Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the accompanying paper (Nemoto, Y., Namba, T., Teru-uchi, T., Ushikubi, F., Morii, N., and Narumiya, S. (1992) J. Biol. Chem. 267, 20916-20920), we have identified rhoA protein as the sole substrate protein for botulinum C3 ADP-ribosyltransferase (C3 exoenzyme) in human blood platelets. Here we examined the role of rhoA protein in platelet functions. C3 exoenzyme added to washed platelets dose- and time-dependently ADP-ribosylated rhoA protein in situ in the cells. Concomitant with this modification, inhibition of thrombin-induced platelet aggregation was observed. This inhibition was not reversed by washing the treated platelets, but was not found when C3 exoenzyme was pretreated with mouse monoclonal anti-C3 exoenzyme antibody. C3 exoenzyme treatment did not affect thrombin-induced inositol 1,4,5-trisphosphate production. Secretion of preloaded [14C]serotonin was delayed by the enzyme treatment, but the extent of the secretion was not influenced. In addition, the enzyme treatment did not change the expression of the glycoprotein IIb-IIIa complex on the platelet surface. The enzyme treatment also suppressed platelet aggregation induced by phorbol myristate acetate. These results suggest that rhoA protein plays a role mainly in the aggregation process downstream from receptor-phospholipase C coupling. This, together with the previous finding that rhoA protein modulates stress fiber formation in cultured fibroblasts (Paterson, H. F., Self, A. J., Garrett, M. D., Just, I., Aktories, K., and Hall, A. (1990) J. Cell Biol. 111, 1001-1007), suggests that rhoA protein regulates the assembly of actin filaments and the avidity of the platelet integrin (glycoprotein IIb-IIIa) in the aggregation process.
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PMID:A rho gene product in human blood platelets. II. Effects of the ADP-ribosylation by botulinum C3 ADP-ribosyltransferase on platelet aggregation. 140 Apr 7

Monoclonal antibody P256, which is specific for glycoprotein IIb-IIIa complex, was found to induce aggregation of normal platelets in plasma. The mechanism of platelet activation induced by this monoclonal antibody was thoroughly studied. The divalent binding to the IIb-IIIa molecule was necessary for triggering aggregation since Fab' fragments did not induce aggregation as did IgG and F(ab')2 fragments; however, F(ab')2 did not induce the release as did the whole IgG. P256-induced aggregation was accompanied by release of all three granule constituents, namely dense granules, alpha-granules and lysosomes, with parallel kinetics showing half-maximum release 50 s after addition of P256. Thromboxane synthesis was initiated at the same time. Using 32P-prelabeled platelets, no variation in level of [32P]phosphatidylinositol 4,5-bisphosphate could be detected in the first minute after P256 addition, indicating no activation of the calcium-independent phospholipase C specific for polyphosphoinositol phospholipid. P256 induced a calcium mobilization as measured by Indo-1 fluorescence of about the third of that measured in the presence of a thrombin concentration giving the same intensity of aggregation. P256 induced phosphorylation of the myosin light chain p20 and of the main substrate of protein kinase C, p43. Addition of aspirin inhibited almost totally calcium mobilization and partially aggregation, release and protein phosphorylations. By contrast, in the absence of external calcium, although no aggregation could occur, the release reaction was only partially reduced. In this activation, the glycoprotein IIb-IIIa complex thus appears to play a role in modulating platelet response, not only via calcium fluxes but also in activating protein kinase C responsible for p43 phosphorylation.
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PMID:Activation of platelets induced by mAb P256 specific for glycoprotein IIb-IIIa. Possible evidence for a role for IIb-IIIa in membrane signal transduction. 236 45

