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)

Okadaic acid and calyculin A, specific and cell permeable inhibitors of protein phosphatase 1 and 2A, inhibited aggregation, secretion and delta [Ca++]i in thrombin stimulated platelets. The inhibitory effect of calyculin A (IC50: 3.6-4.8nM) was about two hundred times more potent than that of okadaic acid (IC50: 0.8-1.3 microM), which is consistent with the difference of the reported Ki values for protein phosphatase 1. These phosphatase inhibitors and PGI2 synergistically enhanced the phosphorylation of 50kDa protein (P50), which is solely related to the inhibition of platelet reaction. These results indicate that serine/threonine protein phosphatase 1 might play a role in platelet activation.
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PMID:The effects of okadaic acid and calyculin A on thrombin induced platelet reaction. 131 73

The addition of either okadaic acid or calyculin A desensitizes human platelets to thrombin. One objective of this study was to determine which step(s) leading to secretion reactions may be affected by these protein phosphatase inhibitors. In a dose-dependent manner, okadaic acid or calyculin A inhibits phosphatidylinositol metabolism and Ca(2+)-transients. In all cases, calyculin A was approximately 10-fold more potent than okadaic acid, and it had maximal effects at a concentration of 1 microM. Although thrombin-induced rises in [Ca2+]i were diminished, an increase in the phosphorylation state of myosin light chains (MLC) was still observed. Changes in this phosphorylation were diminished, however, following the addition of thrombin to calyculin A-treated platelets that were loaded with dimethyl-BAPTA. These data demonstrate that calyculin A and okadaic acid lower agonist-induced Ca(2+)-transients, which in turn prevents responses such as secretion reactions. Calyculin A/okadaic acid-induced phosphorylation events were not diminished in BAPTA-loaded platelets, suggesting that these phosphorylations are Ca(2+)-insensitive. Thus, a second objective of this study was to identify the protein kinase(s) that was(were) responsible for the calyculin A-induced phosphorylations. In a platelet lysate system, calyculin A caused an increase in the incorporation of [32P]phosphate into p50. This phosphorylation event was identical to that observed in the intact platelet and was not mimicked by cAMP, cGMP, Ca2+, or a Ca2+/phospholipid/diacylglycerol mixture. Kinase activity was removed after the lysate was incubated with p13suc1-Sepharose. This suggests that a p13suc1-sensitive protein kinase, e.g., a cell cycle-dependent protein kinase, is responsible for the calyculin A-sensitive phosphorylation events.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibitors of protein phosphatase type 1 and 2A attenuate phosphatidylinositol metabolism and Ca(2+)-transients in human platelets. Role of a cdc2-related protein kinase. 132 63

The involvement of protein phosphatases in regulating platelet activation was studied. The major portion of the phosphorylase phosphatase activity found in platelet lysates appears to be of the type 1 variety. The identification of this enzyme was based on the finding that greater than 80% of protein phosphatase activity was inhibited by the heat-stable inhibitor protein inhibitor 2 and, while only 20% of the phosphorylase phosphatase activity in platelet extracts was inhibited by 2 nM okadaic acid, greater than 95% of the activity was inhibited in the presence of 1 microM okadaic acid. Increases in protein phosphorylations occurred and thrombin-induced release of serotonin was prevented as a result of artificially inhibiting the enzyme with okadaic acid in intact platelets. This implies either that the regulation of okadaic acid sensitive protein phosphatases is necessary for some agonist-induced effects or that okadaic acid sensitive phosphatases are required for maintaining platelets in a responsive state.
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PMID:Thrombin-induced effects are selectively inhibited following treatment of intact human platelets with okadaic acid. 164 61

