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 protein tyrosine phosphatase (PTPase) inhibitor pervanadate (vanadyl hydroperoxide) stimulated protein tyrosine phosphorylation 29-fold more than did thrombin in intact and saponin-permeabilized platelets. Increased tyrosine phosphorylation preceded, or was coincident with, a fall in PtdIns(4,5)P2 levels, production of PtdIns(3,4)P2 and phosphatidic acid, mobilization of intracellular Ca2+, stimulation of protein kinase C-dependent protein phosphorylation, secretion of dense and alpha-granules, increased actin polymerization, shape change and aggregation which required fibrinogen and was mediated by increased surface expression of GPIIb-IIIa. The tyrosine kinase inhibitor RG 50864 totally prevented induction of tyrosine phosphorylation by pervanadate, as well as all other responses measured; in contrast, the inactive structural analogue, tyrphostin #1, had no effect. Dense-granule secretion induced by pervanadate required protein kinase C activity; however, aggregation and alpha-granule secretion were independent of protein kinase C. In saponin-permeabilized platelets pervanadate and thrombin stimulated phospholipase C activity by GTP-independent and GTP-dependent mechanisms respectively. We conclude that PTPases are important regulators of signal transduction in platelets.
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PMID:Activation of signal transduction in platelets by the tyrosine phosphatase inhibitor pervanadate (vanadyl hydroperoxide). 153 May 76

Several systems have been developed to allow for rapid and efficient purification of recombinant proteins expressed in bacteria. The expression of polypeptides in frame with glutathione S-transferase (GST) allows for purification of the fusion proteins from crude bacterial extracts under nondenaturing conditions by affinity chromatography on glutathione agarose (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). This vector expression system has also incorporated specific protease cleavage sites to facilitate proteolysis of the bacterial fusion proteins. In our hands, the cleavage of these fusion proteins at a thrombin cleavage site proceeded slowly. To facilitate the cleavage of fusion proteins, we have introduced a glycine-rich linker (glycine kinker) containing the sequence P.G.I.S.G.G.G.G.G located immediately following the thrombin cleavage site. This glycine kinker greatly increases the thrombin cleavage efficiency of several fusion proteins. The introduction of the glycine kinker into fusion proteins allows for the cleavage of the fusion proteins while they are attached to the affinity resin resulting in a single step purification of the recombinant protein. More than 2 mg of the highly purified protein was obtained from the equivalent of 100 ml of bacterial culture within a few hours when a protein tyrosine phosphatase was employed as a test protein. The vector, pGEX-KG, has also been modified to facilitate cloning of a variety of cDNAs in all reading frames and has been successfully used to express several eukaryotic proteins.
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PMID:Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. 185 37

A significant protein tyrosine phosphatase (PTP) activity was found to be associated with the cytoskeleton of thrombin-stimulated platelets. Translocation of the enzyme became maximal within 1-2 min of thrombin stimulation and was suppressed by cytochalasin D or upon inhibition of aggregation. Immunoblotting as well as immunoprecipitation revealed that a PTP with two SH2 domains (SH-PTP1) displayed the same behaviour, translocation to the cytoskeleton showing the same time course as that observed for pp60c-src. We conclude that SH-PTP1 might represent a critical enzyme in the complex interplay between the various proteins regulating protein tyrosine phosphorylation in the cytoskeletal matrix.
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PMID:Translocation of an SH2-containing protein tyrosine phosphatase (SH-PTP1) to the cytoskeleton of thrombin-activated platelets. 751 33

Thrombin stimulation of platelets induces a transient increase in the specific activity of pp60c-src followed by a redistribution of pp60c-src to the Triton X-100-insoluble, cytoskeleton-rich fraction. Concomitant with the observed increase in pp60c-src activity was a rapid dephosphorylation of tyrosine 527 in 10 to 15% of pp60c-src molecules. In addition, we found that pp60c-src from the Triton-insoluble fraction was phosphorylated on tyrosine 416, the autophosphorylation site which is phosphorylated in activated oncogenic variants of pp60src. Furthermore, in platelets from patients with Glanzmann's thrombasthenia (which are deficient in the integrin receptor GPIIb-IIIa), pp60c-src was not translocated to the Triton-insoluble fraction, and there was a sustained increase in pp60c-src activity following thrombin treatment. These results suggest that pp60c-src is rapidly activated in thrombin-stimulated platelets, potentially by a protein tyrosine phosphatase, before it translocates to a cytoskeletal fraction, where many of its potential substrates are found. The evidence that the cytoskeletal association of pp60c-src is dependent upon engagement of the integrin receptor GPIIb-IIIa suggests that integrin-cytoskeletal complexes may serve to compartmentalize and anchor activated enzymes involved in signal transduction.
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PMID:Redistribution of activated pp60c-src to integrin-dependent cytoskeletal complexes in thrombin-stimulated platelets. 768 Jan

