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)

In human platelets stimulated by thrombin and collagen, diacylglycerol is rapidly produced from phosphatidylinositol. Concurrently, an endogenous protein having a molecular weight of about 40,000 (40K protein) is phosphorylated, and serotonin is released. These reactions are all inhibited by a prior treatment of platelets with prostaglandin E1, dibutyryl cyclic AMP, sodium nitroprusside, or with 8-bromo-cyclic GMP, which are known as potent inhibitors for platelet activation. Ca2+-activated phospholipid-dependent protein kinase (protein kinase C) preferentially phosphorylates 40K protein. As judged by fingerprint analysis, the sites in 40K protein that are phosphorylated during the platelet activation appear to be identical with those phosphorylated by protein kinase C in a purified cell-free system. 12-O-Tetradecanoylphorbol-13-acetate, which directly activates protein kinase C by substituting for diacylglycerol, stimulates 40K protein phosphorylation and release reaction without inducing diacylglycerol formation. Tetracaine, which inhibits protein kinase C by competing with phospholipid, blocks 40K protein phosphorylation and serotonin release without inhibiting the receptor-linked diacylglycerol formation. The results indicate that thrombin and collagen activate platelets in almost similar mechanisms and that protein kinase C may lie on a common pathway which leads to the release of serotonin. However, analysis with indomethacin indicates that the role of thromboxane A2 appears to be more predominant for the action of collagen, and it is suggestive that this arachidonate metabolite activates platelets in an analogous mechanism to thrombin.
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PMID:A role of calcium-activated phospholipid-dependent protein kinase in human platelet activation. Comparison of thrombin and collagen actions. 621 69

Ca2+-activated, phospholipid-dependent protein kinase (C-kinase) in platelets is normally activated by diacylglycerol, which is derived from phosphatidylinositol through its receptor-linked breakdown. Under appropriate conditions this enzyme can also be activated by synthetic diacylglycerol which is directly added to intact platelets. C-Kinase thus activated preferentially phosphorylates an endogenous platelet protein having a molecular weight of approximately 40,000. This protein phosphorylation is merely a prerequisite but not a sufficient requirement for the release of serotonin. Evidence is presented suggesting that Ca2+ mobilization and C-kinase activation are synergistically involved in the physiological response of platelets to extracellular messengers, such as thrombin, collagen and platelet-activating factor.
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PMID:Phosphatidylinositol turnover in platelet activation; calcium mobilization and protein phosphorylation. 621 78

In intact human platelets activated by thrombin, diacylglycerol is produced with the concomitant disappearance of phosphatidylinositol (PI). This reaction is associated with phosphorylation of a protein having a molecular weight of about 40,000 (40 K protein) and serotonin release. All the reactions are inhibited in a parallel manner by incubation of platelets with either dibutyryl cyclic AMP or 8-bromocyclic GMP, prior to the stimulation by thrombin. The inhibition of these reactions is inversely related to phosphorylation of another group of platelet proteins. Since Ca2+-activated, phospholipid-dependent protein kinase (C-Kinase) is activated by diacylglycerol and is responsible for 40 K protein phosphorylation (Kawahara, Y., Takai, Y., Minakuchi, R., Sano, K., & Nishizuka, Y. (1980) Biochem. Biophys. Res. Commun. 97, 309-317), the results suggest that in platelets both cyclic AMP and cyclic GMP may serve as inhibitors of C-Kinase by counteracting the receptor-linked PI breakdown probably through the actions of cyclic nucleotide-dependent protein kinases.
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PMID:Counteraction of calcium-activated, phospholipid-dependent protein kinase activation by adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate in platelets. 627 87

Insulin stimulates the growth and proliferation of a variety of somatic cells in culture, and evidence suggests that insulin is also an important regulator of growth in vivo. In cell culture, insulin interacts synergistically with other hormones and growth factors such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), tumor-promoting phorbol esters, and thrombin, to stimulate progression through the cell cycle of cells that have been arrested in G1 by deprivation for serum. In addition, insulin is required by most cells for optimal long term growth in hormone-supplemented serum-free media. In some cells, such as human skin fibroblasts, the growth-promoting effects of insulin appear to be mediated primarily by its low affinity interaction with receptors for insulin-like growth factor I (IGF-I). In other cells, such as hepatocytes, hepatoma cells, adrenocortical tumor cells, mammary carcinoma cells, and F9 embryonal carcinoma cells, insulin appears to stimulate growth by binding to high affinity insulin receptors. The insulin and IGF-I receptor proteins, like the receptor proteins for other growth-promoting hormones such as EGF and PDGF, are closely associated with tyrosine-specific protein kinase activities. The mechanism by which the binding of insulin to its receptor and activation of the receptor-associated tyrosine protein kinase activity control intracellular protein phosphorylation and dephosphorylation reactions, such as the phosphorylation of ribosomal protein S6, is a subject of considerable current interest. The phosphorylation of ribosomal protein S6 may be related mechanistically to the activation by insulin of protein synthesis, and hence the passage of cells through the G1 phase of the cell cycle. Malignant transformation does not generally result in a total loss of the growth requirement of cells for insulin or insulin-like growth factors, although transformation is accompanied in some cases by a qualitative reduction in the insulin/IGF requirement. Abnormalities in insulin production or sensitivity in vivo are accompanied by abnormalities in growth; thus, insulin appears to be an important regulator of growth in vivo. Some of the growth-promoting effects of insulin in vivo may be attributable to direct action of insulin, while other effects may be caused by the regulatory effect of insulin on somatomedin production, and possibly on somatomedin action.
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PMID:Growth-stimulatory actions of insulin in vitro and in vivo. 637 81