We have characterized a novel ecto-protein kinase activity and a novel ecto-protein phosphatase activity on the membrane surface of human platelets. Washed intact platelets, when incubated with [gamma-32P]ATP in Tyrode's buffer, showed the phosphorylation of a membrane surface protein migrating with an apparent molecular mass of 42 kDa on 5-15% SDS polyacrylamide gradient gels. The 42 kDa protein could be further resolved on 15% SDS gels into two proteins of 39 kDa and 42 kDa. In this gel system, it was found that the 39 kDa protein became rapidly phosphorylated and dephosphorylated, whereas the 42 kDa protein was phosphorylated and dephosphorylated at a much slower rate. NaF inhibited the dephosphorylation of these proteins indicating the involvement of an ecto-protein phosphatase. The platelet membrane ecto-protein kinase responsible for the phosphorylation of both of these proteins was identified as a serine kinase and showed dependency on divalent cations Mg2+ or Mn2+ ions. Ca2+ ions potentiated the Mg(2+)-dependent ecto-protein kinase activity. The ecto-protein kinase rapidly phosphorylated histone and casein added exogenously to the extracellular medium of intact platelets. Following activation of platelets by alpha-thrombin, the incorporation of [32P]phosphate from exogenously added [gamma-32P]ATP by endogenous protein substrates was reduced by 90%, suggesting a role of the ecto-protein kinase system in the regulation of platelet function. The results presented here demonstrate that both protein kinase and protein phosphatase activities reside on the membrane surface of human platelets. These activities are capable of rapidly phosphorylating and dephosphorylating specific surface platelet membrane proteins which may play important roles in early events of platelet activation and secretion.
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PMID:Phosphorylation and dephosphorylation of human platelet surface proteins by an ecto-protein kinase/phosphatase system. 185 Mar 5

To identify the protein kinase that is responsible for catalyzing phosphorylation of actin-binding protein (ABP) in platelets, we have examined the effects of protein kinase C and cAMP-dependent protein kinase on this process. We found that purified platelet protein kinase C from platelets was unable to phosphorylate ABP in vitro. However, a crude platelet kinase preparation phosphorylated ABP in the presence of cAMP, but not in the presence of Ca2+/phosphatidylserine. Fresh platelet plasma membranes incubated with [gamma-32P]ATP phosphorylated ABP in the presence of cAMP and the process was blocked by a cAMP-dependent protein kinase inhibitor; ABP phosphorylation induced by prostaglandin E1 (PGE1) appeared to be reduced by the subsequent addition of thrombin. These results strongly suggest that in situ ABP is phosphorylated by activated cAMP-dependent protein kinase when platelet function is inhibited by PGE1. Furthermore, in the PGE1-treated platelets, ABP was proteolyzed at a slower rate than in control platelets when they were lysed with Triton in the absence of EGTA. Partially purified ABP was proteolyzed by calpain in vitro at a slower rate as well. It was demonstrated that ABP from PGE1-treated platelets recovered its sensitivity to calpain after ABP was incubated with a protein phosphatase that had been purified from platelets. We postulate that ABP is stabilized against proteolysis in response to cAMP-elevating agents and that this blocks cytoskeleton reorganization.
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PMID:In situ phosphorylation of platelet actin-binding protein by cAMP-dependent protein kinase stabilizes it against proteolysis by calpain. 254 93

When platelets are stimulated by the addition of thrombin, a series of temporally linked signaling events are initiated. Some of the early events are needed to engage the integrin glycoprotein (GP) IIb-IIIa in a high-affinity state. This in turn leads to aggregation, which initiates a wave of events distinct from those triggered by thrombin. Platelet responses are sensitive to protein serine/threonine phosphatase inhibitors, but which events are dependent on protein phosphatase activity is not known. In the present studies, the effect of the phosphatase inhibitor calyculin A on aggregation-induced signaling was examined. The addition of 0.2 unit/mL thrombin caused aggregation-dependent redistribution of cytoskeletal proteins (actin binding protein, talin, vinculin, and alpha-actinin), glycoproteins (GPIIb-IIIa, PECAM), and signaling molecules (PI3-kinase, pp60c-src) to the cytoskeletal fraction of platelets. Addition of 1-2 microM calyculin A blocked the ability of 0.2 unit/mL thrombin to induce aggregation and the association of these molecules with the cytoskeleton. Aggregation (60-80% of control) was restored if 1 unit/mL thrombin was added, but there was no corresponding redistribution of actin binding protein, talin, vinculin, alpha-actinin, GPIIb-IIIa, PECAM, PI3-kinase, and pp60c-src to the cytoskeleton. Treatment of platelets with calyculin A resulted in an increase in the phosphorylation state of a membrane skeletal protein of 50 kDa. These data strongly suggest that platelet aggregation is dissociable from aggregation-induced signaling, which is dependent on type 1 and 2A phosphatase activities.
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PMID:Aggregation-dependent signaling in human platelets is sensitive to protein serine/threonine phosphatase inhibitors. 762 26

