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)

To determine if selective activation of individual isozymes of protein kinase C (PKC) might explain the apparently divergent effects of PKC stimulation on platelets, we purified and characterized the isozymes from both platelets and human erythroleukemia (HEL) cells, a cell line that has many features of megakaryocytes. Two peaks of platelet PKC activity were resolved by hydroxylapatite chromatography; immunoblot analysis revealed that these two peaks represented the alpha and beta isozymes of PKC. In contrast, HEL cells produced only a single peak that contained the beta isozyme. None of the other PKC isozymes were detected in these fractions. The cytosol of platelets and HEL cells, however, were both found to contain the PKC-delta isozyme. Northern hybridization analyses and mRNA amplification by the polymerase chain reaction demonstrated the presence of mRNA encoding the alpha, beta, and delta PKC isozymes in platelets, but only the beta and delta isozymes in HEL cells. Phorbol myristate acetate (PMA), thrombin, or an endoperoxide analog induced the phosphorylation of the 47-kDa substrate of PKC (pleckstrin) found in platelets and HEL cells; preincubation of either HEL cells or platelets with PMA reduced the intracellular Ca2+ rise induced by thrombin. Thus, although both HEL cells and platelets contain PKC-beta and the recently described PKC-delta isozymes, the widely distributed alpha isozyme of PKC is absent in HEL cells; however, isozymes other than PKC-alpha are sufficient for some PMA-mediated functions that are similar to those seen in stimulated platelets.
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PMID:Identification and functional characterization of protein kinase C isozymes in platelets and HEL cells. 137 94

Human platelets were found by immunoblot analysis to express protein kinase C (PKC) isozymes alpha, beta, delta, and zeta, but not gamma, epsilon, or eta. Exposure of platelets to thrombin, in the presence of 1 mM calcium, induced increased membrane association of PKC-alpha, -beta, and -zeta, while the subcellular distribution of PKC-delta remained unaltered. Maximal membrane association (2-fold) of PKC-alpha, -beta, and -zeta occurred within 1 min and was sustained for at least 10 min after the addition of thrombin. Similar results were obtained in the presence of the RGDS peptide, which blocks thrombin-induced binding of fibrinogen to its receptor, which indicates that PKC translocation was independent of fibrinogen binding. In the absence of added extracellular calcium, thrombin-induced translocation of PKC-alpha, -beta, and -zeta was transient, reaching a maximum at 1 min and returning to base line by 10 min. In the presence of calcium, thrombin induced a rapid (within 15 s) 8-fold rise in inositol 1,4,5-trisphosphate, which returned to baseline levels within 1 min, and a biphasic increase in sn-1,2-diacylglycerol (DAG), with peaks at 15 s and 2 min, which remained elevated for at least 5 min. Chelation of external calcium abolished the second phase of DAG formation but had no effect on the kinetics or magnitude of the increase in inositol 1,4,5-trisphosphate or the first phase of DAG formation. Two early PKC-dependent functions, serotonin release and 40-kDa protein phosphorylation, were independent of extracellular calcium and sustained DAG. These data demonstrate that in thrombin-stimulated human platelets the duration of the increased PKC membrane association closely parallels that of increased DAG content, and sustained elevations in DAG content and PKC translocation are dependent on extracellular calcium.
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PMID:Translocation of protein kinase C isozymes in thrombin-stimulated human platelets. Correlation with 1,2-diacylglycerol levels. 163 99

Activation of the M3 muscarinic receptor in 1321N1 human astrocytoma cells leads to increased phospholipase D (PLD)-catalyzed hydrolysis of phosphatidylcholine, which is maximal within 1 min of exposure to agonist. Studies examining the kinetics of phosphatidylethanol formation indicate that there is no further PLD activation beyond this time. Thrombin, a mitogen for quiescent 1321N1 cells, also activates PLD only transiently. The PLD response does not recover for up to 1 hr and cells that have been exposed to carbachol or thrombin do not respond to subsequent challenge with the heterologous agonist. In contrast to the desensitization observed with agonists, phorbol-12-myristate-13-acetate induces a sustained stimulation of PLD. In addition, cells pretreated with carbachol or thrombin show a normal response to phorbol-12-myristate-13-acetate, suggesting that the enzymatic activity of PLD is not compromised. Guanosine-5'-O-(3-thio)triphosphate activation of PLD in cell-free homogenates is also unaffected by prior treatment of the cells with agonist. Agonist-stimulated PLD activation in 1321N1 cells is mediated by protein kinase C (PKC). Thrombin and carbachol cause comparable changes in redistribution of both PKC-alpha and PKC-epsilon. The increase in membrane-associated PKC-alpha is transient and is reinitiated by addition of the heterologous agonist, whereas PKC-epsilon remains membrane associated for at least 60 min and is not further increased by addition of the heterologous agonist. We suggest that desensitization of PLD activation results from the down-regulation of an as yet undefined mediator required for agonist receptor coupling to PLD and that PKC-epsilon might participate in this down-regulation.
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PMID:Rapid heterologous desensitization of muscarinic and thrombin receptor-mediated phospholipase D activation. 793 19

