Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P35237 (thrombin inhibitor)
 2,012 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thrombin is a multifunctional protease. Recent studies on cultured neuronal cells have suggested a function for thrombin in the development and maintenance of the nervous system. Thrombin has been found to induce neurite retraction and reverse stellation in neuroblastoma cell lines and rat astrocytes, respectively. The major focus of our study was to investigate the potential role of thrombin in peripheral nervous system development using the rat embryonic dorsal root ganglion model. We found a dose dependent inhibition of neurite outgrowth from explant dorsal root ganglion cultures upon exposure to 2 to 200 nM thrombin. This effect was reversed by the specific thrombin inhibitor, hirudin. A synthetic peptide that imitates the fully active receptor, thrombin receptor activating peptide, was also found to inhibit neurite outgrowth from dorsal root ganglia. bis-Benzimide stained neuronal cultures did not show any evidence of cell death after exposure to thrombin or thrombin receptor activating peptides. Immunohistochemical studies revealed specific staining of the thrombin receptor on neurons, with intense labeling along neurites. Enriched neuronal cultures exposed to thrombin and thrombin receptor activating peptides revealed rapid activation of phospholipase Cgamma-1, a second messenger associated with the thrombin receptor. These findings are the first to describe the localization of the thrombin receptor to dorsal root ganglion neurons. We propose that receptor activation is associated with thrombin induced inhibition of neurite outgrowth.
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PMID:Thrombin induced inhibition of neurite outgrowth from dorsal root ganglion neurons. 966 59

Thrombin, a serine protease, is a potent mitogen for vascular smooth muscle cells (SMCs), but its mechanism of action is not known. Since L-ornithine is metabolized to growth-stimulatory polyamines, we examined whether thrombin regulates the transcellular transport and metabolism of L-ornithine by vascular SMCs. Treatment of SMCs with thrombin initially (0 to 2 hours) decreased L-ornithine uptake, whereas longer exposures (6 to 24 hours) progressively increased transport. Kinetic studies indicated that thrombin-induced inhibition was associated with a decrease in affinity for L-ornithine, whereas stimulation was mediated by an increase in transport capacity. Thrombin induced the expression of both cationic amino acid transporter (CAT)-1 and CAT-2 mRNA. Furthermore, thrombin stimulated L-ornithine metabolism by inducing ornithine decarboxylase (ODC) mRNA expression and activity. The stimulatory effect of thrombin on both L-ornithine transport and ODC activity was reversed by hirudin, a thrombin inhibitor, and was mimicked by a 14-amino acid thrombin receptor-activating peptide. Thrombin also markedly increased the capacity of SMCs to generate putrescine, a polyamine, from extracellular L-ornithine. The thrombin-mediated increase in putrescine production was reversed by N(G)-methyl-L-arginine, a competitive inhibitor of cationic amino acid transport, or by alpha-difluoromethylornithine (DFMO), an ODC inhibitor. DFMO also inhibited thrombin-induced SMC proliferation. These results demonstrate that thrombin stimulates polyamine synthesis by inducing CAT and ODC gene expression and that thrombin-stimulated SMC proliferation is dependent on polyamine formation. The ability of thrombin to upregulate L-ornithine transport and direct its metabolism to growth-stimulatory polyamines may contribute to postangioplasty restenosis and atherosclerotic lesion formation.
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PMID:Thrombin stimulates vascular smooth muscle cell polyamine synthesis by inducing cationic amino acid transporter and ornithine decarboxylase gene expression. 968 62