SH-PTP1 is a protein tyrosine phosphatase (PTP) predominantly expressed in haematopoietic cells and containing two src homology-2 (SH2) domains. Here we report that SH-PTP1 is phosphorylated on both serine and tyrosine residues in response to thrombin or phorbol myristate acetate (PMA), which increased by 60 and 40%, respectively, SH-PTP1 activity. Thrombin-induced phosphorylation of SH-PTP1 is an early signalling event (maximal within 10 s) involving neither integrin signalling, nor calcium, nor release of ADP or thromboxane A2. Moreover, in contrast with PMA, the effect of thrombin on the tyrosine phosphorylation of SH-PTP1 was hardly affected by GF109203X, a specific protein kinase C (PKC) inhibitor. Finally, phosphorylation of SH-PTP1 could be provoked in permeabilized platelets by thrombin or GTP gamma S. This was abolished by pertussis toxin, the specificity of this effect being verified with the megakaryocytic cell line Dami cell. Our data thus identify SH-PTP1 as an in vivo substrate of a putative protein tyrosine kinase linked to the thrombin receptor by a Gi protein. This might offer some clue to unravel the mechanism of thrombin not only in platelets but also in nucleated cells, where its mitogenic effect is known to involve pertussis toxin-sensitive G-proteins, tyrosine phosphorylation and the ras pathway.
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PMID:Tyrosine phosphorylation of an SH2-containing protein tyrosine phosphatase is coupled to platelet thrombin receptor via a pertussis toxin-sensitive heterotrimeric G-protein. 778 4

In this report we have examined the effects of the protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO) on receptor-mediated platelet shape change, secretion and aggregation. PAO was found to inhibit platelet aggregation induced by collagen, thrombin, ADP and epinephrine at IC50 values of 0.35 mumol/l, 2.5 mumol/l, 0.2 mumol/l and 0.3 mumol/l, respectively. Agonist-induced secretion of ATP was inhibited at similar or lower concentrations of PAO. The specificity of the interaction of PAO with platelet proteins was demonstrated by the ability of the disulfhydryl compound 2,3-dimercaptopropanol, which abstracts PAO from proteins to form a stable cyclic adduct, to reverse PAO inhibition of both agonist-induced platelet secretion and aggregation. Dimercaptopropanesulphonic acid, a membrane-impermeable analogue of dimercaptopropanol, did not reverse inhibition of collagen-induced shape change or aggregation by PAO, thereby demonstrating that PAO acted intracellularly. PAO inhibited collagen-induced shape change and internal Ca2+ mobilization but had no effect on these two phenomena when induced by thrombin or ADP. PAO was also unable to prevent arachidonic acid-induced shape change, indicating that PAO acts at a site prior to the phospholipase A2-mediated release of arachidonic acid to inhibit collagen-induced shape change. PAO induced the accumulation of a number of phosphotyrosine-containing proteins and inhibited the collagen-induced phosphorylation of a 40 kD protein. The potency and agonist-specific effects of PAO on platelet activation suggest that this inhibitor will be of value in elucidation of signal transduction pathways involved in receptor-mediated platelet function.
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PMID:Platelet shape change and Ca2+ mobilization induced by collagen, but not thrombin or ADP, are inhibited by phenylarsine oxide. 781 6

Activation of human platelets by thrombin or a thrombin receptor-activating peptide (TRAP) resulted in a decrease in tyrosine phosphorylation of two proteins, P38 and P140. Preincubation of platelets with the tyrosine phosphatase inhibitor orthovanadate prevented the tyrosine dephosphorylation of P38 and P140, and reduced platelet aggregation induced by thrombin receptor activation. When platelets were stimulated under conditions that precluded the activation of glycoprotein IIb/IIIa (dissociation of the complex by EGTA at 37 degrees C) or the binding of fibrinogen (preincubation of platelets with RGDS), tyrosine dephosphorylation of P38 and P140 was not observed. The results indicate that protein tyrosine phosphatase stimulation (a) occurs during platelet activation induced by a physiological stimulus, (b) is a positive regulatory signal for platelet aggregation and (c) is dependent on the activation of the integrin alpha IIb beta 3.
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PMID:Integrin-dependent protein dephosphorylation on tyrosine induced by activation of the thrombin receptor in human platelets. 791 85

