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)

Human plasma heparin cofactor II (HCII) inhibits thrombin by rapidly forming a stable, equimolar complex in the presence of heparin or dermatan sulfate. Cultured human hepatoma-derived cells (PLC/PRF-5) secreted (approximately equal to 200 ng/ml in 3 days) a protein of MW - 72 kD that was immunoisolated and immunoblotted with anti-HCII, co-migrated on SDS-PAGE with human plasma HCII, and formed covalent complexes with thrombin (MW - 101 kD) in the presence but not absence of heparin or dermatan sulfate; these complexes co-migrated with those obtained by incubating thrombin with human plasma under the same conditions. HCII was not detectable (less than 0.13 ng/ml) in post-culture medium from cultured human umbilical vein endothelial cells or human foreskin fibroblasts.
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PMID:Biosynthesis of functionally active heparin cofactor II by a human hepatoma-derived cell line. 299 59

Although clofibrate has been shown to inhibit platelet aggregation that is caused by thrombin, ADP and epinephrine, by blocking the release of arachidonic acid from platelet phospholipids [8], here we have demonstrated that clofibrate enhanced platelet aggregation by arachidonic acid and PLC and reversed the effects of PGE1 on platelet cAMP concentration and on PLC-induced secretion of [14C]-5HT in similar, concentration-dependent manners. Taken together, these findings strongly suggest that the proaggregatory effect of clofibrate is mediated by a lowering of cAMP in platelets.
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PMID:Potentiating effects of clofibrate on prostaglandin-dependent and -independent pathways of human platelet activation: evidence for involvement of cyclic AMP. 628 50

The importance of PLC activation in cell proliferation is evident from the fact that the hydrolysis of PtdIns(4,5)P2 is one of the early events that follow the interaction of many growth factors and mitogens with their respective receptors. However, the importance of PLC activation is not restricted to proliferation; it is one of the most common transmembrane signaling events elicited by receptors that regulate many other cellular processes, including differentiation, metabolism, secretion, contraction, and sensory perception. It is also clear that cell proliferation signaling does not always require PLC, as indicated by the fact that growth factors such as insulin and CSF-1 do not appear to elicit the hydrolysis of PtdIns(4,5)P2, even though the intracellular domains of their receptors carry a PTK domain and the receptors show topologies very similar to those of the PLC-activating growth factors PDGF, EGF, and FGF. The growth factor-dependent activation of PLC is initiated by the formation of a complex between the receptor PTK and PLC-gamma; the formation of this complex is mediated by a specific interaction between a tyrosine phosphate residue on the intracellular domain of PTK and the SH2 domain of PLC-gamma. The receptor PTK subsequently phosphorylates PLC-gamma, of which two distinct isozymes, PLC-gamma 1 and PLC-gamma 2, have been identified. Proliferation of T cells and B cells in response to the aggregation of their respective cell surface receptors is also accompanied by the activation of PLC-gamma isozymes at an early stage. Unlike growth factor receptors, the T cell and B cell receptors lack intrinsic PTK activity but associate with several non-receptor PTKs of the Src and Syk families. Although the specific kinases are not known, one or more of these enzymes phosphorylate and activate PLC-gamma 1 and PLC-gamma 2. Transduction of growth signals by G protein-coupled receptors such as those for thrombin or bombesin also requires PtdIns(4,5)P2 hydrolysis, which, in this instance, is mediated by PLC-beta isozymes. The PLC-beta subfamily consists of four distinct members: PLC-beta 1, PLC-beta 2, PLC-beta 3, and PLC-beta 4. Agonist interaction with specific G protein-coupled receptors causes the dissociation of Gq proteins into G alpha and G beta gamma subunits and the exchange of GDP bound to G alpha for GTP. The resulting GTP-bound G alpha subunit then activates PLC-beta isoforms by binding to the carboxyl-terminal region of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphoinositide-specific phospholipase C and mitogenic signaling. 749 69