Diglycerides play an important role in a number of agonist-induced signal transduction pathways. We have recently demonstrated that alpha-thrombin induces a rapid increase in the level of diglyceride mass in the nucleus and a selective increase in nuclear PKC-alpha [Leach, K.L., Ruff, V.A., Jarpe, M.B., Fabbro, D., Adams, L.D., & Raben, D.M. (1992) J. Biol. Chem. 267, 21816-21822]. In the present report, we examined the potential source of the induced nuclear diglycerides by examining the molecular species profiles of both the induced diglycerides and nuclear phospholipids by capillary gas chromatography. The molecular species profiles of the nuclear diglycerides generated resemble the species profiles of PC, and not PI species, at all times. In addition, while our previous data indicated that the molecular species of whole-cell phospholipids did not change in response to alpha-thrombin, nuclear PE was altered in a dramatic and selective manner in response to this agonist. These results demonstrate that PC hydrolysis is the predominant, if not exclusive, source of the alpha-thrombin-induced nuclear diglycerides in these fibroblasts.
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PMID:Alpha-thrombin-induced nuclear sn-1,2-diacylglycerols are derived from phosphatidylcholine hydrolysis in cultured fibroblasts. 828 82

Troglitazone and pioglitazone, antidiabetic thiazolidinediones, are known to improve insulin resistance. However, the effect of these drugs on platelet aggregation remains unclear. The chemical structure of troglitazone contains vitamin E. Accordingly, we studied the effect of troglitazone, pioglitazone, and vitamin E on thrombin-induced platelet aggregation, metabolism of phosphoinositide, protein phosphorylation, protein kinase C (PKC)-alpha and -beta, and phosphatidylinositol (PI) 3-kinase activation in vitro in human platelets. Maximum platelet aggregation by ADP, collagen, and thrombin decreased in the presence of 0.1-1 micromol/l troglitazone and 500 nmol/l vitamin E for 60 min compared with controls. However, pioglitazone did not inhibit ADP-, collagen-, or thrombin-induced platelet aggregation. Pretreatment with troglitazone and vitamin E, but not with pioglitazone, resulted in decreases in thrombin-induced phosphatidic acid production, hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C, and 47-kDa protein phosphorylation. Thrombin-induced PKC-alpha and -beta activation in membrane fraction was suppressed by pretreatment with troglitazone and vitamin E, but not with pioglitazone. Separately, troglitazone and pioglitazone stimulated PI 3-kinase activity, but thrombin-induced PI 3-kinase activation was suppressed by pretreatment with troglitazone and pioglitazone for 60 min. These results suggest that troglitazone and vitamin E, but not pioglitazone, have a potent inhibitory effect on platelet aggregation via suppression of the thrombin-induced activation of phosphoinositide signaling in human platelets. Finally, the chemical structure of vitamin E may contribute to the inhibitory effect of troglitazone on platelet aggregation in human platelets.
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PMID:Differential effect of the antidiabetic thiazolidinediones troglitazone and pioglitazone on human platelet aggregation mechanism. 972 40

Renal mesangial cells express the 14 kDa secretory phospholipase A2-type IIA (sPLA2-IIA) in response to interleukin-1beta (IL-1beta). In order to understand the regulation of cytokine-induced sPLA2-IIA induction in more detail, we investigated whether phorbol ester-activated protein kinase C (PKC) has an influence on the IL-1beta-induced expression of sPLA2-IIA. We found that treatment of mesangial cells with the biologically active phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate inhibited IL-1beta induction of sPLA2-IIA mRNA, protein, and activity, whereas the inactive compound 4alpha-phorbol 12,13-didecanoate was without effect. An 8-hr pretreatment with PMA, which led to down-regulation of PKC-alpha and -delta isoenzymes, still inhibited sPLA2-IIA induction. Only after down-regulation of PKC-epsilon isoenzyme by 24-hr preincubation with PMA were we able to reconstitute the IL-1beta-induced sPLA2-IIA expression. Thrombin as a physiological activator of PKC in mesangial cells exerted similar effects as PMA and inhibited sPLA2-IIA expression. The selective PKC inhibitor calphostin C potentiated IL-1beta induction of sPLA2-IIA mRNA levels and partially reconstituted the thrombin-induced inhibition of sPLA2-IIA mRNA and activity. These data show that IL-1beta induction of sPLA2-IIA can be modulated by PKC and that the epsilon-isoenzyme of the PKC family is the most likely candidate mediating the suppression of cytokine-induced sPLA2-IIA expression in mesangial cells.
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PMID:Modulation of cytokine-induced expression of secretory phospholipase A2-type IIA by protein kinase C in rat renal mesangial cells. 1057 Dec 49

