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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Platelets exposed to thrombin or thrombin receptor agonist peptide (SFLLRN) activate phospholipase C and protein kinase C (PKC), and accumulate 3-phosphorylated phosphoinositides (3-PPI) as a function of the activation and relocalization of two cytoskeletally-associated phosphoinositide 3-kinases (PI 3-K): p85/PI 3-K and PI 3-Kgamma. We now report that exposure of platelets to PKC-activating beta-phorbol myristate acetate (betaPMA) does not stimulate PI 3-Kgamma, but rather stimulates p85/PI 3-K, which associates with the cytoskeleton. Wortmannin is an inhibitor of both PI 3-Ks, known to act with more potency on p85/PI 3-K. betaPMA-stimulated 3-PPI accumulation is more sensitive to wortmannin (IC50 = 1.3 nM) than is SFLLRN- or thrombin-stimulated 3-PPI accumulation (IC50 = 10 nM). The activity of p85/PI 3-K in immunoprecipitates or in cytoskeletal fractions is inhibited more potently by exposure of platelets to wortmannin than is the activity of PI 3-Kgamma. betaPMA or SFLLRN promotes the conversion of platelet integrin alphaIIb/beta3 into a fibrinogen-binding form required for platelet aggregation. Activation of alphaIIb/beta3 in response to betaPMA or SFLLRN is inhibited by wortmannin with an IC50 of 1 nM in each case. Wortmannin inhibits neither activation of alphaIIb/beta3 by ligand-induced binding site antibody (anti-LIBS6 Fab) nor anti-LIBS6 Fab-induced platelet aggregation in the presence of fibrinogen, indicating that this type of "outside-in" signaling by alphaIIb/beta3 is largely PI 3-K-independent. We conclude that p85/PI 3-K, in preference to PI 3-Kgamma, contributes to activation of alphaIIb/beta3 when the thrombin receptor or PKC is stimulated.
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PMID:Phosphoinositide 3-kinase gamma and p85/phosphoinositide 3-kinase in platelets. Relative activation by thrombin receptor or beta-phorbol myristate acetate and roles in promoting the ligand-binding function of alphaIIbbeta3 integrin. 862 20

This study focused on the variations in phosphoinositide metabolism depending upon alphaIIbbeta3-integrin/fibrinogen interaction without previous activation of platelet agonist receptors. We found that adhesion of resting human platelets to immobilized fibrinogen stimulates phosphatidic acid production and a concomitant decrease in phosphatidylinositol 4',5'-bisphosphate. These results, and the absence of a transphosphatidylation reaction, argue in favor of the activation of a phospholipase C. Moreover, we observed the accumulation of phosphatidylinositol 3',4'-bisphosphate in adherent platelets as a consequence of the activation of a phosphatidylinositol 3-kinase. This effect was inhibited by ADP scavengers. Our results demonstrate that in adherent platelets, whereas phosphatidylinositol 3-kinase activation is controlled by both alphaIIbbeta-integrin engagement and released ADP, phospholipase C stimulation is triggered only by alphaIIbbeta-integrin/fibrinogen interaction.
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PMID:alphaIIb beta 3-integrin mediated adhesion of human platelets to a fibrinogen matrix triggers phospholipase C activation and phosphatidylinositol 3',4'-biphosphate accumulation. 876 10

