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)

Activation of human platelets by the peptide YFLLRNP has been shown to induce shape change but not secretion, Ca2+ mobilization, or pleckstrin phosphorylation (Rasmussen, U.B., Gachet, C., Schlesinger, Y., Hanau, D., Ohlmann, P., Van Obberghen-Schilling, E., Pouyssegur, J., Cazenave, J.P., and Pavirani, A. (1993) J. Biol. Chem. 268, 14322-14328). YFLLRNP was added to washed human platelets that had been pretreated with EGTA at 37 degrees C or preincubated with the fibrinogen receptor antagonist RGDS to preclude the activation of the integrin alpha IIb beta 3 (fibrinogen receptor). YFLLRNP induced shape change and stimulated the tyrosine phosphorylation of proteins of 62, 68, and 130 kDa within 7 s. Tyrosine phosphorylation of these proteins reached maximum levels (2-3-fold) 15-30 s after addition of YFLLRNP and decreased subsequently. The chelation of intracellular Ca2+ by BAPTA-AM decreased basal tyrosine protein phosphorylation but did not inhibit the increase of tyrosine phosphorylation of P62, P68, and P130 or the shape change induced by YFLLRNP. Preincubation of platelets with the tyrosine kinase inhibitors genistein or tyrphostin A23 completely inhibited platelet shape change and protein tyrosine phosphorylation induced by YFLLRNP. The inactive structural analogs daidzein and tyrphostin A1 were barely inhibitory. P62, P68, and P130, which exhibited increased tyrosine phosphorylation upon stimulation with YFLLRNP, were found in the cytoskeleton. P130 was not identical to vinculin or the focal adhesion kinase pp125FAK. The results indicate that stimulation of G-protein-coupled thrombin receptors rapidly induces protein tyrosine kinase activation through a Ca(2+)- and integrin-independent mechanism. Protein tyrosine kinase activation and tyrosine phosphorylation of novel protein substrates seem to play an essential role in the induction of platelet shape change.
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PMID:Platelet shape change induced by thrombin receptor activation. Rapid stimulation of tyrosine phosphorylation of novel protein substrates through an integrin- and Ca(2+)-independent mechanism. 783 59

Thrombus generation is central to thrombosis at vascular lesion sites, including post-PCTA acute reocclusion and chronic restenosis. Thrombin stimulates platelet activation, monocyte and neutrophil chemotaxis, and endothelial production of prothrombotic factors. The varied physiologic effects of thrombin are due to the widespread presence of thrombin receptors in many cell types. The receptor is uniquely activated: thrombin binds to the receptor at the thrombin anion-binding exosite, the receptor ligand ("tethered ligand") apparently being a sequence of 6 amino acids (SFLLRN). Thus, peptides corresponding to the sequence of the tethered ligand can stimulate almost all functions of native thrombin itself. Several intracellular signaling pathways have been identified as important in the restenosis process: the G protein-related pathway, cyclic adenosine monophosphate (cAMP) mediator pathway, and tyrosine kinase activation pathway. In situ hybridization has demonstrated an increase in thrombin receptor mRNA throughout the period of neointimal and vascular lesion development. The mechanism of this increase is unknown, but may be mediated by multiple inflammatory modulators. Several strategies have been tested in animal models for inhibiting thrombin: (1) Hirudin not only prevents thrombin from cleaving fibrinogen, but also prevents thrombin receptor activation. (2) Thrombin receptor antagonist peptides block platelet aggregation effects of thrombin. (3) Mono- and polyclonal antibodies inhibit thrombin receptor activation. (4) Antisense oligonucleotides block thrombin receptor expression.
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PMID:Role of the thrombin receptor in restenosis and atherosclerosis. 786 77

Recombinant p56lck tyrosine kinase was purified to near homogeneity from a baculovirus/insect cell expression system. Treatment with thrombin proteolytically removed the C-terminal 54 amino acids from p56lck. Processed enzyme migrated on sodium dodecyl sulfate (SDS) gels with a M(r) approximately 6,000 lower than intact enzyme. Analytical ultracentrifugation of intact and processed p56lck gave M(r)'s of 62,600 and 56,200, respectively, confirming that the thrombin treated enzyme existed in solution as a processed polypeptide and that there was no anomalous migration in SDS gels due to thrombin treatment. Simultaneous multispeed analysis of sedimentation equilibrium data demonstrated that both intact and processed enzyme can dimerize with a weak binding constant in the range of 200-300 microM. Purified intact p56lck incorporated 2 mol of [32P]P(i) per mole of enzyme. Purified processed p56lck incorporated only 1 mol of [32P]P(i) per mole of enzyme. The loss of 1 mol of [32P]P(i) per mole of enzyme after thrombin deletion of the C-terminus demonstrates that p56lck undergoes autophosphorylation at the C-terminus. The data are consistent with autophosphorylation at tyrosine 505, which has previously been thought to be a regulatory phosphorylation site, but which now must also be considered as an autophosphorylation site.
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PMID:Dimerization of native and C-terminally proteolyzed p56lck tyrosine kinase. 787 93