Increasing evidence indicates several roles for thrombin-like serine proteases and their cognate inhibitors (serpins) in normal development and/or pathology of the nervous system. In addition to its prominent role in thrombosis and/or hemostasis, thrombin inhibits neurite outgrowth in neuroblastoma and primary neuronal cells in vitro, prevents stellation of glial cells, and induces cell death in glial and neuronal cell cultures. Thrombin is known to act via a cell surface protease-activated receptor (PAR-1), and recent evidence suggests that rodent neurons express PAR-1. Previously, we have shown that the thrombin inhibitor, protease nexin-1, significantly prevents neuronal cell death both in vitro and in vivo. Here we have examined the effects of human alpha-thrombin and the presence and/or activation of PAR-1 on the survival and differentiation of highly enriched cultures of embryonic chick spinal motoneurons. We show that thrombin significantly decreased the mean neurite length, prevented neurite branching, and induced motoneuron death by an apoptosis-like mechanism in a dose-dependent manner. These effects were prevented by cotreatment with hirudin, a specific thrombin inhibitor. Treatment of the cultures with a synthetic thrombin receptor-activating peptide (SFLLRNP) mimicked the deleterious effects of thrombin on motoneurons. Furthermore, cotreatment of the cultures with inhibitors of caspase activities completely prevented the death of motoneurons induced by either thrombin or SFLLRNP. These findings indicate that (1) embryonic avian spinal motoneurons express functional PAR-1 and (2) activation of this receptor induces neuronal cell degeneration and death via stimulation of caspases. Together with previous reports, our results suggest that thrombin, its receptor(s), and endogenous thrombin inhibitors may be important regulators of neuronal cell fate during development, after injury, and in pathology of the nervous system.
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PMID:Thrombin perturbs neurite outgrowth and induces apoptotic cell death in enriched chick spinal motoneuron cultures through caspase activation. 971 58

HDs racI Thrombin causes profound metabolic and morphological changes in cultured neural cells via activation of the thrombin receptor, also called protease-activated receptor 1 (PAR1). PAR1 mRNA is present in the rat brain, but the role of this receptor in the nervous system remains elusive. The expression of PAR1 and the potent thrombin inhibitor protease nexin-1 (PN-1) was investigated in the developing rat brain and spinal cord and after peripheral nerve lesion. As seen by in situ hybridization, the PAR1 mRNA signal in the late embryonic and early postnatal nervous system was widespread, but generally of low intensity whereas in the adult it was more pronounced and confined to particular neuronal cells. These include the mesencephalic dopaminergic neurons, several thalamic and brainstem nuclei, the mitral cells in the olfactory bulb and the Purkinje cells in the cerebellum. In the spinal cord, PAR1 mRNA was abundant in motoneurons and a particularly high expression was detected in the preganglionic neurons of the autonomic nervous system. High PAR1 mRNA expression was also found in the dorsal root ganglia. Interestingly, strong immunoreactivity for the protease inhibitor PN-1 was present in spinal motoneuron cell bodies, although its transcript was undetectable there. In response to sciatic nerve transection, the signal intensity of PAR1 mRNA as seen by Northern analysis increased in the proximal and the distal part of the lesioned nerve and in the denervated muscle, whereas the PN-1 mRNA signal strongly increased only in the distal part of the nerve but remained unchanged in the proximal part and in the muscle. After facial nerve transection, PAR1 mRNA expression substantially decreased in facial motoneurons. No PAR1 transcript was detected in reactive astrocytes. Similar to PAR1, PN-1 mRNA which was expressed in interneurons within the facial nucleus was also decreased following facial nerve transection.
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PMID:Changes in the expression of protease-activated receptor 1 and protease nexin-1 mRNA during rat nervous system development and after nerve lesion. 975 Nov 32

Activity-dependent selective reduction of synaptic efficacy is expressed in an in vitro system involving mouse spinal cord and muscle cells. Thrombin or electrical stimulation of the innervating axons induces a decrease in neuromuscular synapse strength, and a specific thrombin inhibitor, hirudin, blocks the electrically evoked down-regulation of synapse effectiveness. We further demonstrate that a thrombin receptor-activating peptide (TRAP), SFLLRNPNDKYEPF, produces a decrement of synapse strength. Both TRAP and electrically evoked synapse decrement are prevented by the specific protein kinase C blocker calphostin C, and the TRAP-evoked synapse decrement is unaffected by a specific protein kinase A blocker, H-89. Thus, we propose that muscle activity, thrombin release, and thrombin receptor and PKC activation are initial steps in the process of the activity-dependent synapse reduction expressed in our system.
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PMID:The thrombin receptor mediates functional activity-dependent neuromuscular synapse reduction via protein kinase C activation in vitro. 1002 79