It has recently been reported that protein-tyrosine kinase activity is required for thrombin-induced growth in vascular smooth muscle cells (VSMC). In the present study, we have identified several phosphoproteins that are tyrosine-phosphorylated in response to thrombin in quiescent VSMC. These proteins are insulin-like growth factor-1 receptor beta-subunit (IGF-IR beta), insulin receptor substrate-1 (IRS-1), and phospholipase C-gamma 1 (PLC-gamma 1). Thrombin-stimulated phosphorylation of these proteins was rapid; it was maximal at 1 min and reduced thereafter. Thrombin also activated mitogen-activated protein kinases (MAPK) in quiescent VSMC in a biphasic manner with a rapid and larger peak at 10 min (6-fold) followed by a sustained smaller second peak at 2 h (2-fold). Inhibition of protein-tyrosine kinase activity by the use of two structurally different protein-tyrosine kinase inhibitors, genistein and herbimycin A, significantly blocked the thrombin-induced tyrosine phosphorylation of IGF-1R beta, IRS-1, and PLC-gamma 1 and decreased thrombin-stimulated DNA synthesis. In contrast, however, inhibition of protein-tyrosine kinase activity had no effect on thrombin activation of MAPK. Collectively, these findings suggest a role for tyrosine phosphorylation of IGF-IR beta, IRS-1, and PLC-gamma 1 in thrombin-induced mitogenic signaling events in VSMC. Furthermore, while protein tyrosine phosphorylation is essential for thrombin-induced DNA synthesis, it is not required for thrombin-stimulated MAPK activation. Since thrombin rapidly activated Src in VSMC, Src may be involved in the cross-talk between the G-protein-coupled receptor agonist and a tyrosine kinase receptor such as IGF-1R.
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PMID:Thrombin stimulates phosphorylation of insulin-like growth factor-1 receptor, insulin receptor substrate-1, and phospholipase C-gamma 1 in rat aortic smooth muscle cells. 749 60

The sulphydryl reagent phenylarsine oxide (PAO) (1 microM) inhibited completely formation of inositol phosphates in human platelets induced by collagen or by cross-linking of the platelet low affinity Fc receptor, F c gamma RIIA, but did not alter the response to the G protein receptor agonist thrombin. PAO also inhibited completely tyrosine phosphorylation of PLC gamma 2 in collagen and Fc gamma RIIA-stimulated cells, although tyrosine phosphorylation of other proteins including the tyrosine kinase syk was relatively unaffected. PAO (1 microM) also inhibited completely tyrosine phosphorylation of PLC gamma 1 induced by platelet derived growth factor (PDGF) in NIH-3T3 fibroblasts but only partially reduced phosphorylation of the PDGF receptor. These results provide further evidence that collagen and Fc gamma RIIA cross-linking activate platelets through a pathway distinct from that used by thrombin and suggest that PAO may be a selective inhibitor of PLC gamma relative to PLC beta isozymes.
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PMID:Phenylarsine oxide inhibits tyrosine phosphorylation of phospholipase C gamma 2 in human platelets and phospholipase C gamma 1 in NIH-3T3 fibroblasts. 762 42

Two membrane-associated phosphoinositide-specific phospholipase Cs (mPI-PLC-1 and mPI-PLC-2) and a cytosolic enzyme (cPI-PLC) that were activated by brain G-protein beta gamma subunits have been isolated from human platelets. The truncation of mPI-PLC-1 that was mediated by mu-calpain induced much higher activation by beta gamma subunits (Banno, Y., Asano, T., and Nozawa, Y. (1994) FEBS Lett. 340, 185-188). On the basis of size and immunological cross-reactivity, mPI-PLC-1 (155 kDa) was PLC-beta 3, and mPI-PLC-2 (100 kDa) was its truncated form. The cPI-PLC (140 kDa) was recognized by the antibody selective for internal sequences of PLC-beta 3 but not by the antibody raised against its carboxyl terminus, indicating that it may be related to PLC-beta 3. Treatment of human platelets with A23187 and dibucaine, activators of calpain, caused cleavage of actin-binding protein and talin in a time-dependent manner. At the same time, decrease of PLC-beta 3 (155 and 140 kDa) and concomitant increase of the 100-kDa product of cleavage were observed on immunoblots with the antibody to internal sequences of PLC-beta 3. Furthermore, stimulation of platelets by natural agonists, thrombin and collagen, caused the cleavage of PLC-beta 3 (155 and 140 kDa) and an increase of 100 kDa PLC-beta 3 in a time- and dose-dependent manner. The cleavage of these PLC-beta 3 enzymes was completely blocked by calpain inhibitor, calpeptin, indicating that the PLC-beta 3 modification may be a consequence of platelet activation leading to activation of calpain. This is the first demonstration that PLC-beta 3 is indeed cleaved by calpain upon platelet activation by physiological agonists. The cleavage of PLC-beta 3 evoked by thrombin and collagen but not ADP was correlated with irreversible aggregation, suggesting that the PLC-beta 3 modification may play a role in secondary irreversible aggregation in agonist-stimulated human platelets.
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PMID:Endogenous cleavage of phospholipase C-beta 3 by agonist-induced activation of calpain in human platelets. 787 93

