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)

1. The effect of okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A), on human platelets has been investigated. 2. Okadaic acid exerts a general increase in phosphorylation of platelet proteins but did not induce aggregation or secretion of 5-hydroxytryptamine (5-HT). Okadaic acid, however, did inhibit thrombin-induced functional responses. 3. Maximally effective concentrations of prostacyclin, to elevate adenosine 3'-5'-cyclic monophosphate (cyclic AMP), or phorbol dibutyrate, to activate protein kinase C, inhibited the formation of inositol phosphates by thrombin by approximately 60%. When used in combination, prostacyclin and phorbol dibutyrate reduced the levels of inositol phosphates induced by thrombin to 11%. 4. Okadaic acid (1 microM) decreased thrombin-induced formation of inositol phosphates by approximately 55% and increased the inhibitory action of prostacyclin or phorbol dibutyrate. Okadaic acid had no further effect when prostacyclin and phorbol dibutyrate were used in combination. 5. These results suggest that protein kinases A and C act to inhibit phospholipase C by distinct mechanisms and that their action is reversed by PP1 and/or PP2A.
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PMID:Okadaic acid inhibits activation of phospholipase C in human platelets by mimicking the actions of protein kinases A and C. 162 49

Platelet signal transduction involves not only reversible phosphorylation of proteins on both tyrosine and serine/threonine residues, but also mechanisms of cross-talk to coordinate different pathways. We have, therefore, investigated the effect of okadaic acid, a potent inhibitor of serine/threonine protein phosphatases type 1 and type 2A (PP1 and PP2A), to better understand the interplay that must exist between serine/threonine and tyrosine phosphorylations during platelet activation. Okadaic acid drastically inhibits thrombin-induced platelet aggregation, secretion, and thromboxane synthesis. The inhibition is accompanied by a marked increase in the phosphorylation of at least 5 proteins (230, 210, 74, 57, and 50 to 52 kDa). However, protein kinase C activity is not modified because thrombin-and phorbol-12-myristate-13-acetate-induced phosphorylation of pleckstrin is still occurring, although slightly decreased. Inhibition of platelet function and extent of the phosphorylation of the 5 substrates in the presence of okadaic acid are concentration and time dependent, suggesting a relation between the accumulation of one or more phosphoproteins and the inhibitory effect of okadaic acid. Okadaic acid inhibits thrombin-induced tyrosine phosphorylation in a concentration-dependent manner. According to Brautigan and Pinault, the inhibition of protein phosphatases in kidney cells resulted in the activation of a 55-kDa-tyrosine phosphatase and the tyrosine phosphatase activity was synergistically increased when okadaic acid acted in concert with prostaglandin I2 (PGI2). Interestingly, in agreement with these results, the okadaic acid-induced phosphorylation of the 50-kDa substrate, which occurs without a cyclic adenosine monophosphate increase in platelets, has the same molecular weight as the platelet membrane tyrosine phosphatase isolated by Dawicki and Steiner. Furthermore, we also found that thrombin-induced tyrosine phosphorylation was markedly inhibited in the presence of low concentrations of both okadaic acid and PGI2, therefore explaining the synergistic inhibition of platelet aggregation and secretion. The results greatly support the notion of a cross-talk between stimulation of serine/threonine kinases (in response to inhibition of serine/threonine PP) and inhibition of tyrosine phosphorylations and emphasize the role of the 50-kDa substrate in regulating platelet activation.
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PMID:Serine/threonine dephosphorylation may be involved in tyrosine phosphorylation: a new mode of signal transduction in platelets. 894 16