1. Several thrombin cellular effects are dependent upon stimulation of proteinase activated receptor-1 (PAR-1) localized over the cellular surface. Following activation by thrombin, a new N-terminus peptide is unmasked on PAR-1 receptor, which functions as a tethered ligand for the receptor itself. Synthetic peptides called thrombin receptor activating peptides (TRAPs), corresponding to the N-terminus residue unmasked, reproduce several thrombin cellular effects, but are devoid of catalytic activity. We have evaluated the bronchial response to intravenous administration of human alpha-thrombin or a thrombin receptor activating peptide (TRAP-9) in anaesthetized, artificially ventilated guinea-pigs. 2. Intravenous injection of thrombin (100 microkg(-1)) caused bronchoconstriction that was recapitulated by injection of TRAP-9 (1 mg kg(-1)). Animal pretreatment with the thrombin inhibitor Hirulog (10 mg kg(-1) i.v.) prevented thrombin-induced bronchoconstriction, but did not affect bronchoconstriction induced by TRAP-9. Both agents did not induce bronchoconstriction when injected intravenously to rats. 3. The bronchoconstrictor effect of thrombin and TRAP-9 was subjected to tolerance; however, in animals desensitized to thrombin effect, TRAP-9 was still capable of inducing bronchoconstriction, but not vice versa. 4. Depleting animals of circulating platelets prevented bronchoconstriction induced by both thrombin and TRAP-9. 5. Bronchoconstriction was paralleled by a biphasic change in arterial blood pressure, characterized by a hypotensive phase followed by a hypertensive phase. Thrombin-induced hypotension was not subject to tolerance and was inhibited by Hirulog; conversely, hypertension was subject to tolerance and was not inhibited by Hirulog. Hypotension and hypertension induced by TRAP-9 were neither subject to tolerance nor inhibited by Hirulog. 6. Our results indicate that thrombin causes bronchoconstriction in guinea-pigs through a mechanism that requires proteolytic activation of its receptor and the exposure of the tethered ligand peptide. Platelet activation might be triggered by the thrombin effect.
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PMID:Bronchoconstrictor effect of thrombin and thrombin receptor activating peptide in guinea-pigs in vivo. 1007 41

Thrombin, a serine protease, plays a central role in the initiation of thrombotic events. We report the design, synthesis, and antithrombotic efficacy of XU817 (7), a nonpeptide 5-(amidino) indole thrombin inhibitor. Utilizing the co-crystal structure of XU817 bound in the active site of thrombin we were able to synthesize analogs with enhanced thrombin affinity.
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PMID:Design and synthesis of potent and selective 5,6-fused heterocyclic thrombin inhibitors. 1023 Jun 12

The mechanism of airway remodeling in asthmatic patients is poorly understood. Thrombin is a multifunctional protease that, in addition to its critical role in thrombotic processes, has also been described as inducing cellular and molecular events relevant to tissue remodeling. The present investigation was undertaken to evaluate the activity of thrombin in the sputum of asthmatic patients and its potential role in airway remodeling. The study population comprised 8 healthy subjects and 14 stable patients with bronchial asthma. The concentrations of thrombin, thrombin-antithrombin complex (TAT), and tissue factor were measured in the sputum of all subjects. The concentrations of thrombin (p = 0. 007), TAT (p = 0.01), and tissue factor (p = 0.02) in sputum were significantly higher in asthmatic patients than in healthy controls. The proliferative effects that sputum from asthmatic patients (p = 0. 01) and thrombin (p = 0.03) have on cultured human smooth muscle cells was inhibited significantly in the presence of recombinant hirudin, a specific thrombin inhibitor. Significant statistical correlation was observed between the degree of bronchial responsiveness and the sputum concentrations of thrombin (r = -0.8; p = 0.02) and TAT (r = -0.9; p = 0.01). The results of this study showed that increased thrombin generation occurs in the airway of patients with asthma and that it may play a role in the pathogenesis of airway remodeling. Further studies should be carried out to assess whether these findings are also observed in other airway diseases.
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PMID:Thrombin in the airways of asthmatic patients. 1038 63