Human platelets stimulated by epinephrine undergo enhanced turnover of phosphatidylinositol 4,5-bisphosphate, accumulate inositol trisphosphate, diacylglycerol, and phosphatidic acid, and phosphorylate a 47-kDa protein. All of these phenomena indicate stimulation of phospholipase C. These responses are blocked completely by inhibitors of alpha 2-adrenergic receptors (yohimbine), cyclooxygenase (aspirin or indomethacin), phospholipase A [2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (ONO-RS-082)], Na+/H+ exchange [ethylisopropylamiloride (EIPA)], fibrinogen binding to glycoprotein IIb/IIIa (antibody A2A9), Ca2+/Mg+ binding (EDTA), or removal of fibrinogen. Epinephrine evokes (i) an increased turnover of ester-linked arachidonic acid in aspirin treated platelets that is inhibited by ONO-RS-082, EDTA, yohimbine, or the absence of fibrinogen and (ii) a rapid cytoplasmic alkalinization that is inhibited partially by blockage of cyclooxygenase activity and completely by A2A9 or EIPA. In contrast, when incubated with subaggregatory concentrations of the prostaglandin H2/thromboxane A2 analogue [(15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic acid (U46619) and epinephrine, aspirin-treated platelets show a potentiation of phospholipase C activation that is unaffected by the above inhibitors. We propose that epinephrine, in promoting exposure of glycoprotein IIb/IIIa sites for fibrinogen binding, leads to a cytoplasmic alkalinization, which, in conjunction with local shifts in Ca2+, promotes low-level activation of phospholipase A. The resulting free arachidonic acid is converted to cyclooxygenase products, which, potentiated by epinephrine, activate phospholipase C. This further amplifies the initial stimulatory response.
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PMID:Activation of phospholipases A and C in human platelets exposed to epinephrine: role of glycoproteins IIb/IIIa and dual role of epinephrine. 302 70

Glycoprotein IIb/IIIa has been proposed as the platelet receptor for high density lipoproteins (HDL3). We characterized the HDL3-induced second messenger response in normal and glycoprotein IIb/IIIa-deficient platelets. In normal platelets physiological concentrations of HDL3 induced the time-dependent generation of phosphatidic acid in the absence of phosphoinositide turnover. The rise in phosphatidic acid preceded that of diacyglycerol which was inconsistent with phospholipase C/diacylglycerol kinase pathway being the source of phosphatidic acid and suggested the involvement of phospholipase D. In the presence of butanol, HDL3 stimulated the accumulation of phosphatidylbutanol, an unequivocal indicator of phospholipase D activity. No increase in phosphatidic acid, diacylglycerol, and phosphatidylbutanol was observed upon addition of HDL3 to glycoprotein IIb/IIIa-deficient platelets. We conclude that phosphatidic acid is generated in HDL3-stimulated platelets by phospholipase D and that glycoprotein IIb/IIIa is the receptor involved in this process.
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PMID:HDL3 activates phospholipase D in normal but not in glycoprotein IIb/IIIa-deficient platelets. 785 58

Megakaryocytopoiesis is governed in the bone marrow microenvironment by cellular interactions that include various adhesion receptor systems and pericellular proteolysis for proper regulation of cell motility and differentiation. In order to define the role of cell surface molecules required for these processes, we searched for protease receptors on these cells. In an in vitro system utilizing different cell lines of the megakaryoblastic lineage (MEG-01, Dami), low level surface expression of the urokinase (uPA) receptor was noted. Following stimulation with phorbolester (PMA), a 3-6 fold higher expression of uPA receptor over a period of up to 5 days could be observed by fluorescent activated cell-sorting as well as by direct ligand-binding of amino-terminal fragment of uPA or vitronectin. Together with elevated expression of alpha IIb beta 3-integrin (glycoprotein IIb/IIIa complex), double immuno-fluorescence staining of stimulated cells confirmed the increased cell surface localization of uPA receptor. Semi-quantitative RT-PCR, ligand blot analysis and measurement of cell-bound proteolytic activity revealed a differentiation-dependent upregulation of the uPA receptor expression in megakaryoblastic cell lines as in monocytic cells. Due to its glycolipid anchorage, incubation with phosphatidylinositol-specific phospholipase C reduced uPA receptor-mediated ligand binding by about 60%, uPA receptor mRNA was expressed in cultured megakaryocytes derived from bone marrow, whereas no uPA receptor mRNA was detectable in platelets. These results indicate a differentiation-dependent increase in the expression of uPA receptor in megakaryoblastic cells. The characteristics of surface expression and functionality of the receptor on megakaryocytic cells may influence their maturation by regulating cellular communication in the bone marrow micro-environment.
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PMID:The urokinase-receptor (CD87) is expressed in cells of the megakaryoblastic lineage. 906 8