The plasminogen activator inhibitor PAI-1 is markedly elevated in vivo and in vitro upon exposure to the inflammatory mediators tumor necrosis factor alpha (TNF alpha), interleukin-1 (IL-1), and bacterial lipopolysaccharide. Here we report that the isoflavone compound genistein prevents the increase in synthesis of PAI-1 induced by these inflammatory mediators in human endothelial cells in vitro, and partially reduces the basal PAI-1 production by these cells. These effects of genistein were accompanied by a decrease in PAI-1 mRNA and in a suppression of the PAI-1 transcription rate as shown by run-on assay. A specific action of genistein, probably by inhibiting a tyrosine protein kinase, is likely, because the structural genistein analogue daidzein, which has a low tyrosine protein kinase inhibitor activity, did not inhibit PAI-1 synthesis. Vanadate, a tyrosine protein phosphatase inhibitor, increased PAI-1 production. The effect of genistein on PAI-1 synthesis was rather selective. Herbimycin A also reduced PAI-1 synthesis, but several other tyrosine protein kinase inhibitors, namely tyrphostin A47, methyl-2,5-dihydroxy-cinnamate, and compound 5, were unable to do so. All these tyrosine protein kinase inhibitors reduced basic fibroblast growth factor (b-FGF)-induced [3H]thymidine incorporation in endothelial cells. This indicates that the effect of genistein on PAI-1 transcription proceeds independently of its effect on mitogenesis. In contrast to TNF-alpha-induced PAI-1 production, the transcription and synthesis of urokinase-type plasminogen activator (u-PA) was not inhibited by genistein. A TNF-alpha-mutant (Trp32Thr86TNF alpha) that specifically recognizes the 55-kD TNF-receptor, mimicked the effects of TNF alpha on both PAI-1 and u-PA. Because genistein affected PAI-1, but not u-PA induced by this mutant, involvement of different TNF-receptors cannot underlie the difference in the effects of genistein on PAI-1 and u-PA synthesis. Because genistein also inhibited PAI-1 induction by thrombin and IL-4, it is likely that genistein does not act on a TNF alpha-receptor-coupled protein kinase but on the signal transduction pathway enhancing PAI-1 transcription. Our results suggest that the TNF alpha-induced signal transduction pathway of PAI-1 transcription involves a genistein-sensitive step that is not involved in the induction of u-PA by TNF alpha. Given the limited sensitivity to several other tyrosine protein kinase inhibitors, this genistein-sensitive step may be a potential target for pharmacologic intervention to reduce elevated plasma PAI-1 levels.
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PMID:Genistein reduces tumor necrosis factor alpha-induced plasminogen activator inhibitor-1 transcription but not urokinase expression in human endothelial cells. 794 70

