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)

Triton-insoluble cytoskeletons prepared from thrombin-activated platelets were found to potentiate the activation of prothrombin (prothrombinase activity). Cytoskeletons prepared from red cells or lymphoblasts contained no prothrombinase activity. The platelet prothrombinase activity was dependent on cytoskeletal-associated Factor Va, and exogenously added Factor Xa and prothrombin. Cytoskeletons contained 38% of the total platelet prothrombinase activity. Both platelets and cytoskeletons displayed half-maximal activities at similar prothrombin concentrations. The role of lipids in the cytoskeletal prothrombinase activity was investigated. Cytoskeletons were found to contain 3.8% of the total platelet phospholipids, consisting of the following lipids expressed as percentage of total present in platelets: 6.0% sphingomyelin, 3.8% phosphatidylcholine, 2.9% phosphatidyl-ethanolamine, 4.4% phosphatidylinositol, and 2.2% phosphatidylserine. The cytoskeletal prothrombinase activity and the lipid phosphorus content of cytoskeletons decreased after treatment of cytoskeletons with various doses of phospholipase C. Incubation of cytoskeletons with the highest concentrations tested (10 micrograms/ml) resulted in a 72% loss of phosphatidylserine and 84% loss of cytoskeletal prothrombinase activity. Cytoskeletal prothrombinase activity destroyed by phospholipase C treatment could be restored to control levels by treatment of hydrolyzed cytoskeletons with total cytoskeletal lipid or mixtures of phosphatidylserine/phosphatidylcholine (25:75% by weight). These results suggest that the cytoskeletal prothrombinase complex in addition to containing Factor Va, as has been previously shown (15), contains a lipid cofactor activity consisting in part of phosphatidylserine.
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PMID:The platelet cytoskeleton contains elements of the prothrombinase complex. 653 31

The hypercoagulable state in pregnancy is partly caused by the increased activity of factor VII in plasma. We demonstrate here that this activity is reduced to levels similar to those in plasma from non-pregnant women by highly purified phospholipase C from Bacillus cereus, i.e. the activity increase is due to a circulating complex of factor VII and a phospholipase C-sensitive compound. Phospholipase C had no effect on the levels of factor II and X in blood from pregnant women. This novel form of activated factor VII is not inhibited by an antiserum to the protein component of thromboplastin (apoprotein III). By gel filtration of plasma from pregnant women on Sephadex G-100 the phospholipase C-sensitive complex was partly separated from non-phospholipase sensitive factor VII also present in the same plasma.
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PMID:The coagulation factor VII in pregnancy. 660 66

Alveolar lavage cells from normal sheep were found to be composed of over 95% macrophages. When the cells were cultured, fibrinolytic and thromboplastin-like activities could be detected within 2-4 hours of incubation. As the number of cultured cells was increased the two activities in the conditioned medium increased proportionately. The cells were separated into two distinct subpopulations by means of a sedimentation velocity cell fractionation technique. The macrophage subpopulations were examined for differences in size, morphology, esterase staining and ability to release plasminogen activator and procoagulant activity respectively. These activities were confined to the large cell subpopulation. The fibrinolytic activity was shown to be plasminogen-dependent and could be inhibited by DFP. On the basis of this the fibrinolytic activity has been designated as plasminogen activator. The procoagulant activity was shown to be thromboplastin in nature because it was Factor VII dependent, inactivated by phospholipase C and not inhibited by DFP. The procoagulant activity has been designated as macrophage thromboplastin. The two activities could be distinguished on the basis of DFP inhibition.
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PMID:Plasminogen activator and thromboplastin activity from sheep alveolar macrophages. 668 4

Purified phospholipase C from Bacillus cereus caused a significant loss in the procoagulant activity of thromboplastin preparations from man, rabbit, sheep, cow, rat and mouse. However, marked differences were observed with respect to the degree of inactivation. Rat, mouse, bovine and one type of rabbit preparations (prepared from acetone powdered brain) were markedly more sensitive to attack by phospholipase C than were preparations of human, sheep and standard rabbit preparations. The relative amounts of the individual phospholipids in thromboplastin preparations showed only minor variations among the species. The effect of phospholipase C on each of these phospholipids in the various thromboplastin preparations showed some significant differences.
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PMID:In vitro effect of phospholipase C from Bacillus cereus on tissue thromboplastin from different species. 681 11

Tissue thromboplastin is the most potent physiological trigger of blood coagulation and is probably involved in the pathogenesis of several forms of intravascular coagulation. Phospholipase C from Bacillus cereus is an effective inhibitor of thromboplastin. As part of an investigation into the possible use of phospholipase C as a therapeutic or prophylactic agent in thrombosis and other forms of intravascular coagulation, we have previously studied its effect in rats. We now report on the toxicity in rabbits (along with some data on cats and monkeys). Estimated LD50 for rabbits was about 0.45-0.65 mg/kg (as compared to 1.70 mg/kg for rats). No respiratory or circulatory changes were observed, but phospholipase C caused a significant increase of several plasma enzymes in rabbits.
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PMID:Toxicity of phospholipase C in rabbits. 713 8