The Rho-GDP guanine nucleotide dissociation inhibitor (GDI) complexes with the GDP-bound form of Rho and inhibits its activation. We investigated the role of protein kinase C (PKC) isozymes in the mechanism of Rho activation and in signaling the loss of endothelial barrier function. Thrombin and phorbol 12-myristate 13-acetate induced rapid phosphorylation of GDI and the activation of Rho-A in human umbilical venular endothelial cells. Inhibition of PKC by chelerythrine chloride abrogated the thrombin-induced GDI phosphorylation and Rho activation. Depletion of PKC prevented the thrombin-induced GDI phosphorylation and Rho activation, thereby indicating that these events occurred downstream of phorbol ester-sensitive PKC isozyme activation. The depletion of PKC or inhibition of Rho by C3 toxin also prevented the thrombin-induced decrease in transendothelial electrical resistance (a measure of increased transendothelial permeability), thus indicating that PKC-induced barrier dysfunction was mediated through Rho-dependent pathway. Using inhibitors and dominant-negative mutants, we found that Rho activation was regulated by PKC-alpha. Moreover, the stimulation of human umbilical venular endothelial cells with thrombin induced rapid association of PKC-alpha with Rho. Activated PKC-alpha but not PKC-epsilon induced marked phosphorylation of GDI in vitro. Taken together, these results indicate that PKC-alpha is critical in regulating GDI phosphorylation, Rho activation, and in signaling Rho-dependent endothelial barrier dysfunction.
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PMID:Protein kinase C-alpha signals rho-guanine nucleotide dissociation inhibitor phosphorylation and rho activation and regulates the endothelial cell barrier function. 1130 97

Human glioblastoma cell line A172 expressed protease-activated receptor-1 and -2 (PAR-1 and PAR-2). We investigated the effects of the stimulation of these receptors by receptor-activating agonist peptides on the Ca2+ signaling, protein kinase C translocation, cell morphology and cell proliferation in A172. Both PAR-1 agonist SFLLRN and PAR-2 agonist SLIGKV induced an increase in [Ca2+]i. The prior treatment of A172 with PAR-2 agonist SLIGKV did not influence the [Ca2+]i response to PAR-1 agonist SFLLRN or thrombin, however, the prior treatment with PAR-1 agonist SFLLRN or thrombin completely abolished the second response to PAR-2 agonist SLIGKV. Treatment with each agonist peptide produced thinner and fewer processes in A172. The PAR-2 agonist inhibited the proliferation of A172 significantly while PAR-1 agonist did not. PKC-alpha and gamma were translocated from cytosol to membrane with either PAR-1 or PAR-2 stimulation, however, L was specifically translocated with SFLLRN, and lambda with SLIGKV, respectively. These results indicated that PAR-1 and PAR-2 stimulation produced a similar [Ca2+]i response and morphological changes in A172 glioblastoma while the effects on the cell proliferation and activation of PKC isozymes were distinct, suggesting that different signal transduction pathways were activated by these receptors. The uni-directional cross desensitization implies a functional linkage between PAR-1 and PAR-2 receptors.
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PMID:The effects of stimulating protease-activated receptor-1 and -2 in A172 human glioblastoma. 1131 68

Serine/threonine (Ser/Thr) protein phosphatases (PPs) are implicated in the recovery from endothelial barrier dysfunction caused by inflammatory mediators. We hypothesized that Ser/Thr PPs may regulate protein kinase C (PKC), a critical signaling molecule in barrier dysfunction, in the promotion of barrier recovery. Western analysis indicated that bovine pulmonary microvascular endothelial cells (BPMECs) expressed the three major Ser/Thr PPs, PP1, PP2A, and PP2B. Pretreatment with 100 ng/ml of FK506 (a PP2B inhibitor) but not with the PP1 and PP2A inhibitors calyculin A or okadaic acid potentiated the thrombin-induced increase in PKC phosphotransferase activity. FK506 also potentiated thrombin-induced PKC-alpha but not PKC-beta phosphorylation. FK506 but not calyculin A or okadaic acid inhibited recovery from the thrombin-induced decrease in transendothelial resistance. Neither FK506 nor okadaic acid altered the thrombin-induced resistance decrease, whereas calyculin A potentiated the decrease. Downregulation of PKC with phorbol 12-myristate 13-acetate rescued the FK506-mediated inhibition of recovery, which was consistent with the finding that the thrombin-induced phosphorylation of PKC-alpha was reduced during the recovery phase. These results indicated that PP2B may play a physiologically important role in returning endothelial barrier dysfunction to normal through the regulation of PKC.
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PMID:Protein phosphatase 2B inhibitor potentiates endothelial PKC activity and barrier dysfunction. 1150 79

The intracellular serine/threonine kinase protein kinase C (PKC) has an important role in the genesis of pulmonary edema. This review discusses the PKC-mediated mechanisms that participate in the pulmonary endothelial response to agents involved in lung injury characteristic of the respiratory distress syndrome. Thus the paradigms of PKC-induced lung injury are discussed within the context of pulmonary transvascular fluid exchange. We focus on the signal transduction pathways that are modulated by PKC and their effect on lung endothelial permeability. Specifically, alpha-thrombin, tumor necrosis factor (TNF)-alpha, and reactive oxygen species are discussed because of their well-established roles in both human and experimental lung injury. We conclude that PKC, most likely PKC-alpha, is a primary supporter for lung endothelial injury in response to alpha-thrombin, TNF-alpha, and reactive oxygen species.
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PMID:Protein kinase C modulates pulmonary endothelial permeability: a paradigm for acute lung injury. 1257 83


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