The association of protein Ser/Thr phosphatase type 1(PP1) and type 2A (PP2A) with the cytoskeleton (Triton X-100 insoluble residue) during human platelet activation was investigated. In unstimulated platelets, 40% of total PP1-like activity was present in the Triton-insoluble cytoskeleton, while only 10% of the total PP2A-like activity was present in this fraction. Stimulation with 1 U/ml thrombin produced a 1.8-fold increase in PP1-like activity and a 7-fold increase in PP2A-like activity, respectively, in the cytoskeletal fraction, under aggregating conditions. Immunoblot analysis revealed that thrombin treatment increased association of PP1 catalytic subunit isozymes (PP1 alpha, PP1 gamma, PP1 delta) and PP2A catalytic subunit with the cytoskeleton, with concomitant decrease of these enzymes in Triton-soluble fractions. The amounts of cytoskeleton-associated PP1 and PP2A depended on the dose of thrombin which could activate platelets. Agonist-induced redistribution of PP1 and PP2A into the cytoskeleton was inhibited by OP-41483 (a prostaglandin I2 analog). Interaction of PP2A with cytoskeletal proteins strongly correlates with aggregation, whereas the association of PP1 with cytoskeleton can be detected upon platelet activation, even in the absence of aggregation. Co-extraction of protein kinase C and myosin light chain kinase with the cytoskeleton eventually translocated to the cytoskeleton, but only during aggregation. These results suggest that differential translocation of PP1 and PP2A to the cytoskeleton is involved in platelet activation, and their association with cytoskeletal proteins may regulate phosphorylation levels together with protein kinases in platelets.
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PMID:Differential association of protein Ser/Thr phosphatase types 1 and 2A with the cytoskeleton upon platelet activation. 897 31

Stimulation of platelets by collagen leads to activation of a tyrosine kinase cascade resulting in secretion and aggregation. We have recently shown that this pathway involves rapid tyrosine phosphorylation of an Fc receptor gamma chain, which contains an immunoreceptor tyrosine-based activation motif (ITAM), enabling interaction with the tandem SH2 domains of the tyrosine kinase Syk. Activation of Syk lies upstream of tyrosine phosphorylation of phospholipase Cgamma2. In the present study we sought to test directly the role of the ITAM/Syk interaction and the role of the Src-related kinases in collagen receptor signaling using mouse megakaryocytes. We demonstrate that the calcium-mobilizing action of a collagen-related peptide (CRP) is kinase-dependent, inhibited by the microinjection of the tandem SH2 domains of Syk and abolished in Syk-deficient mice. Furthermore, the CRP response is abolished by the Src family kinase inhibitor PP1 and inhibited in Fyn-deficient mice. In contrast, the calcium response to the G-protein-linked receptor agonist thrombin is not significantly altered under these conditions. These results provide direct evidence of the functional importance of Fyn and Syk in collagen receptor signaling and support the megakaryocyte as a model for the study of proteins involved in this pathway.
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PMID:Syk and Fyn are required by mouse megakaryocytes for the rise in intracellular calcium induced by a collagen-related peptide. 934 87

The binding of collagen to platelet glycoprotein VI (GPVI) leads to the subsequent activation of phospholipase Cgamma2 through a pathway that is dependent on the Fc receptor gamma (FcR gamma) chain and the tyrosine kinase p72syk. We have investigated the role of platelet Src-family kinases in this signalling pathway. The selective Src-family kinase inhibitor PP1 prevented collagen-stimulated increases in whole-cell tyrosine phosphorylation and tyrosine phosphorylation of the FcR gamma chain and p72syk. A similar set of observations was made for a collagen-related peptide (CRP), which binds to GPVI but not to the integrin alpha2beta1 (GPIa/IIa). These effects were seen at a concentration of PP1 that inhibited platelet aggregation, dense granule release and Ca2+ mobilization induced by CRP, but not aggregation and Ca2+ mobilization mediated by the G-protein-coupled receptor agonist thrombin. After stimulation by CRP or collagen, the Src-family kinases p59fyn and p53/56lyn became associated with several tyrosine-phosphorylated proteins including the FcR gamma chain. This was not true of the other platelet Src-family kinases. The association between the FcR gamma chain and p59fyn was also seen under basal conditions, and was stable only in the weak detergent Brij96 but not in Nonidet P40, suggesting a non-SH2-dependent interaction. These results provide strong evidence for the involvement of p59fyn and p53/56lyn in signalling via GPVI, with p59fyn possibly acting upstream of FcR gamma chain phosphorylation.
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PMID:Evidence for the involvement of p59fyn and p53/56lyn in collagen receptor signalling in human platelets. 993 17