Glomerular accumulation of extracellular matrix (ECM) is the common pathologic feature following glomerular injury, and the alteration in the synthesis and degradation of ECM may be involved in the glomerular accumulation of ECM. Glomerular fibrin formation occurs in various forms of human and experimental glomerulonephritis, and it may play an important role in progressive glomerular injury. Thrombin, a multifunctional serine proteinase that is generated at the site of vascular injury, has central functions in hemostasis and it also shows various biologic effects. In this study, it is hypothesized that thrombin may alter the production and the degradation of type IV collagen, which is an important component of ECM in the glomeruli. Human mesangial cells (HMC) were cultured, and the levels of type IV collagen, tissue inhibitor of metalloproteinase-1 (TIMP-1), and matrix metalloproteinase-2 (MMP-2) in the culture supernatants were measured by enzyme immunoassay using specific antibodies. MMP-2 activity was also evaluated by zymography using polyacrylamide/ sodium dodecyl sulfate gel-containing gelatin. Thrombin increased the production of type IV collagen and TIMP-1 in a dose-and time-dependent manner, but it did not increase MMP-2. Thrombin also stimulated the gene expressions of the type IV collagen and TIMP-1 in HMC in a dose- and time-dependent manner. Thrombin treated with diisopropylfluorophosphate, a serine proteinase inhibitor, did not show any of these effects. Hirudin, a natural thrombin inhibitor, and anti-transforming growth factor-beta-neutralizing antibody inhibited the stimulating effect of thrombin. These findings suggest that thrombin may contribute to the excessive accumulation of ECM and progression of glomerulosclerosis through an increase of type IV collagen production and a decreased matrix degradation presumably via a transforming growth factor-beta-dependent mechanism.
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PMID:Thrombin stimulates synthesis of type IV collagen and tissue inhibitor of metalloproteinases-1 by cultured human mesangial cells. 1040 7

The N-terminal 16K fragments of rat and human PRLs possess angiostatic activity. 16K PRL has also been detected in vivo in both humans and rats. Based on an in vitro study, cathepsin D, an acid protease, has been implicated in the generation of rat 16K PRL. However, the proteolytic cleavage of human PRL has not been demonstrated. Our objective was to identify an enzyme that is capable of forming an angiostatic human 16K PRL. To confirm the angiostatic action of rat 16K PRL, the fragment was generated by incubating 23K PRL with rat mammary microsomal fraction at pH 3.2. Upon incubation with human umbilical vein endothelial cells (HUVEC), rat 16K PRL, but not 23K PRL, inhibited basal- and basic fibroblast growth factor-stimulated cell proliferation. Intact rat and human PRLs were then incubated with cathepsin D or acidified microsomal pellets of MCF-7 human breast cancer cells. Analysis by SDS-PAGE showed cleavage of rat, but not human, PRL. Next, hormones were incubated with thrombin at pH 7.4. As shown by SDS-PAGE, digestion of both human and rat PRL by thrombin resulted in the formation of 16K fragments. PRL contained within human amniotic fluid was also cleaved by thrombin. Enzyme specificity was supported by prevention of cleavage by the thrombin inhibitor hirudin. When tested with HUVEC, the human 16K PRL was devoid of angiostatic activity. The activity of this fragment in the Nb2 lymphoma bioassay was 10- to 15-fold lower than that of 23K PRL. Mass spectrometry revealed that the fragment has a mass of 16,878.30+/-15.8 Daltons. Subsequent N-terminal sequencing showed that the thrombin cleavage occurred between amino acid residues 53 (Lys) and 54 (Ala), resulting in the formation of a C-terminal, not an N-terminal, 16K fragment. We conclude that, unlike rat PRL, human PRL is resistant to cleavage by cathepsin D. Thrombin at a physiological pH can generate a C-terminal 16K fragment of human PRL that is not angiostatic and retains little mitogenic activity. We suggest that the precise nature of endogenous 16K PRL fragments that are present in human tissues and body fluids should be carefully examined.
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PMID:Proteolysis of human prolactin: resistance to cathepsin D and formation of a nonangiostatic, C-terminal 16K fragment by thrombin. 1046 85


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