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

Membrane-associated phosphoinositide-phospholipase C (PI-PLC)-beta (150 kDa) and its truncated forms (100 kDa and 45 kDa) were purified from human platelets. The 100 kDa PI-PLC-beta was found to be activated to a greater extent by brain G-protein beta gamma subunits compared to the intact 150 kDa enzyme. Furthermore, treatment with mu-calpain of the intact PI-PLC-beta (150 kDa) caused a marked augmentation of its activation by beta gamma subunits. This enhanced PLC activation by beta gamma subunits was due to truncation by mu-calpain, producing a 100 kDa PI-PLC, but not by another protease, thrombin.
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PMID:Proteolytic modification of membrane-associated phospholipase C-beta by mu-calpain enhances its activation by G-protein beta gamma subunits in human platelets. 813 42

The regulatory mechanism(s) of a phosphoinositide-specific phospholipase C, PLC-delta 1, was investigated using a clone of stably overexpressed PLC-delta 1 (PLC delta 30 cells) in Chinese hamster ovary cells. Thrombin stimulation of PLC delta 30 cells exhibited 6.5-fold increase in total inositol phosphates (InsP), which was significantly higher than that in the vector-transfected (V1) cells (2.0-fold). AIF-4 increased InsP accumulation in both V1 and PLC delta 30 cells, and pertussis toxin partially blocked InsP accumulation in thrombin-stimulated PLC delta 30 cells. Guanosine thiotriphosphate (GTP gamma S) markedly potentiated thrombin-stimulated InsP generation in permeabilized PLC delta 30 cells compared with V1 cells, suggesting possible involvement of a G-protein (s) in the activation of PLC-delta 1. In PLC delta 30 cells, ionomycin-induced significant InsP generation and thrombin-stimulated InsP generation were completely inhibited by addition of EGTA. Furthermore, the stimulatory effects of thrombin plus GTP gamma S in PLC delta 30 cells were more sensitive to change in free calcium concentration than in V1 cells. Suppression by 12-O-tetradecanoylphorbol 13-acetate of thrombin-stimulated InsP accumulation was not affected by increasing Ca2+ concentration. These results indicate that thrombin-induced PLC-delta 1 activation is regulated via both G-protein(s) and calcium.
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PMID:Thrombin-mediated phosphoinositide hydrolysis in Chinese hamster ovary cells overexpressing phospholipase C-delta 1. 819 39

Human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors are linked to phosphoinositide-specific phospholipase C (PI-PLC) via a G protein tentatively identified as a member of the Gq class. In contrast, platelet thrombin receptors appear to activate PI-PLC via other unidentified G proteins. Platelets from most dogs are TXA2 insensitive (TXA2-); i.e., they do not aggregate irreversibly or secrete although they bind TXA2, but they respond normally to thrombin. In contrast, a minority of dogs have TXA2-sensitive (TXA2+) platelets that are responsive to TXA2. To determine the mechanism responsible for TXA2- platelets, we evaluated receptor activation of PI-PLC. Equilibrium binding of TXA2/PGH2 receptor agonists, [125I]BOP and [3H]U46619, and antagonist, [3H]SQ29,548, revealed comparable high-affinity binding to TXA2-, TXA2+, and human platelets. U46619-induced PI-PLC activation was impaired in TXA2- platelets as evidenced by reduced (a) phosphorylation of the 47-kD substrate of protein kinase C, (b) phosphatidic acid (PA) formation, (c) rise in cytosolic calcium concentration, and (d) inositol 1,4,5 trisphosphate (IP3) formation, while thrombin-induced PI-PLC activation was not impaired. GTPase activity stimulated by U46619, but not by thrombin, was markedly reduced in TXA2- platelets. Antisera to Gq class alpha subunits abolished U46619-induced GTPase activity in TXA2-, TXA2+, and human platelets. Direct G protein stimulation by GTP gamma S yielded significantly less PA and IP3 in TXA2- platelets. Immunotransfer blotting revealed comparable quantities of Gq class alpha-subunits in all three platelet types. Thus, TXA2- dog platelets have impaired PI-PLC activation in response to TXA2/PGH2 receptor agonists secondary to G protein dysfunction, presumably involving a member of the Gq class.
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PMID:Thromboxane-insensitive dog platelets have impaired activation of phospholipase C due to receptor-linked G protein dysfunction. 822 62


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