Neutrophil elastase (NE) and cathepsin G are two serine proteinases released concomitantly by stimulated polymorphonuclear neutrophils. We previously demonstrated that while NE by itself does not activate human platelets, it strongly enhances the weak aggregation induced by a threshold concentration of cathepsin G (threshold of cathepsin G) (Renesto, P., and Chignard, M. (1993) Blood 82, 139-144). The aim of this study was to delineate the molecular mechanisms involved in this potentiation process. Two main pieces of data prompted us to focus on the activation of the platelet fibrinogen receptor, the alphaIIbbeta3 integrin. First, previous studies have shown this integrin to be particularly prone to proteolytic regulation of its function. Second, we found that the potentiating activity of NE on the threshold of cathepsin G-induced platelet aggregation was strictly dependent on the presence of exogenous fibrinogen. Using flow cytometry analysis, NE was shown to trigger a time-dependent binding of PAC-1 and AP-5, two monoclonal antibodies specific for the activated and ligand-occupied conformers of alphaIIbbeta3. Furthermore, the potentiated aggregation was shown to result from an increased capacity of platelets to bind fibrinogen. Indeed, the combination of NE and threshold of cathepsin G increased the binding of PAC-1 approximately 5.5-fold over basal values measured on nontreated platelets, whereas this binding raised only by approximately 3-fold in threshold of cathepsin G-stimulated platelets (p < 0.05). By contrast, phosphatidic acid accumulation, pleckstrin phosphorylation, and calcium mobilization produced by the combination of NE and threshold of cathepsin G were not significantly different from those measured with threshold of cathepsin G alone (p > 0.05), indicating that the phospholipase C/protein kinase C pathway is not involved in the potentiation of aggregation. The foregoing data, as well as the requirement of catalytically active NE to trigger alphaIIbbeta3 activation and potentiate threshold of cathepsin G-initiated platelet aggregation, led us to examine whether the structure of this integrin was affected by NE. Immunoblot and flow cytometry analysis revealed a limited proteolysis of the carboxyl terminus of the alphaIIb subunit heavy chain (alphaIIbH), as judged by the disappearance of the epitope for the monoclonal antibody PMI-1. Mass spectrometry studies performed on a synthetic peptide mapping over the cleavage domain of alphaIIbH predicted the site of proteolysis as located between Val837 and Asp838. Treatment by NE of ATP-depleted platelets or Chinese hamster ovary cells expressing human recombinant alphaIIbbeta3 clearly established that activation of the integrin was independent of signal transduction events and was concomitant with the proteolysis of alphaIIbH. In support of this latter observation, a close correlation was observed between the kinetics of proteolysis of alphaIIbH on platelets and that of expression of the ligand binding activity of alphaIIbbeta3 (r2 = 0.902, p </= 0. 005). However, only a subpopulation ( approximately 25%) of the proteolyzed alphaIIbbeta3 appeared to fully express the ligand binding capacity. Altogether, these results demonstrate that NE up-regulates the fibrinogen binding activity of alphaIIbbeta3 through a restricted proteolysis of the alphaIIb subunit, and that this process is relevant for the potentiation of platelet aggregation.
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PMID:Human neutrophil elastase proteolytically activates the platelet integrin alphaIIbbeta3 through cleavage of the carboxyl terminus of the alphaIIb subunit heavy chain. Involvement in the potentiation of platelet aggregation. 911 Oct 81

We have previously demonstrated that thrombin possesses an active yet cryptic Arg-Gly-Asp (RGD) site which upon exposure induces endothelial cell (EC) adhesion via alpha nu beta 3 integrin [Bar-Shavit et al. (1991): J Cell Biol 112:335]. This was achieved in the presence of cell surface-associated heparan sulfate proteoglycans (HSPG) and exceedingly low concentrations of plasmin [Bar-Shavit et al. (1993): J Cell Biol 123:1279]. A portion of the cell surface-associated HSPG (glypican) is anchored via a covalently linked glycosyl-phosphatidylinositol (PI) residue, which can be released by treatment with glycosyl-PI-specific phospholipase C (PI-PLC). We report here that exposure of either bovine aortic EC, smooth muscle cells (SMC), or wild-type CHO cells to PI-PLC released HSPG involved in the conversion of thrombin to an adhesive molecule. The adhesion-promoting activity of the released HSPG was abolished following treatment with heparinase but not chondroitinase ABC. Incubation of thrombin with heparan sulfate-deficient CHO cells or cells that were pretreated with PI-PLC failed to induce its conversion to an adhesive molecule, indicating that glypican was playing a major role in this conversion. Moreover, affinity-purified glypican, but not syndecan or fibroglycan, elicited efficient conversion of plasmin-treated thrombin into an adhesive molecule. Antibodies raised against the RGD site in thrombin failed to interact with native thrombin, prothrombin, or the RGD site in other adhesive proteins such as vitronectin, fibrinogen, or fibronectin. Anti-thrombin-RGD antibodies which blocked the adhesion-promoting activity of thrombin were also capable of recognizing thrombin that was first incubated with a suboptimal concentration of plasm in in the presence of PI-PLC-released HSPG. Heparin, heparan sulfate, and PI-PLC-released HSPG had no effect on other cellular properties of thrombin such as receptor binding and growth-promoting activity. Altogether we have demonstrated that the heparin binding domain in thrombin plays a specific role in promoting thrombin adhesive properties and that membrane-associated glypican is likely to be the major physiological inducer of this property.
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PMID:Specific involvement of glypican in thrombin adhesive properties. 917 91

Convulxin, a very potent aggregating protein from rattlesnake venom, was purified by a new procedure and its heterodimeric structure alpha 3 beta 3 was confirmed. The polypeptide N-terminal sequences of convulxin subunits were determined by Edman degradation. They are very similar and appear homologous to botrocetin from Bothrops jararaca venom and to rattlesnake lectin from Crotalus atrox venom, both being classified among the C-type lectin family. The binding of 125I-labelled convulxin to blood platelets has also been analysed under equilibrium conditions. These studies indicated that convulxin binds to platelets with a high affinity (Kd = 30 pM) on a small number of binding sites (1000 binding sites per cell). The high-affinity binding of convulxin appears specific to platelets, since it is not observed on other cell types such as neutrophils and erythrocytes. Also, the high-affinity binding of convulxin to membranes platelet is not inhibited by alpha-thrombin, fibrinogen, collagen, laminin binding inhibitor, RGDS peptide, adenosine diphosphate, platelet-activating factor-acether, serotonin or epinephrine. This, together with the recent observation that platelet activation by convulxin is partially mediated by phospholipase C and involves other mechanisms as well, indicates that convulxin may interact with a specific platelet acceptor (receptor) protein which has yet to be characterized.
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PMID:Convulxin, a potent platelet-aggregating protein from Crotalus durissus terrificus venom, specifically binds to platelets. 927 71