In human platelets, thrombin activates Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) and mobilizes Ca2+ concomitantly, whereas 12-O-tetradecanoylphorbol-13-acetate (TPA) may be intercalated into membranes and directly activates protein kinase C without mobilization of Ca2+ in sufficient quantities. A series of experiments with TPA and Ca2+-ionophore (A23187) indicates that activation of protein kinase C is a prerequisite requirement for release of serotonin, and that this enzyme activation and Ca2+ mobilization act synergistically to elicit a full cellular response. Both cyclic AMP and cyclic GMP inhibit activation of protein kinase C by prohibiting the signal-dependent breakdown of inositol phospholipid to produce diacyl-glycerol, but none of these cyclic nucleotides prevents the TPA-induced activation of this enzyme.
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PMID:Synergistic functions of phorbol ester and calcium in serotonin release from human platelets. 640 48

When human platelets were stimulated by synthetic diacylglycerol such as 1-oleoyl-2-acetyl-glycerol, which was a potent activator in vitro of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) (Mori, T., Takai, Y., Yu, B., Takahashi, J., Nishizuka, Y., and Fujikura, T. (1982) J. Biochem. (Tokyo) 91, 427-431), a protein having Mr approximately 40,000 (40-kilodalton protein) was rapidly phosphorylated, just as it was by natural extracellular messengers such as thrombin. Fingerprint analysis appeared to indicate that protein kinase C was indeed responsible for this 40-kilodalton protein phosphorylation in intact platelets. Under these conditions, neither inositol phospholipid breakdown nor endogenous diacylglycerol formation was observed, indicating that the synthetic diacylglycerol intercalated into the membrane and directly activated protein kinase C without interaction with cell surface receptors. During this process, the diacylglycerol was converted in situ to the corresponding phosphatidate, 1-oleoyl-2-acetyl-glyceryl-3-phosphoric acid. Experiments with the synthetic diacylglycerol and Ca2+ ionophore A23187 suggested that the protein phosphorylation catalyzed by protein kinase C was a prerequisite requirement for the release of serotonin, and that the receptor-linked protein phosphorylation and Ca2+ mobilization acted synergistically to elicit the full physiological cellular response.
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PMID:Synergistic functions of protein phosphorylation and calcium mobilization in platelet activation. 640 88

After human platelets have been rendered permeable to small molecules by high voltage electric discharges, addition of buffered micromolar concentrations of Ca2+ causes an ATP-dependent secretion of dense granule serotonin [Knight & Scrutton (1980) Thromb. Res. 20, 437-446]. In the present study, platelets permeabilized by this technique were found to show an up to 10-fold increase in their sensitivity to Ca2+ after exposure to thrombin. In permeabilized platelets, as in the intact cells, release of serotonin was associated with the Ca2+-dependent phosphorylation of 47 000 and 20 000 Da polypeptides (P47 and P20). Thrombin markedly increased the phosphorylation of P47 in the presence of 0.1-1.0 microM-Ca2+ free but had a much smaller effect on phosphorylation of P20. Thrombin also stimulated the formation of 1,2-diacylglycerol in the presence of 0.1 microM-Ca2+ free and was even more effective with 1.0 microM-Ca2+ free, suggesting that receptor-activated hydrolysis of phosphoinositides to 1,2-diacylglycerol was preserved in permeabilized platelets and was potentiated by low intracellular concentrations of Ca2+. The increase in phosphorylation of P47 on addition of thrombin may therefore be accounted for by the stimulatory action of 1,2-diacylglycerol on Ca2+-activated, phospholipid-dependent protein kinase. However, in both the presence and absence of thrombin, higher Ca2+ concentrations were required for optimal secretion than for maximal phosphorylation of both P47 and P20, indicating that additional actions of Ca2+ and thrombin, perhaps also mediated by 1,2-diacylglycerol formation, may be involved in the release of serotonin.
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PMID:Potentiation by thrombin of the secretion of serotonin from permeabilized platelets equilibrated with Ca2+ buffers. Relationship to protein phosphorylation and diacylglycerol formation. 647 19

