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. Troponin C and calmodulin were not digested by thrombin at a significant rate in the presence of Ca2+. 2. In the presence of EGTA, troponin C was digested by thrombin to yield three peptides, TH1 (residues 1--120), TH3 (residues 1--100) and TH2 (residues 121--159). 3. In the presence of EGTA calmodulin was digested by thrombin giving two peptides, TM1 (residues 1--106) and TM2 (residues 107--148). 4. The electrophoretic mobilities of peptides TH1 and TM1 were increased at pH 8.6 by Ca2+ both in the presence and absence of urea. The mobilities of peptides TH2 and TM2 were unaltered under these conditions. 5. Peptides TH1, TH2 and tM1 formed complexes with troponin I on polyacrylamide gels at pH 8.6 in the presence of Ca2+. 6. The phosphorylation of troponin I by cyclic AMP-dependent protein kinase was significantly inhibited by peptides TH1 and TH3 and to a lesser extent by peptide TM1. 7. The calmodulin peptide TM1 activated myosin light-chain kinase when present in large molar excess. Peptide TM2 did not activate the enzyme.
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PMID:Biological activities of the peptides obtained by digestion of troponin C and calmodulin with thrombin. 689 66

Using a rabbit anti-human prekallikrein antibody crossed immunoelectrophoresis (CIE) and Laurell rocket antigen determinations were done in plasma of subjects with Fletcher (prekallikrein, PKA), Fitzgerald (high molecular weight kininogen), Hageman (XII), and PTA (XI) deficiencies as well as in patients with activation of coagulation (intravascular coagulation syndromes). Abnormal CIE patterns were seen in the Fletcher and Fitzgerald deficient plasmas and also in some of the patients with intravascular coagulation. In vitro studies of plasma treated with thrombin, plasmin, and contact activating agents indicated that abnormal CIE patterns and increased PKA antigen levels were indicative of activation of the Hageman factor dependent pathway and not the result of plasma clotting by thrombin. In vivo activation of the Hageman factor dependent pathway frequently results in an abnormal CIE and a low PKA antigen level.
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PMID:Immunoelectrophoretic studies of prekallikrein in human plasma. 691 63

Ca2+-activated, phospholipid-dependent protein kinase recently found in rat brain (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., & Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695) is present in large quantities in human platelets. The activation of this enzyme appears to be initiated by unsaturated diacylglycerol and intimately related to phosphatidylinositol hydrolysis which is induced by thrombin. The enzyme is selectively and profoundly inhibited by several phospholipid-interacting compounds such as imipramine, verapamil, and tetracaine, which concomitantly inhibit aggregation and release reaction in parallel manners. It is suggestive that this protein kinase may be involved in the transmembrane control of intracellular events eventually leading to platelet activation.
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PMID:Possible involvement of Ca2+-activated, phospholipid-dependent protein kinase in platelet activation. 741 28

In human platelets a proline-directed kinase distinct from the ERK MAP kinases is stimulated by both thrombin and the thrombin receptor agonist peptide SFLLRN and may be involved in the activation of Ca(2+)-dependent cytosolic phospholipase A2 (Kramer, R. M., Roberts, E. F., Hyslop, P. A., Utterback, B. G., Hui, K. Y., and Jakubowski, J.A. (1995) J. Biol. Chem. 270, 14816-14823). Here we show that this kinase is identical with or closely related to p38 (the mammalian homolog of HOG1 from yeast), a recently discovered protein kinase typically activated by inflammatory cytokines and environmental stress. Further, we demonstrate that activation of this kinase by thrombin is transient (with maximal stimulation at 1 min), is accompanied by tyrosine phosphorylation, and precedes the activation of the ERK kinases. This is the first report to show that p38 kinase is activated by thrombin and to suggest a role for this MAP kinase in the thrombin-mediated signaling events during platelet activation.
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PMID:Thrombin induces activation of p38 MAP kinase in human platelets. 749 91