1Convulxin (Cvx) is a well-characterized platelet aggregating glycoprotein isolated from Crotalus durissus terrificus and C. d. cascavella venoms. In the present report we show that Cvx induces tyrosine phosphorylation of human platelet proteins, including phospholipase C-gamma 2 (PLC gamma 2), and also stimulates [3H]arachidonic acid ([3H]AA) mobilization, pleckstrin phosphorylation, and an increase in the cytosolic Ca2+ concentration ([Ca2+]in) due to both Ca2+ entry and internal Ca2+ mobilization. Staurosporine, a potent protein kinase inhibitor, and genistein, a specific inhibitor of protein tyrosine kinases (PTK), were used to evaluate the role of protein tyrosine phosphorylation (PTP) in the signal transduction evoked by Cvx. Staurosporine and genistein inhibited in a dose-dependent manner platelet aggregation induced by Cvx. Both inhibitors significantly blocked to near basal levels breakdown of phosphatidylinositol 4,5-bisphosphate from [myo-2-3H]inositol-labeled platelets and the production of [3H]AA metabolites from [3H]AA-labeled platelets after challenge with Cvx. Cvx provokes an increase in [Ca2+]in in Fura-2-loaded platelets that was abolished by concentrations of staurosporine which also inhibited Cvx-induced platelet aggregation. In addition, Cvx stimulates a rapid increase in tyrosine phosphorylation of human platelets proteins with molecular masses of 40, 72/74, 78/80, 105, 120, and 145 kDa, followed by dephosphorylation. Furthermore, Cvx stimulates a rapid tyrosyl phosphorylation of a 145-kDa molecular mass protein that was identified as PLC gamma 2. PTP induced by Cvx was not inhibited when platelets were stimulated in the presence of indomethacin, apyrase, EDTA, or RGDS peptide. These results indicate that PTP is chronologically proximal to Cvx binding to platelets, and is independent of aggregation or fibrinogen binding to the integrin alpha IIb beta 3. On the other hand, the dephosphorylation step is inhibited by RGDS peptide or EDTA, suggesting that integrin alpha IIb beta 3 is envolved in this step. The profile obtained with Cvx resembles that obtained in platelets adherent to an immobilized ligand, such as immobilized collagen, in which PTP is independent on integrin alpha IIb beta 3. Thus, we suggest that Cvx is an example of a protein with adhesion molecule-like properties; i.e., it is an adhesin. In conclusion, our results show that Cvx induces multiple signaling pathways in platelets via a PTK-dependent pathway involving PLC gamma 2 tyrosyl phosphorylation, with the subsequent platelet responses. Cvx is unique among platelet soluble agonists because under test tube stirring conditions it induces a PTP profile independently of integrin alpha IIb beta 3.
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PMID:Convulxin induces platelet activation by a tyrosine-kinase-dependent pathway and stimulates tyrosine phosphorylation of platelet proteins, including PLC gamma 2, independently of integrin alpha IIb beta 3. 960 58

Congenital defects in platelet function are associated with bleeding manifestations of variable intensity and arise by diverse mechanisms. Defects in platelet-vessel wall interaction (disorders of adhesion) may arise because of qualitative or quantitative abnormalities in plasma von Willebrand factor (von Willebrand disease) or in platelet glycoprotein Ib, the binding site on platelets for von Willebrand factor (Bernard-Soulier syndrome). Disorders characterized by abnormal platelet-platelet interaction (disorders of aggregation) arise because of absence of plasma fibrinogen (congenital afibrinogenemia) or because of qualitative or quantitative abnormalities in platelet glycoprotein IIb-IIIa complex (Glanzmann's thrombasthenia). Patients with abnormalities in platelet secretion and signal transduction are a heterogeneous group characterized by impaired aggregation responses and secretion of granule contents. A small proportion of these patients have deficiency of granule stores (storage pool deficiency [SPD]) or impaired production of thromboxane A2; in most, the mechanisms underlying the platelet dysfunction are unknown. Evidence is accumulating that at least some patients have abnormalities in early signal transduction events. Abnormalities in phospholipase C activation, G-protein activation, and other events (eg, protein phosphorylation) have been documented. Platelets play a major role in blood coagulation, and in Scott syndrome, there is an abnormality in platelet contribution to the mechanisms leading to thrombin generation. In most patients with inherited platelet dysfunction, the underlying mechanisms remain to be delineated. Future studies of these patients should yield valuable new information on normal platelet mechanisms.
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PMID:Congenital disorders of platelet function: disorders of signal transduction and secretion. 970 60