Two potent inhibitors of protein phosphatase type 1 (PP1) and type 2A (PP2A), calyculin A (CAL-A) and okadaic acid (OKA), inhibited human platelet aggregation induced by thrombin, collagen and 9,11-epithio-11,12-methano-thromboxane A2 (STA2). IC50 values of CAL-A and OKA for STA2-induced aggregation were 53 nM and 3.5 microM, respectively. These drugs also inhibited thrombin-induced [14C]serotonin secretion of platelets. CAL-A and OKA elicited phosphorylation of certain proteins with an apparent M(r) (x 10(-3) of 200, 60, 50 and 20 light chain of myosin (MLC). Agonist-induced 47,000 M(r) protein phosphorylation was strongly inhibited by these compounds, whereas phosphorylation of 20,000 M(r) MLC was enhanced. The increase in 50,000 M(r) protein phosphorylation by CAL-A and OKA was observed in the presence of agonists, and the 50,000 M(r) phosphorylation may be involved in the inhibition of platelet activation by these compounds. Subcellular analysis of the phosphatase activity in human platelets showed that MLC phosphatase activity was present mainly (approx. 78%) in the cytosolic fraction. Chromatography of human platelet extract on heparin-Sepharose resolved two peaks of MLC phosphatase activity: PP2A in 0.1 M NaCl eluate and PP1 in 0.5 NaCl eluate. PP2A and PP1 isozymes (PP1 alpha, PP1 gamma and PP1 delta) have also been identified in human platelets, by cross-reactivity with polyclonal antibodies against PP2A and PP1 isozymes, respectively. These results suggest that PP1 and/or PP2A may play an important role in the process of platelet activation by regulating levels of phosphorylation of certain proteins.
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PMID:Calyculin A and okadiac acid inhibit human platelet aggregation by blocking protein phosphatases types 1 and 2A. 801 29

Platelets activated by various agonists produce vesicles (microparticles; MPs) from the plasma membrane. However, the mechanism of this MP formation remains to be elucidated. To investigate the possible involvement of protein phosphorylation and cytoskeletal reorganization in MP formation, the effects of various inhibitors on MP formation were investigated. Flow cytometry was employed to detect the amount of MP formation by using monoclonal antibodies against glycoprotein (GP) IIb-IIIa (NNKY 1-32) or GPIIb (Tab). The relationship between changes in cytoskeletal architecture and MP formation in the platelets activated by thrombin plus collagen was observed by scanning electron microscopy (SEM). MPs were observed in the vicinity of the terminals of pseudopods, suggesting that MPs may be related by budding of the pseudopods. Cytochalasin D (10 microM) inhibited MP formation from the activated platelets almost completely. Moreover, SEM of the cytochalasin D-treated platelets revealed the absence of shape change, pseudopod formation and MPs. These findings suggest that cytoskeletal reorganization is necessary for MP formation. Since cytoskeletal reorganization is considered to be regulated by a dynamic phosphorylation-dephosphorylation process, we investigated the effects of the protein phosphatase inhibitors, calyculin A (CLA) and okadaic acid (OA), on MP formation. Flow cytometry showed that these two inhibitors doubled MP formation in activated platelets. SEM of the platelets treated with CLA or OA demonstrated more prominent shape change and pseudopod formation in these platelets than in those without inhibitor. From these results, we conclude that cytoskeletal reorganization, which is controlled by phosphorylation, is involved in MP formation.
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PMID:The role of protein phosphorylation and cytoskeletal reorganization in microparticle formation from the platelet plasma membrane. 816 55

Thrombin dramatically activated p72syk in a time- and dose- dependent fashion in extracts of resting porcine platelets in the presence of EDTA. Separation analysis using Sephacryl S-300 column chromatography has demonstrated that p72syk may exist as large (complex) and small (monomer) forms in resting platelets, and activation of p72syk was only observed in the fraction of large form. Pretreatment with ATP scavenger, GDP beta S and protein phosphatase inhibitors had no effect on this activation. Furthermore, washed immuno-precipitates of large form p72syk were also activated by thrombin or fibrinogen. These results suggest that p72syk may associate with thrombin receptor or other agonist receptors and there may be a novel activation mechanism of non-receptor type protein-tyrosine kinase, which does not require the modification by other protein kinases, protein phosphatases and GTP binding proteins.
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PMID:Activation of p72syk by thrombin in a cell-free system. 816 76


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