Rap 1b is a 22-kDa low molecular mass GTP-binding protein which is both a member of the Ras superfamily and a substrate for cAMP-dependent protein kinase. Recently, evidence has been presented to show that Rap 1b is incorporated into the detergent-extracted cytoskeleton of platelets during thrombin-induced activation. The aims of this study were to compare the incorporation of Rap 1b into the detergent-extracted cytoskeleton after activation with different agonists, to examine the role of extracellular calcium on the incorporation of Rap 1b into the cytoskeleton, to investigate the relationship between the association of Rap 1b and other proteins with the cytoskeleton, and to determine the effect of phosphorylation of Rap 1b incorporation into the cytoskeleton. Platelets were activated with thrombin, A23187, phorbol myristate acetate, ADP, epinephrine, and collagen in the presence and absence of calcium. The time dependence of Rap 1b incorporation into the detergent-extracted cytoskeleton was then measured. When platelets were activated by thrombin in the presence of extracellular calcium, conditions which permit aggregation, incorporation of Rap 1b into the detergent-extracted cytoskeleton was biphasic. Approximately 20% of the total cellular Rap 1b incorporated into the cytoskeleton within seconds and was followed by a slower second phase of incorporation. In contrast, when platelets were activated by thrombin in the absence of calcium, conditions which inhibit aggregation, or by the other agents in the presence or absence of calcium, only the initial phase of Rap 1b incorporation into the cytoskeleton was measured. The incorporation of Rap 1b paralleled the incorporation of membrane glycoproteins (GP) IIb/IIIa and PECAM-1, but not the incorporation of pp60c-src. The GTPase-activating protein for Ras (Ras-GAP) did not associate with the detergent-extracted cytoskeleton. Two-dimensional isoelectric focusing SDS-polyacrylamide gel electrophoresis of the total cellular and cytoskeletal Rap 1b showed that unphosphorylated as well as phosphorylated isoforms of Rap 1b were incorporated into the cytoskeleton in the same molar ratio as was present in the intact cell. Furthermore, the rates of incorporation of phosphorylated and unphosphorylated Rap 1b into the cytoskeleton were similar. These experiments show that Rap 1b can regulate events that take place within seconds after activation, such as the initial formation of the cytoskeleton, as well as longer term changes in the cytoskeleton that occur in response to thrombin-induced aggregation. Furthermore, phosphorylation could modulate the (unknown) functions of Rap 1b as a component of the cytoskeleton.
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PMID:Incorporation of Rap 1b into the platelet cytoskeleton is dependent on thrombin activation and extracellular calcium. 751 36

We have compared the desensitization of two receptors, the thrombin receptor which displays dual coupling to both pertussis toxin-sensitive (Gi) and -insensitive (Gq) proteins and the serotonin type 2 (5-HT2) receptor which selectively couples to Gq. In the case of the thrombin receptor, cleavage induces activation and irreversible receptor modification followed by rapid (T1/2 = 3 min) and extensive desensitization of the receptor's ability to modulate phospholipase C (Gq). 5-HT-induced desensitization of its receptor is markedly slower (T1/2 = 10 min) and by 60 min only 50% of the phospholipase C response is lost. This effect occurs with a parallel disappearance of 5-HT receptors from the cell surface. Whole cell phosphorylation studies showed that the thrombin receptor is rapidly phosphorylated upon activation. In contrast, the 5-HT2 receptor displays a low basal level of phosphorylation which is not increased upon agonist treatment. The cytoplasmic tail of the 5-HT2 receptor which contains several protein kinase consensus sequences was found not to be involved in receptor activation or desensitization. However, a chimeric receptor having the core of the 5-HT2 receptor and the cytoplasmic tail of the thrombin receptor was able to undergo 5-HT-induced desensitization and phosphorylation. These results indicate that (i) both 5-HT2 and thrombin receptors have unique shut-off mechanisms, and (ii) that sequences in the carboxyl terminus of the thrombin receptor are sufficient to trigger rapid uncoupling of the receptor from its G protein(s) and downstream effector(s).
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PMID:Distinct mechanisms regulate 5-HT2 and thrombin receptor desensitization. 753 66

Previous studies demonstrated that the thrombin-induced permeability of endothelial cell monolayers is reduced by the elevation of cGMP. In the present study, the presence of cGMP-dependent protein kinase (cGMP-PK) immunoreactivity and activity in various types of human endothelial cells (ECs) and the role of cGMP-PK in the reduction of thrombin-induced endothelial permeability was investigated. cGMP-PK type I was demonstrated in freshly isolated ECs from human aorta and iliac artery as well as in cultured ECs from human aorta, iliac vein, and foreskin microvessels. Addition of the selective cGMP-PK activator 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) to these ECs caused phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), an established cGMP-PK substrate, which is localized at cell-cell contact sites of confluent ECs. cGMP-PK type I expression decreased during serial passage of ECs, which correlated with a diminished ability of 8-pCPT-cGMP to induce VASP phosphorylation. Preincubation of aorta and microvascular EC monolayers with 8-pCPT-cGMP caused a 50% reduction of the thrombin-stimulated permeability, as determined by measuring the peroxidase passage through EC monolayers on porous filters. Furthermore, the thrombin-induced rise in cytoplasmic [Ca2+]i was strongly attenuated by the cGMP-PK activator in fura 2-loaded aorta ECs. In contrast, cGMP-PK could not be demonstrated in freshly isolated and cultured human umbilical vein ECs. Incubation of umbilical vein ECs with 8-pCPT-cGMP did not cause VASP phosphorylation and had no effect on the thrombin-induced increases in cytoplasmic Ca2+ and endothelial permeability.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression of cGMP-dependent protein kinase I and phosphorylation of its substrate, vasodilator-stimulated phosphoprotein, in human endothelial cells of different origin. 755 43