Activation of phospholipase C (PLC) is a central component of the signal transduction process in numerous cells, including platelets. U73122 has been widely used as a selective PLC inhibitor. In the present study, the effects of U73122 on platelet function have been further examined. Platelets were stimulated with collagen (via PLC-gamma), the stable thromboxane mimetic U46619 (via PLC-beta), or phorbol myristate acetate (PMA) via protein kinase C (PKC). Consistent with inhibition of PLC, U73122 inhibited platelet aggregation and [3H]-serotonin release in response to collagen and U46619 in a concentration-dependent manner. Similarly, U73122 blocked collagen-induced release of thromboxane A2. U73122 also inhibited U46619-induced [32P]phosphatidic acid production and phosphorylation of the major PKC substrate, pleckstrin. U73122 had no effect on PMA-induced pleckstrin phosphorylation, [3H]-serotonin release, or intracellular vacuole formation. However, U73122 did inhibit PMA-induced platelet aggregation and fibrinogen binding. Overall, these results suggest that U73122, in addition to its inhibition of PLC, also affects PKC-independent events that interfere with platelet aggregation.
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PMID:The phospholipase C inhibitor U73122 inhibits phorbol ester-induced platelet activation. 1021 45

Tyrosine phosphorylation of the non-receptor tyrosine kinases pp72syk and pp125FAK and of the gamma2 isoform of phospholipase C (PLCgamma2) in human platelets stimulated with the lectin Concanavalin A was investigated. Concanavalin A induced the rapid tyrosine phosphorylation of pp72syk and PLCgamma2 with a similar kinetics, while tyrosine phosphorylation of pp125FAK occurred in a later phase of platelet activation. When compared with other platelet agonists, Concanavalin A revealed to be at least as potent as collagen in inducing tyrosine phosphorylation of PLCgamma2 and pp125FAK, while tyrosine phosphorylation of pp72syk induced by the lectin was much stronger than that induced by thrombin or collagen. Concanavalin A-induced tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK was not dependent on platelet aggregation as it occurred normally even in the absence of sample stirring and when fibrinogen binding to integrin alphaIIb-beta3 was inhibited by the peptide RGDS. Tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK required the binding of the lectin to the platelet surface, but was not observed in platelets treated with succinyl-Concanavalin A, a derivative of the lectin that interacts with the same receptors but does not promote clustering of membrane glycoproteins. Moreover, the aggregation-independent tyrosine phosphorylation of pp125FAK and pp72syk induced by Concanavalin A required the expression of integrin alphaIIb-beta3 on the platelet surface as it was strongly inhibited in platelets from patients affected by Glanzmann thrombasthenia. By contrast, tyrosine phosphorylation of PLCalpha2 occurred normally also in thrombasthenic platelets stimulated with Concanavalin A. These results demonstrate that, even in the absence of aggregation, the clustering of integrin alphaIIb-beta3 induced by Concanavalin A on the platelet surface directly promotes tyrosine phosphorylation of pp72syk and pp125FAK and provide further evidence that the oligomerization of the fibrinogen receptor promoted by its natural ligand during platelet aggregation may be responsible for the tyrosine phosphorylation of these proteins induced by physiological agonists.
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PMID:Clustering of integrin alphaIIb-beta3 differently regulates tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK in concanavalin A-stimulated platelets. 1034 3

It has recently been suggested that the concomitant activation of two distinct G protein-coupled receptors (G(i) and G(q)) is essential for platelet aggregation: in fact, the thromboxane A2 synthetic agonist, U46619, which causes the selective activation of Gq, is not able to elicit fibrinogen receptor exposure unless ADP or epinephrine is present. In the present study we demonstrate that a direct Gq activation is not required for platelet aggregation and that the activation of an enzyme downstream of Gq, such as phospholipase C (PLC) or protein-kinase C (PKC), is sufficient for such a process. In fact, platelet aggregation occurred in response to the snake venom toxin convulxin, which activates the PLC isoform PLCgamma2 or to cytosolic PKC activator phorbol 12-myristate 13-acetate (PMA) provided a Gi protein-coupled receptor was activated by ADP or epinephrine. The evidence that the PKC inhibitor, Ro 31-8220 did not suppress platelet aggregation in response to convulxin plus ADP or epinephrine led us to conclude that PLC and PKC are both involved in platelet aggregation, although not concomitantly, provided a Gi protein-coupled receptor is activated.
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PMID:Concomitant activation of Gi protein-coupled receptor and protein kinase C or phospholipase C is required for platelet aggregation. 1057 Oct 57


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