We have investigated the effects of the tyrosine kinase inhibitors, genistein and methyl 2,5-dihydroxycinnamate, on [3H]arachidonic acid release from human platelets. Both tyrosine kinase inhibitors blocked, in a dose-dependent manner, the release of arachidonic acid stimulated by thrombin or suspensions of collagen fibres. Blockade by the tyrosine kinase inhibitors occurred early in the arachidonate release time course. Both genistein and methyl 2,5-dihydroxycinnamate also inhibited tyrosine phosphorylation in platelets. The inhibitors were specific in that they did not affect protein kinase C activity, ATP levels or mobilization of Ca2+ from internal stores. These findings suggest a role for tyrosine kinase activity in the regulation of phospholipase A2 in platelets stimulated by the physiological ligands, thrombin and collagen.
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PMID:The tyrosine kinase inhibitors, genistein and methyl 2,5-dihydroxycinnamate, inhibit the release of (3H)arachidonate from human platelets stimulated by thrombin or collagen. 787 44

Several neurotransmitters that act through G protein-linked receptors have been shown to affect the growth rate of dividing cells. An analysis of the early signaling events that mediate this response revealed some novel activities for G protein-linked receptors. Activation of D2 receptors heterologously expressed in CHO cells also stimulates the synthesis of DNA, which results in increased proliferation. Pertussis toxin pretreatment abolishes D2 agonist-stimulated mitogenesis, which indicates the need for a G protein. D2 receptor-stimulated mitogenesis occurs in the presence of a membrane-soluble cyclic AMP analog and, in Chinese hamster ovary cells with a mutated protein kinase A, which is resistant to the growth effects of cyclic AMP. Therefore, the proliferative response is independent of changes in cyclic AMP. It was determined that a number of other signaling pathways commonly used by Gi-linked receptors are not involved in the D2-mediated mitogenic response. These include arachidonic acid release, stimulation of protein kinase C, stimulation of inositol phosphates, opening of K+ channels and activation of amiloride sensitive Na+/H+ exchange. D2 receptor-stimulated mitogenesis is blocked by genistein, a tyrosine kinase inhibitor, at the same concentrations that block thrombin-stimulated mitogenesis. In fact, dopamine and thrombin stimulate a rapid increase in tyrosine phosphorylation of a number of substrates in the transfected Chinese hamster ovary cells. These results reveal a novel signaling event for D2 dopamine receptors, activation of tyrosine phosphorylations. They suggest the importance of these events for D2 dopamine receptor-stimulated mitogenesis.
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PMID:D2 dopamine receptor stimulation of mitogenesis in transfected Chinese hamster ovary cells: relationship to dopamine stimulation of tyrosine phosphorylations. 790 93

The functional significance of phospholipase D (PLD) could most easily be investigated using selective inhibitors. We have isolated a family of fungal metabolites, ketoepoxides, that inhibit chemotactic peptide (formyl-Met-Leu-Phe)-stimulated PLD activation and superoxide generation in granulocytes in the low micromolar range (SCH 49210 having an IC50 of 1.6 microM). Unlike receptor-mediated PLD activation, ketoepoxides were poor inhibitors of phorbol ester-induced PLD activity in granulocytes (IC50 = 43 microM for SCH 49210). Ketoepoxides did not inhibit platelet-derived growth factor-stimulated PLD activity in fibroblasts at up to 50 microM. We also tested the effect of ketoepoxides on in vitro epidermal growth factor receptor and neu tyrosine kinase activities. SCH 49210 (and 49209) did not inhibit the tyrosine kinases at up to 100 microM. These results suggest that ketoepoxides do not inhibit PLD activation due to effects on tyrosine kinase activity. fMLP-induced phospholipase A2 (PLA2) activation is also inhibited by ketoepoxides in the low micromolar range (SCH 49210 having an IC50 of 3.2 microM), but the ketoepoxides were poorer inhibitors of Ca2+ ionophore A23187-induced PLA2 (SCH 49210 having an IC50 of 83 microM). As a measure of phospholipase C (PLC) activity, the generation of inositol-1,4,5 triphosphate in thrombin-stimulated platelets was measured. The ketoepoxides did not inhibit PLC activation indicating that, unlike the aminosteroid U73122, ketoepoxides exhibit some selectivity among receptor-linked phospholipases. The ketoepoxides were also effective inhibitors of tumor cell invasion, as measured by penetration of HT1080 human fibrosarcoma cells into a reconstituted basement membrane matrix. Interestingly, both PLD inhibition and anti-tumor invasion activity correlate closely. These ketoepoxides are, therefore, potential anti-metastatic compounds and may be useful probes to study the role of PLD in cell function.
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PMID:Novel ketoepoxides block phospholipase D activation and tumor cell invasion. 791 2