Discordant xenogeneic organ transplantation is a potential solution to the critical shortage of suitable donor organs. However, clinical application of xenotransplantation with physiologically suitable organs such as those from the pig, is currently limited by the lack of agents to prevent antibody and complement-mediated hyperacute rejection of the transplanted organ. We have used retrovirus-mediated gene transfer to express the terminal complement inhibitor protein, human CD59, in neonatal porcine aortic endothelial cells (nPAEC). Human CD59 was constitutively expressed in nPAECs at levels similar to that of native CD59 in human umbilical vein endothelial cells. The protein was tethered to the cell surface by a glycosyl-phosphatidylinositol anchor, as demonstrated by its removal following treatment with phosphatidylinositol-specific phospholipase C. In a model of antibody-dependent complement activation, nPAECs expressing human CD59 were protected from membrane pore formation and cell lysis by complement derived from either human or baboon sera. Conversely, nPAECs expressing CD59 were not protected from lysis by rabbit or dog complement, indicating that recombinant CD59 retained its species-restricted inhibitory activity. Additionally, CD59 expressed on nPAECs inhibited the C5b-9-dependent generation of membrane prothrombinase activity. Collectively, these data establish that stable expression of human CD59 on xenotypic (porcine) endothelial cells renders these cells resistant to both the cytolytic and procoagulant effects of human complement. We propose that expression of recombinant human CD59 on porcine donor organs may prevent complement-mediated lysis and activation of endothelial cells that leads to hyperacute rejection.
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PMID:Protection of porcine aortic endothelial cells from complement-mediated cell lysis and activation by recombinant human CD59. 751

The membrane-associated CD14 receptor (mCD14) is a monocyte/macrophage differentiation antigen, and it has been demonstrated to serve as a receptor for bacterial lipopolysaccharide (LPS; endotoxin). Binding of LPS to mCD14 has been shown to be associated with LPS-induced macrophage, monocyte, and neutrophil activation in humans. In this report, we describe the presence and function of an mCD14-like receptor on bovine alveolar macrophages (bAM). An immunofluorescence technique and flow cytometric analysis indicated binding of anti-human CD14 monoclonal antibodies (MAb) My4, 3C10, and 60bd to bAM. Binding of anti-CD14 MAb (3C10 and MY4) was reduced over 20% by pretreatment of bAM with phosphatidylinositol-specific phospholipase C (0.5 to 1.0 U/ml), indicating that bovine mCD14 is a glycosyl phosphatidylinositol-anchored protein. In addition, pretreatment of bAM with anti-CD14 MAb decreased binding of 125I-labeled LPS to macrophages, suggesting that bovine mCD14 serves as a receptor for LPS. A cDNA probe based on the human sequence for CD14 was used in Northern (RNA) blot analysis, and hybridization to human monocyte CD14 yielded the expected 1.5-kb band. Hybridization to bovine mRNA yielded a 1.5-kb band plus an unexpected 3.1-kb band. Constitutive expression of bovine CD14 mRNA was observed, and the expression level was modestly elevated in bAM stimulated for 24 h with LPS (1 ng/ml) in the presence of bovine serum. The function and activation of bAM were assessed by quantitation of tissue factor (TF) expression on the cells using an activated factor X-related chromogenic assay and S-2222 substrate. LPS (1 ng/ml)-mediated upregulation of TF expression on bAM was dependent on the presence of bovine serum components, and TF expression was inhibited by anti-CD14 MAb. In addition, TF mRNA levels in LPS-stimulated bAM were decreased by pretreatment of cells with anti-CD14 MAb (MAb 60bd, 10 micrograms/ml).
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PMID:CD14 and tissue factor expression by bacterial lipopolysaccharide-stimulated bovine alveolar macrophages in vitro. 752 35

The mitogenic effect of activated coagulation factor X (factor Xa) was examined in cultured aortic smooth muscle cells (VSMC) from Wistar-Kyoto rats (WKY). Factor Xa stimulated DNA synthesis and cell growth in VSMC, not through the phospholipase C-protein kinase C pathway because increase of inositol monophosphate (IP) accumulation and intracellular Ca2+ concentration was not observed, but probably via the PDGF receptor tyrosine kinase pathway since the pathway's components, Ras, Raf-1, MAPK (both 42 and 44 kD), and the transcription factors, c-Fos and c-Jun, were activated. These appeared to be effected by the serine protease activity of factor Xa, since in the presence of serine protease inhibitors such as PMSF, leupeptin, benzamidine, TAP anticoagulant, and TLCK, the latter three being specific inhibitors of the factor Xa, active site, the effects were completely blocked. Anti-factor Xa mAb, 5224, which specifically negated the activity of factor Xa, also inhibited completely the mitogenic effect of factor Xa, but not that of thrombin. Addition of PDGF did not affect the effect of factor Xa, which, however, was inhibited by anti-PDGF-AB antibody. This observation and the activation of PDGF receptor tyrosine kinase pathway suggested that the factor Xa might exert its effect via PDGF-like function. Direct measurement confirmed that factor Xa stimulated the release of PDGF from VSMC. Factor Xa, therefore, exerts serine protease activity on VSMC, causing somehow the release of PDGF, that in turn acts on the PDGF receptor tyrosine kinase; the pathway is then turned on, leading eventually to DNA synthesis and cell proliferation.
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PMID:Coagulation factor Xa stimulates platelet-derived growth factor release and mitogenesis in cultured vascular smooth muscle cells of rat. 882 16