We have reported that platelets exposed to thrombin or thrombin receptor-directed ligand activate phospholipase C and rapidly accumulate phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) and phosphatidylinositol (3,4)-bisphosphate (PtdIns(3,4)P2) as a function of the activation of phosphoinositide (PI) 3-kinases in a GTP-binding protein-dependent manner. In such platelets, serine- and threonine-directed phosphorylation of pleckstrin also occurs and has been attributed to protein kinase C activation. We now report that the phosphorylation of pleckstrin is partially dependent upon PI 3-kinase. Pleckstrin phosphorylation in response to thrombin receptor stimulation is progressively susceptible to inhibition by wortmannin, a potent and specific inhibitor of platelet PI 3-kinases. PI 3-kinase thus seems to play a gradually increasing role in promoting pleckstrin phosphorylation. The IC50 for wortmannin in inhibiting SFLLRN-stimulated 3-phosphorylated phosphoinositide accumulation is 10 nM, and that (i.e. 50% of maximum inhibition) for inhibiting pleckstrin phosphorylation is 15 nM. Synthetic PtdIns(3,4,5)P3, when added to saponin-permeabilized (but not intact) platelets, causes wortmannin-insensitive phosphorylation of pleckstrin. PtdIns(3,4,5)P3 also overcomes the inhibition by wortmannin of thrombin- or guanosine 5'-3-O-(thio)trisphosphate-stimulated pleckstrin phosphorylation. In contrast, PtdIns(4,5)P2 or inositol (1,3,4,5)-tetrakisphosphate are ineffective in these respects. The pattern of phosphorylation of pleckstrin activated by PtdIns(3,4,5)P3 is not distinguishable from that of pleckstrin phosphorylated in intact platelets exposed to protein kinase C-activating beta-phorbol myristate acetate, mimicking diacylglycerol. Activation of protein kinase(s) by PtdIns(3,4,5)P3 thus offers a route for pleckstrin phosphorylation in vivo that is an alternative to activation of phospholipase C-->diacylglycerol-->protein kinase C.
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PMID:Phosphatidylinositol (3,4,5)-trisphosphate stimulates phosphorylation of pleckstrin in human platelets. 755 10

The brain-enriched p21cdc42/rac1-activated serine/threonine kinase, p65PAK, was identified and purified on the basis of overlays with [gamma-32P]GTP-Cdc42 onto SDS-fractionated proteins (Manser, E., Leung, T., Salihuddin, H., Zhao, Z.-S., and Lim, L. (1994) Nature 367, 40-46). In this study, the ubiquitously expressed p21cdc42/rac1 binding protein with relative molecular weight of 62,000 was purified from rat testes and shown to contain peptides related to PAK. It has thus been designated as the gamma-PAK isoform (alpha- and beta-isoforms being brain enriched). Isolation of gamma-PAK cDNAs show that the kinase is highly conserved with alpha-PAK in both the p2 binding and kinase domains. The purified protein exhibited kinase activity that was activated by GTP-Cdc42 or GTP-Rac1 in vitro. In platelets, a p62 in situ renaturable kinase was recognized by antibodies raised against gamma-PAK. This thrombin-activated protein kinase appears to coprecipitate with another kinase of M(r) 86,000, suggesting that PAK may be part of a thrombin-responsive signaling complex.
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PMID:Identification and molecular cloning of a p21cdc42/rac1-activated serine/threonine kinase that is rapidly activated by thrombin in platelets. 759 96

The link between increased usage of beta-adrenoceptor agonists and worsening of asthma symptoms has raised interest in the effects of agents such as salbutamol on airway wall remodelling, and particularly airway smooth muscle proliferation. In the present study we have investigated the role of increases in intracellular cAMP in the inhibitory effect of salbutamol on airway smooth muscle proliferation. The inhibitory effects of a combination of submaximally effective concentrations of salbutamol (10 nM) and the non-selective phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 100 microM) on thrombin (0.3 U/mL)-induced mitogenesis in human cultured airway smooth muscle cells was greater than that for either agent alone. In addition, agents known to increase cAMP-dependent protein kinase activity including forskolin (10 microM), 8-bromoadenosine-3',5'-cyclic monophosphate (100 microM), and prostaglandin E2 (1 microM) have an inhibitory effect on thrombin (0.3 U/mL)-induced induced proliferation. Furthermore, the cAMP antagonist, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (300 microM) significantly reduced the inhibitory effect of salbutamol (10 nM) on thrombin (0.3 U/mL)-induced DNA synthesis. In IBMX (100 microM)-pretreated cells, salbutamol (100 nM) increased intracellular cAMP levels via stimulation of a beta 2-adrenoceptor. Salbutamol (10 microM), at concentrations supramaximally effective for inhibition of mitogenesis, had no effect on thrombin (0.3 U/mL)-induced increases in intracellular calcium levels. Therefore, our results suggest that the previously reported inhibition of mitogen-induced proliferation in human cultured airway smooth muscle cells by the beta 2-adrenoceptor agonist, salbutamol (100 nM), is at least partly due to elevation of intracellular cAMP, while there is no effect of salbutamol on initial mitogen-induced increases in intracellular calcium.
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PMID:Salbutamol inhibits the proliferation of human airway smooth muscle cells grown in culture: relationship to elevated cAMP levels. 759 43


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