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1.
J Immunol Res ; 2019: 5836476, 2019.
Article in English | MEDLINE | ID: mdl-31355297

ABSTRACT

BACKGROUND: Mastocytosis is a condition characterized by the expansion and accumulation of mast cells (MCs) in various organs. The symptoms are related to the increased release of MC-derived mediators that exert local and distant effects. MCs are a source and target of phospholipase enzymes (PLs), which catalyze the cleavage of membrane phospholipids releasing lipid mediators (e.g., diacylglycerols (DAGs) and the endocannabinoid (EC) 2-arachidonoylglycerol (2-AG)). To date, there are no data on the role of these lipid mediators in mastocytosis. Here, we analyzed plasma levels of PLA2, PLC, DAG, ECs, and EC-related N-acylethanolamines in patients with mastocytosis. METHODS: In 23 patients with mastocytosis and 23 healthy individuals, we measured plasma PLA2 and PLC activities, DAG, 2-AG, anandamide (AEA), palmitoylethanolamide (PEA), and oleoylethanolamide (OEA). RESULTS: Plasma PLA2 and PLC activities were increased in mastocytosis patients compared to controls. Concentrations of DAG (18:1 20:4 and 18:0 20:4), two second messengers produced by PLC, were higher in mastocytosis compared to controls, whereas the concentrations of their metabolite, 2-AG, were not altered. AEA was decreased in mastocytosis patients compared to controls; by contrast, AEA congener, PEA, was increased. PLA2 and PLC activities were increased only in patients with mediator-related symptoms. Moreover, PLC activity was positively correlated with disease severity and tryptase concentrations. By contrast, AEA was negatively correlated with tryptase concentrations. CONCLUSIONS: PLs and some lipid mediators are altered in patients with mastocytosis. Our results may pave the way for investigating the functions of these mediators in the pathophysiology of mastocytosis and provide new potential biomarkers and therapeutic targets.


Subject(s)
Diglycerides/metabolism , Endocannabinoids/blood , Ethanolamines/blood , Mastocytosis/metabolism , Phospholipases A2/metabolism , Type C Phospholipases/metabolism , Adult , Aged , Arachidonic Acids/blood , Biomarkers/blood , Diglycerides/blood , Female , Humans , Male , Mastocytosis/blood , Mastocytosis/enzymology , Mastocytosis/pathology , Middle Aged , Phospholipases A2/blood , Polyunsaturated Alkamides/blood , Tryptases/blood , Type C Phospholipases/blood
2.
Clin Pharmacol Drug Dev ; 8(5): 628-636, 2019 07.
Article in English | MEDLINE | ID: mdl-30240132

ABSTRACT

CSL112 (Apolipoprotein A-I [human]) is an intravenous preparation of apolipoprotein A-I (apoA-I), formulated with phosphatidylcholine (PC) and stabilized with sucrose, in development to prevent early recurrent cardiovascular events following acute myocardial infarction (AMI). This phase 1 study was designed to determine if moderate renal impairment (RI) influenced the pharmacokinetics (PK) and safety of CSL112. Thirty-two subjects, 16 with moderate RI (estimated glomerular filtration rate [eGFR] ≥ 30 and < 60 mL/min/1.73 m2 ) and 16 age-, sex-, and weight-matched subjects with normal renal function (eGFR ≥ 90 mL/min/1.73 m2 ) were randomized 3:1 to receive a single infusion of CSL112 2 g (n = 6) or placebo (n = 2), or CSL112 6 g (n = 6) or placebo (n = 2). PK sampling was at prespecified times from 48 hours prior to 144 hours following infusions, with final safety assessments at 90 days. Renal and hepatic safety, and adverse events (AEs) were monitored throughout the study. Plasma apoA-I and PC PK profiles were similar between renal function cohorts at both doses. For CSL112 6 g mean ± SD apoA-I AUC0-last was 7670 ± 1900 and 9170 ± 2910 mg·h/dL in normal renal function and moderate RI subjects, respectively. Renal apoA-I clearance was <1% of CSL112 dose. In moderate RI, sucrose clearance was slower; however, approximately 70% was excreted within 48 hours in both renal function cohorts. No CSL112-related serious AEs or clinically significant renal or hepatic safety changes were observed. Dose adjustment of CSL112 is not required in subjects with moderate RI, supporting its further investigation in AMI patients with moderate RI.