An increase in cytoplasmic free calcium, [Ca2+]i, is thought to be the trigger for secretory exocytosis in many cells. In blood platelets, large rises in [Ca2+]i can cause secretion and calcium has been regarded as the final common activator not only for secretion but also for shape-change and aggregation. We have shown that while thrombin and platelet-activating factor (PAF) normally elevate [Ca2+]i, they can also stimulate shape-change and secretion even when the [Ca2+]i rise is suppressed. The present results strongly implicate diacylglycerol, produced by stimulus-dependent breakdown of phosphoinositide, in this calcium-independent activation. Exogenous diacylglycerol activates a protein kinase (C-kinase) in platelets as do PAF, thrombin and collagen. 12-O-tetradecanoyl phorbol-13-acetate (TPA) also activates C-kinase and is a potent stimulus for secretion and aggregation. It is shown here that the exogenous diacylglycerol 1-oleoyl-2-acetyl-glycerol (OAG) and TPA evoke similar secretion and aggregation without elevating [Ca2+]i above the basal level of 0.1 microM. The pattern of secretion resembles that produced by collagen and thrombin when [Ca2+]i remains at basal levels. Modest increases in [Ca2+]i, insufficient to stimulate secretion, markedly accelerate the responses to TPA and OAG.
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PMID:Diacylglycerol and phorbol ester stimulate secretion without raising cytoplasmic free calcium in human platelets. 662 85

Phosphorylation of the 20,000 molecular weight (MW) light chain of platelet myosin is associated with the activation of platelets and subsequent release of platelet granules, and the protein kinase catalysing this phosphorylation has been identified as the Ca2+/calmodulin-dependent enzyme, myosin light chain kinase. Tumour-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA), which activate Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), can also cause platelet aggregation and phosphorylation of a 20,000-MW peptide in blood platelets. It was therefore of interest to ascertain whether the 20,000-MW peptide phosphorylated in platelets was the light chain of myosin and whether TPA-induced phosphorylation of the 20,000-MW peptide could be differentiated from thrombin-induced phosphorylation. We now report that TPA-induced activation of platelets is associated with the phosphorylation of the 20,000-MW light chain of myosin, that it appears to be mediated mainly through protein kinase C and that the site phosphorylated in the myosin light chain is distinct from that phosphorylated by myosin light chain kinase.
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PMID:Phorbol ester-induced activation of human platelets is associated with protein kinase C phosphorylation of myosin light chains. 668 54

Secretion of platelet granule constituents is closely associated with the phosphorylation of a cytosol polypeptide of Mr = 47,000 that we have called P47 (Haslam, R. J., Lynham, J. A., and Fox, J. E. B. (1979) Biochem. J. 178, 397-406). This polypeptide is a substrate of Ca2+-activated phospholipid-dependent protein kinase (Kawahara, Y., Takai, Y., Minakuchi, R., Sano, K., and Nishizuka, Y. (1980) Biochem. Biophys. Res. Commun. 97, 309-317). Two-dimensional gel electrophoresis of protein from human platelets that had been preincubated with 32Pi demonstrated the presence under control conditions of 2-3 major forms of P47 that contained very little 32P (pI values, 6.6-6.8) and, after induction of secretion with thrombin, their replacement by 7-9 highly labeled phosphorylated forms of P47 (pI values, 6.1-6.5). Native phosphorylated P47 was purified from thrombin-stimulated 32P-labeled platelets by ammonium sulfate fractionation and column chromatography on DEAE-cellulose, phenyl-Sepharose, and hydroxylapatite. The final 32P-labeled product was obtained in a yield of 20-25% and was purified about 400-fold relative to platelet lysate. This material was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis but, like the starting material, contained 7-9 separable phosphorylated components with different pI values. Purified phosphorylated P47 had a sedimentation coefficient (s20,w) of 3.57 S and a Stokes radius of 3.33 nm from which an Mr = 49,000 and a frictional ratio (f/f0) of 1.4 were calculated. These findings and failure to detect multimers after treatment of the protein with dimethyl suberimidate indicate that P47 normally exists as a monomer. The 32P-labeled phosphate present in purified P47 had the chemical stability of serine or threonine phosphoesters and analysis indicated the presence of 83% phosphoserine and 17% phosphothreonine. Limited proteolysis of purified 32P-labeled P47 by Staphylococcus aureus V8 protease generated a major unlabeled fragment (Mr = 23,500) and up to six labeled fragments (Mr = 24,700-14,800), the relative amounts of the latter depending on the extent of proteolysis. The same labeled fragments were obtained after proteolysis of each of the major phosphorylated components of P47, suggesting that these represent different phosphorylation states of variants of the same protein and that most or all of the phosphorylation sites are on a single 14,800-Da segment of the protein. The availability of pure native phosphorylated P47 should facilitate investigation of the physiological role of this protein in platelets.
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PMID:Purification and characterization of the 47,000-dalton protein phosphorylated during degranulation of human platelets. 688 23


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