The aim of this study was to systematically examine the inhibitory mechanisms of rutin, a well-known flavonoid in platelet aggregation. In this study, rutin concentration-dependently (250 and 290 microM) inhibited platelet aggregation in human platelets stimulated by agonists (i.e., collagen). Rutin (250 and 290 microM) did not significantly interfere with the binding of FITC-triflavin to the glycoprotein IIb/IIIa complex in human platelets. Rutin (250 and 290 microM) markedly inhibited intracellular Ca(2+) mobilization and thromboxane A(2) formation in human platelets stimulated by collagen. Rapid phosphorylation of a platelet protein of M(r) 47000 (P47), a marker of protein kinase C activation, was triggered by collagen (1 microg/mL). This phosphorylation was markedly inhibited by rutin (250 and 290 microM). On the other hand, rutin (250 and 290 microM) did not significantly increase the formations of cyclic AMP and nitric oxide/cyclic GMP in platelets. In conclusion, these results indicate that the antiplatelet activity of rutin may involve the following pathways: rutin inhibited the activation of phospholipase C, followed by inhibition of protein kinase C activity and thromboxane A(2) formation, thereby leading to inhibition of the phosphorylation of P47 and intracellular Ca(2+) mobilization, finally resulting in inhibition of platelet aggregation.
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PMID:Mechanisms involved in the antiplatelet activity of rutin, a glycoside of the flavonol quercetin, in human platelets. 1523 45

The cytosolic calcium concentration, (Ca(2+))(c), represents in many types of cells a versatile regulatory system involved in several signal transduction pathways of cell activation (1), and is tightly regulated in human platelets. Under basal conditions, platelets maintain the (Ca(2+))(c) at about 80 nM by actively sequestering calcium into the intracellular stores or by extruding it into the extracellular medium. The binding of specific agonists to their receptors induces a rapid increase of the (Ca(2+))(c), which can reach the micromolar range, depending on the type and the dose of agonist used. This effect is owing to Ca(2+) influx from the extracellular medium to the cytosol and to the release of Ca(2+) from internal stores (1,2) This latter effect is mediated by a phospholipase C-dependent mechanism; phospholipase C hydrolyzes plasma membrane phosphati-dylinositol 4,5-bisphosphate and produces diacylglycerol and inositol 1,4,5-trisphosphate (3). Inositol 1,4,5-trisphosphate causes the release of Ca(2+) from the dense tubular system by interacting with a specific receptor that behaves as a Ca(2+) channel by Itself. Much less is known about the Ca(2) influx across the plasma membrane Several studies suggested a role for membrane glycoproteins, such as glycoprotein IIb-IIIa complex (GPIIb-IIIa) in controlling calcium homeostasis in platelets. GPIIb-IIIa is the platelet receptor for fibrinogen and other adhesive proteins and belongs to the integrin superfamily (4). It represents a high affinity binding site for Ca(2+) in the plasma membrane (5) and may regulate the Ca(2+) homeostasis in human platelets (6), by acting as a channel by itself (7) or by interacting with a closely adjacent channel (8). Moreover, some authors reported a role for GPIIb-IIIa occupation in the regulation of phospholipase C activation (9).
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PMID:Lectin-induced calcium mobilization in human platelets : use of fluorescent probes. 2137 81