Collagen-related peptide (CRP), a collagen homologue, induces platelet activation through a tyrosine kinase-dependent pathway, leading to sequential tyrosine phosphorylation of Fc receptor (FcR) gamma-chain, Syk, and phospholipase C-gamma2. Here we report that CRP and the platelet low affinity immune receptor FcgammaRIIA stimulate tyrosine phosphorylation of the T cell adapter SLP-76, whereas the G protein-coupled receptor agonist thrombin induces only minor tyrosine phosphorylation. This suggests that SLP-76 has a specific role downstream of receptors that signal via an immunoreceptor tyrosine-based activation motif. Immunoprecipitation studies demonstrate association of SLP-76 with SLAP-130, Vav, Fyn, Lyn, and the FcR gamma-chain in CRP-stimulated platelets. Several of these proteins, including SLP-76, undergo tyrosine phosphorylation in in vitro kinase assays performed on SLP-76 immunoprecipitates. Tyrosine phosphorylation of all of these proteins in the in vitro kinase assay was abrogated by the Src family kinase inhibitor PP1, suggesting that it is mediated by either Fyn or Lyn. The physiological significance of this is uncertain, however, since tyrosine phosphorylation of SLP-76 in vivo is not altered in either Fyn- or Lyn-deficient platelets. CRP stimulation of Syk-deficient platelets demonstrated that in vivo tyrosine phosphorylation of SLP-76 is downstream of Syk. The absence of Syk in the SLP-76 immunoprecipitates raises the possibility that another protein is responsible for bringing SLP-76 to Syk. Candidates for this include those proteins that co-immunoprecipitate with SLP-76, including the FcR gamma-chain. Tyrosine phosphorylation of PLC-gamma2 and Ca2+ mobilization is markedly attenuated in SLP-76-deficient platelets following CRP stimulation, suggesting that the adapter plays a critical role in the regulation of the phospholipase. The increase in tyrosine phosphorylation of SLAP-130 in response to CRP is also inhibited in SLP-76-deficient platelets, placing it downstream of SLP-76. This work identifies SLP-76 as an important adapter molecule that is regulated by Syk and lies upstream of SLAP-130 and PLC-gamma2 in CRP-stimulated platelets.
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PMID:Tyrosine phosphorylation of SLP-76 is downstream of Syk following stimulation of the collagen receptor in platelets. 1002 22

G protein-coupled receptors (GPCRs) initiate Ras-dependent activation of the Erk 1/2 mitogen-activated protein kinase cascade by stimulating recruitment of Ras guanine nucleotide exchange factors to the plasma membrane. Both integrin-based focal adhesion complexes and receptor tyrosine kinases have been proposed as scaffolds upon which the GPCR-induced Ras activation complex may assemble. Using specific inhibitors of focal adhesion complex assembly and receptor tyrosine kinase activation, we have determined the relative contribution of each to activation of the Erk 1/2 cascade following stimulation of endogenous GPCRs in three different cell types. The tetrapeptide RGDS, which inhibits integrin dimerization, and cytochalasin D, which depolymerizes the actin cytoskeleton, disrupt the assembly of focal adhesions. In PC12 rat pheochromocytoma cells, both agents block lysophosphatidic acid (LPA)- and bradykinin-stimulated Erk 1/2 phosphorylation, suggesting that intact focal adhesion complexes are required for GPCR-induced mitogen-activated protein kinase activation in these cells. In Rat 1 fibroblasts, Erk 1/2 activation via LPA and thrombin receptors is completely insensitive to both agents. Conversely, the epidermal growth factor receptor-specific tyrphostin AG1478 inhibits GPCR-mediated Erk 1/2 activation in Rat 1 cells but has no effect in PC12 cells. In HEK-293 human embryonic kidney cells, LPA and thrombin receptor-mediated Erk 1/2 activation is partially sensitive to both the RGDS peptide and tyrphostin AG1478, suggesting that both focal adhesion and receptor tyrosine kinase scaffolds are employed in these cells. The dependence of GPCR-mediated Erk 1/2 activation on intact focal adhesions correlates with expression of the calcium-regulated focal adhesion kinase, Pyk2. In all three cell types, GPCR-stimulated Erk 1/2 activation is significantly inhibited by the Src kinase inhibitors, herbimycin A and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-D-3,4-pyrimidine (PP1), suggesting that Src family nonreceptor tyrosine kinases represent a point of convergence for signals originating from either scaffold.
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PMID:Pleiotropic coupling of G protein-coupled receptors to the mitogen-activated protein kinase cascade. Role of focal adhesions and receptor tyrosine kinases. 1031 9