A series of site-specific mutants of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLCBc) was prepared in which the glutamic acid residue at position 146 was replaced with glutamine, aspartic acid, histidine, and leucine to elucidate what role Glu146 might play in catalysis. An expression system for the native enzyme in Escherichia coli was first developed to provide PLCBc that was fused via an intervening factor Xa protease recognition sequence at its N-terminus to maltose binding protein (MBP). This MBP-PLCBc fusion protein was isolated at levels of 50-70 mg/L of culture; selective trypsin digestion of the MBP-PLCBc fusion protein followed by chromatographic purification yielded recombinant PLCBc at levels of ca. 10 mg/L. Polymerase chain reaction (PCR) mutagenesis on the PLCBc gene (plc) was then used to replace the Glu146 codon with those for glutamine (E146Q), aspartic acid (E146D), histidine (E146H), and leucine (E146L). The catalytic efficiency of the E146Q mutant was 1.6% that of native PLCBc, while the other mutants each possessed activities of 0.2-0.3% of the wild type. The kcat/Km vs pH profiles for both E146Q and native PLCBc have ascending acidic limbs, suggesting that Glu146 does not serve as the general base in the hydrolysis reaction. As measured by circular dichroism, all of the mutant proteins contained less helical structure and underwent denaturation at lower temperatures than the wild type in the order: wild type > E146Q > E146D approximately E146H approximately E146L. Atomic absorption analyses indicated that the mutant proteins also exhibited lower Zn2+ content than the wild type. Thus, the Glu146 residue in PLCBc stabilizes the secondary and tertiary structure of the enzyme and serves as a critical ligand for Zn2, but it does not appear to have any specific catalytic role.
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PMID:Expression and site-directed mutagenesis of the phosphatidylcholine-preferring phospholipase C of Bacillus cereus: probing the role of the active site Glu146. 884 Nov 44

Intraglomerular activation of the coagulation cascade is a common feature of mesangioproliferative glomerulonephritis. Besides thrombin, very little is known about the cellular effects of other components of the coagulation system. This study investigated the effect of activated factor X (FXa) on cultured human mesangial cells. This serine protease induced a significant and dose-dependent increase in DNA synthesis. In addition to its mitogenic effect, FXa caused a striking upregulation of platelet-derived growth factor (PDGF) A and B chain gene expression. Next, the intracellular mitogenic signaling pathways activated by FXa were investigated. FXa induced a rapid spike in cytosolic calcium concentration followed by a sustained plateau. This response was not influenced by the downregulation of thrombin receptors. In addition, FXa stimulated a significant upregulation of different tyrosine-phosphorylated proteins. One of these phosphorylated cellular proteins was represented by the c-jun N-terminal kinase, a member of the mitogen-activated protein kinase family. To evaluate the role of FXa enzymatic activity and of PDGF autocrine secretion, FXa-induced DNA synthesis was studied in the presence of leupeptin, a specific serine protease inhibitor, and neutralizing anti-PDGF antibody. To investigate the role of tyrosine kinase (TK) activation on FXa mitogenic effect, FXa-stimulated thymidine uptake was evaluated in the presence of genistein and herbimycin A, two powerful and specific TK inhibitors. FXa-elicited DNA synthesis was also examined after protein kinase C (PKC) downregulation by prolonged incubation with phorbol-12-myristate-13-acetate to study the influence of the phospholipase C-PKC axis. The proliferative effect of FXa required its proteolytic activity, and the activation of TK was only partially dependent on PKC activation while it was PDGF independent. Finally, it was shown by reverse transcription-PCR that mesangial cells do not express the signaling splicing variant of the putative FXa receptor, effector protease receptor-1. In conclusion, the present study demonstrated that FXa is a powerful mitogenic factor for human mesangial cells, and it induces its cellular effect not through effector protease receptor-1, but most likely by binding a protease-activated receptor and activating phospholipase C-PKC and TK signaling pathways.
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PMID:Activated coagulation factor X: a novel mitogenic stimulus for human mesangial cells. 1131 47


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