We show the presence of the tyrosine kinase JAK2 in human platelets and demonstrate that it undergoes phosphorylation on tyrosine residues on challenge with the G protein receptor stimulus, thrombin, or the tyrosine phosphatase inhibitor, peroxovanadate. Thrombin-induced phosphorylation of JAK2 is inhibited by two structurally distinct inhibitors of tyrosine kinases, staurosporine and the tyrphostin ST271. The protein kinase C (PKC) inhibitor, Ro 31-8220, and intracellular Ca2+ chelator, BAPTA-AM, also inhibit thrombin-induced phosphorylation of JAK2, while the phorbol ester, phorbol dibutyrate (PDBu), and Ca2+ ionophore, A23187, induce tyrosine phosphorylation of JAK2. These results suggest that tyrosine phosphorylation of JAK2 stimulated by thrombin may be mediated downstream of phosphoinositide metabolism.
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PMID:Phosphorylation of JAK2 in thrombin-stimulated human platelets. 792 97

Agonist stimulation of platelets induces multiple waves of tyrosine phosphorylation, several of which are dependent on the integrin alpha IIb beta 3. At least two classes of protein tyrosine kinases are activated during various stages of platelet activation, 1) Src family tyrosine kinases are activated during an early phase of platelet activation by an integrin-independent mechanism and 2) pp125FAK is activated during a late stage of platelet activation, and it is dependent on platelet aggregation mediated by fibrinogen binding to alpha IIb beta 3. In this report, we examined the mechanism of agonist-induced phosphorylation and activation of the tyrosine kinase pp72syk, which is known to couple with immune response receptors in B cells and mast cells. pp72syk was found to be regulated by both agonist and integrin receptors in a pattern distinct from that of pp60src and pp125FAK. Specifically, thrombin induced the tyrosine phosphorylation and activation of pp72syk independent of platelet aggregation. However, full activation of pp72syk required integrin engagement since treatment with antibodies that block fibrinogen binding to alpha IIb beta 3 reduced pp72syk phosphorylation by 40%. Furthermore, fibrinogen binding to alpha IIb beta 3 stimulated directly with an anti-beta 3 antibody activated pp72syk 3-fold and stimulated its tyrosine phosphorylation. Thus, pp72syk is the only platelet tyrosine kinase identified to date that can be directly activated through integrin ligation. In addition, we found that the activation of pp72syk is dependent upon the state of actin polymerization and that pp72syk redistributes to actin-rich cytoskeletal complexes in an aggregation-dependent manner. These results suggest a role for pp72syk in both early, integrin-independent tyrosine phosphorylation events as well as those dependent upon subsequent integrin engagement.
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PMID:Regulation of the protein tyrosine kinase pp72syk by platelet agonists and the integrin alpha IIb beta 3. 796 45

Cytoskeleton reorganization has been suggested to play an important role in platelet signal transduction. A number of signalling molecules are found to relocalize to this fraction upon thrombin stimulation. In this paper, we show that PLC-gamma 1, a key enzyme of the inositol lipid metabolism, is also translocated to the platelet cytoskeleton upon thrombin stimulation. Interestingly, its translocation is very rapid and transient, and correlates with the increase in PLC activity previously measured in the cytoskeleton by our group. Using a potent tyrosine kinase inhibitor, tyrphostin AG-213, we show a significant inhibition of the translocation of PLC-gamma 1, indicating an involvement of tyrosine kinases in its relocation. Thus, our results demonstrate for the first time a rapid and transient tyrosine kinase-dependent translocation of PLC-gamma 1 to the cytoskeleton of thrombin-stimulated platelets.
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PMID:Rapid and transient translocation of PLC-gamma 1 to the cytoskeleton of thrombin-stimulated platelets. Evidence for a role of tyrosine kinases. 798 23

During endothelial cell activation, alpha-thrombin elicits both Ca2+ release from internal stores and influx of external Ca2+ across the plasma membrane. The mechanisms of alpha-thrombin-induced Ca2+ entry into endothelial cells are unclear. Therefore, effects of the specific tyrosine kinase inhibitor herbimycin A on protein tyrosine phosphorylation and on intracellular Ca2+ transients were studied in alpha-thrombin-stimulated human umbilical vein endothelial cells. alpha-Thrombin caused significant tyrosine phosphorylation of mainly two proteins and evoked typical biphasic changes of free cytosolic Ca2+ concentration. We show that 24 h pretreatment with herbimycin A inhibited alpha-thrombin-induced endothelial protein tyrosine phosphorylation. Moreover, herbimycin A significantly attenuated alpha-thrombin-induced Ca2+ influx but not release from internal stores. The data suggest that protein tyrosine phosphorylation by alpha-thrombin is involved in the regulation of alpha-thrombin-induced Ca2+ influx into endothelial cells.
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PMID:Thrombin-induced Ca2+ influx and protein tyrosine phosphorylation in endothelial cells is inhibited by herbimycin A. 806 Mar 52


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