular surfaces. 1049 1


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