SH2 domain containing phosphatase-2 (SHP-2) has an important regulatory role in a variety of cell types. However, little is known concerning its function in platelets. We show here that, in thrombin-stimulated human platelets, SHP-2 undergoes a time-dependent association with platelet endothelial cell adhesion molecule-1 (PECAM-1) and four low molecular weight phosphoproteins which are attenuated by the Src kinase inhibitor PP1. The low molecular weight proteins, which may be transmembrane proteins, are shown to bind exclusively to the N-terminal SH2 domain of SHP-2 and are therefore possible activators of the phosphatase. In addition, SHP-2 phosphatase activity is shown to be increased following thrombin stimulation or cross-linking of PECAM.
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PMID:Thrombin-induced association of SHP-2 with multiple tyrosine-phosphorylated proteins in human platelets. 1050 11

Interstitial fibrin deposition is a common histologic feature of tubulointerstitial diseases, which suggests that the coagulation system is activated. Thrombin, generated during the activation of the coagulation cascade, is a powerful activating factor for different cell types. Although proximal tubular cells are potential targets for this coagulation factor, no information is available on the effect of thrombin on these cells. Thus, the expression of protease-activated receptor-1 (PAR-1), the main thrombin receptor, was investigated in human proximal tubular cells (hPTC) in vivo and in vitro. A diffuse expression of PAR-1 was observed by immunohistochemistry along the basolateral membrane of PTC in normal human kidney. This observation was confirmed in vitro in cultured hPTC. Because tubular damage and monocyte infiltration are two hallmarks of tubulointerstitial injury, the effect of thrombin on DNA synthesis and monocyte chemotactic peptide-1 (MCP-1) gene and protein expression was evaluated in cultured hPTC. Thrombin induced a significant and dose-dependent increase in thymidine uptake and a striking upregulation of MCP-1 mRNA expression and protein release into the supernatant. Although PAR-1 is a G protein-coupled receptor, its activation in hPTC, as in other cell systems, resulted in a transient increase in cellular levels of tyrosine-phosphorylated proteins. An increased level of tyrosine-phosphorylated c-src suggested the activation of this cytoplasmic tyrosine kinase in response to thrombin and its potential role in thrombin-induced protein-tyrosine phosphorylation. Interestingly, thrombin-induced DNA synthesis and MCP-1 gene expression were completely blocked by genistein, a specific tyrosine kinase inhibitor, but not by its inactive analogue daidzein, demonstrating a central role for tyrosine kinase activation in the thrombin effects on hPTC. Moreover, the specific src inhibitor PP1 abolished the thrombin effect on DNA synthesis. In conclusion, thrombin might represent a powerful regenerative and proinflammatory stimulus for hPTC in acute and chronic tubulointerstitial diseases.
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PMID:Regenerative and proinflammatory effects of thrombin on human proximal tubular cells. 1082 Jan 65

The Src homology (SH)2 domain-containing protein-tyrosine phosphatase SHP-1 is tyrosine phosphorylated in platelets in response to the glycoprotein VI (GPVI)-selective agonist collagen-related peptide (CRP), collagen, and thrombin. Two major unidentified tyrosine-phosphorylated bands of 28 and 32 kDa and a minor band of 130 kDa coprecipitate with SHP-1 in response to all three agonists. Additionally, tyrosine-phosphorylated proteins of 50-55 and 70 kDa specifically associate with SHP-1 following stimulation by CRP and collagen. The tyrosine kinases Lyn, which exists as a 53 and 56-kDa doublet, and Syk were identified as major components of these bands, respectively. Kinase assays on SHP-1 immunoprecipitates performed in the presence of the Src family kinase inhibitor PP1 confirmed the presence of a Src kinase in CRP- but not thrombin-stimulated cells. Lyn, Syk, and SLP-76, along with tyrosine-phosphorylated 28-, 32-, and 130-kDa proteins, bound selectively to a glutathione S-transferase protein encoding the SH2 domains of SHP-1, suggesting that this is the major site of interaction. Platelets isolated from motheaten viable mice (mev/mev) revealed the presence of a heavily tyrosine-phosphorylated 26-kDa protein that was not found in wild-type platelets. CRP-stimulated mev/mev platelets manifested hypophosphorylation of Syk and Lyn and reduced P-selectin expression relative to controls. These observations provide evidence of a functional role for SHP-1 in platelet activation by GPVI.
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PMID:Evidence of a role for SHP-1 in platelet activation by the collagen receptor glycoprotein VI. 1087 5


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