Yersinia enterocolitica infection in humans causes a broad spectrum of diseases ranging from acute bowel disease to extraintestinal manifestations such as reactive arthritis, erythema nodosum and uveitis. During the last decade a fascinating part of the molecular biology of the pathogenicity of human pathogenic Yersinia species has been unraveled. Pathogenicity factors such as protein tyrosine phosphatase, protein kinase, thrombin- and collagen-binding factors have been identified and characterized on the molecular level. In contrast to many animal models for human enteropathogenic microorganisms, experimental Y. enterocolitica infection in rodents resembles yersiniosis in humans and thus offers extraordinary opportunities to study the sequential steps of the infectious process. Rabbits are suitable animals in which to study Yersinia-induced enteritis (enterotoxin-mediated) and the humoral immune response after oral infection. The role of Peyer's patches (PP) in the entry of enteropathogenic Yersinia species has been elucidated in mice and rabbits. M cells are probably the primary target cells of invading Yersiniae. Surprisingly, after penetration of the mucosal epithelial cell layer Yersinia bacilli were visualized to be exclusively extracellular in PP tissue. Obviously neutrophils within PP were unable to phagocytize the invading microorganisms. Presently, it is not clear how the microorganisms disseminate from PP into lymph nodes, spleen, liver and lung of mice where they form abscesses and granuloma-like lesions. Immunohistologically the involvement of macrophages and T cells could be demonstrated in Yersinia-induced lesions of mice. Direct evidence for the role of T cells and cytokine-activated macrophages in the host defense reaction against a primary Yersinia infection in mice could be obtained from experiments including adoptive transfer of Yersinia-specific T cells and in vivo neutralization of TNF-alpha and IFN-gamma. The experimental rat model turned out to be a suitable model for studying Yersinia-induced aseptic arthritis. Lewis- and SHR rats proved to be arthritis-susceptible. Arthritogenicity of Yersinia for rats appeared to be restricted to Y. enterocolitica of serotype 08 and correlated with the virulence potential of this serotype. Surprisingly, expression of YadA, the collagen-binding factor, was not necessary for arthritis induction. A close association between both susceptibility to arthritis induction and Yersinia infection could be demonstrated in various rat strains. Depletion of alpha/beta T-cell receptor (alpha beta-TCR)-positive T cells by treatment with alpha beta-TCR-specific antibody revealed that T cells were required for clearance of the pathogen.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Experimental Yersinia enterocolitica infection in rodents: a model for human yersiniosis. 836 22

Human platelets possess a specific membrane-bound leukotriene (LT) C4 synthase, which catalyzes the conversion of LTA4 to LTC4. Stimulation of the receptors for thrombin, collagen or thromboxane A2 provoked inhibition of this enzyme, as judged by suppressed transformation of exogenous LTA4 to LTC4. Similarly, direct activation of protein kinase (PK) C with nanomolar concentrations of 4 beta-phorbol 12-myristate 13-acetate (PMA) inhibited the production of LTC4. Kinetic studies demonstrated that the inhibition induced by thrombin and PMA was non-competitive. Elevation of intracellular cAMP levels with carbacyclin did not affect basal LTC4 formation, but abolished the attenuation of platelet LTC4 synthase activity induced by the thromboxane receptor agonist U-46619. The unselective protein kinase inhibitor staurosporine prevented both receptor-mediated and PMA-induced suppression of LTC4 formation. In contrast, two selective PKC inhibitors, Ro 31-8220 and GF 109203X, reversed the inhibitory effect provoked by PMA, but failed to prevent thrombin-induced inhibition. Furthermore, the protein tyrosine phosphatase inhibitor, sodium orthovanadate, induced dose-dependent inhibition of LTC4 production in platelet sonicates. In conclusion, receptor-mediated activation of human platelets leads to decreased LTC4 synthase activity via phosphoregulation. Although the present results demonstrate that platelet LTC4 synthase can be regulated via PKC-dependent events, alternative mechanisms appears to be involved in the physiological regulation of this enzyme. The findings suggest the possible importance of protein tyrosine phosphorylations in this process.
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PMID:Receptor-mediated regulation of leukotriene C4 synthase activity in human platelets. 853 97

We previously cloned a cDNA encoding a protein tyrosine phosphatase (PTP) containing sequence homology to protein 4.1, designated PTPMEG. Recombinant protein and amino- and carboxyl-terminal peptides were used to obtain polyclonal antibodies against PTPMEG to identify endogenous PTPMEG in A172 cells and to show that the enzyme is primarily localized to the membrane and cytoskeletal fractions of these cells. We prepared recombinant protein in Sf9 and COS-7 cells to further characterize it. The protein was phosphorylated in both cell types on serine and threonine residues. The multiple sites of phosphorylation were all within the intermediate domain of the protein between amino acids 386 and 503. This region also contains two PEST sequences and two proline-rich motifs that may confer binding to Src homology 3 domains. The recombinant protein was cleaved by trypsin and calpain in this region and thereby activated 4-8-fold as assayed using Raytide as substrate. We immunoprecipitated the protein from human platelets with both amino- and carboxyl-terminal antipeptide antibodies to assess the state of the enzyme in these cells. The full-length molecule was found in extracts from unstimulated platelets, whereas extracts from both calcium ionophore- and thrombin-treated platelets contained proteolyzed and activated forms of the enzyme, indicating that proteolysis by calpain is evoked in response to thrombin. Prior incubation of platelets with calpeptin, an inhibitor of calpain, blocked the agonist-induced proteolysis.
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PMID:The properties of the protein tyrosine phosphatase PTPMEG. 891 Mar 69


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