Subject(s)
Lipoproteins, HDL/pharmacokinetics , Renal Insufficiency/metabolism , Adult , Aged , Apolipoprotein A-I/blood , Apolipoprotein A-I/urine , Double-Blind Method , Female , Glomerular Filtration Rate , Humans , Kidney/physiology , Lipoproteins, HDL/adverse effects , Lipoproteins, HDL/pharmacology , Male , Middle Aged , Renal Insufficiency/blood , Renal Insufficiency/physiopathology , Renal Insufficiency/urine , Sucrose/blood , Sucrose/urine , Type C Phospholipases/blood
3.
Redox Biol ; 11: 663-672, 2017 04.
Article in English | MEDLINE | ID: mdl-28160743

ABSTRACT

Activated platelets generate an eicosanoid proposed to be 8-hydroxy-9,10-dioxolane A3 (DXA3). Herein, we demonstrate that significant amounts of DXA3 are rapidly attached to phosphatidylethanolamine (PE) forming four esterified eicosanoids, 16:0p, 18:0p, 18:1p and 18:0a/DXA3-PEs that can activate neutrophil integrin expression. These lipids comprise the majority of DXA3 generated by platelets, are formed in ng amounts (24.3±6.1ng/2×108) and remain membrane bound. Pharmacological studies revealed DXA3-PE formation involves cyclooxygenase-1 (COX), protease-activated receptors (PAR) 1 and 4, cytosolic phospholipase A2 (cPLA2), phospholipase C and intracellular calcium. They are generated primarily via esterification of newly formed DXA3, but can also be formed in vitro via co-oxidation of PE during COX-1 co-oxidation of arachidonate. All four DXA3-PEs were detected in human clots. Purified platelet DXA3-PE activated neutrophil Mac-1 expression, independently of its hydrolysis to the free eicosanoid. This study demonstrates the structures and cellular synthetic pathway for a family of leukocyte-activating platelet phospholipids generated on acute activation, adding to the growing evidence that enzymatic PE oxidation is a physiological event in innate immune cells.


Subject(s)
Blood Platelets/metabolism , Dioxolanes/blood , Integrins/blood , Lipids/blood , Phosphatidylethanolamines/blood , Calcium/blood , Cyclooxygenase 1/blood , Eicosanoids/blood , Gene Expression Regulation , Humans , Integrins/biosynthesis , Macrophage-1 Antigen/genetics , Neutrophils/metabolism , Oxidation-Reduction , Phospholipases A2, Cytosolic/blood , Platelet Activation/genetics , Receptor, PAR-1/blood , Receptors, Thrombin/blood , Thrombin/metabolism , Type C Phospholipases/blood
4.
Cell Death Dis ; 4: e806, 2013 Sep 19.
Article in English | MEDLINE | ID: mdl-24052074

ABSTRACT

Phosphatidylcholine-specific phospholipase C (PC-PLC) is a key factor in apoptosis and autophagy of vascular endothelial cells (VECs), and involved in atherosclerosis in apolipoprotein E⁻/⁻ (apoE⁻/⁻) mice. But the endogenous regulators of PC-PLC are not known. We recently found a small chemical molecule (6-amino-2, 3-dihydro-3-hydroxymethyl-1, 4-benzoxazine, ABO) that could inhibit oxidized low-density lipoprotein (oxLDL)-induced apoptosis and promote autophagy in VECs, and further identified ABO as an inhibitor of annexin A7 (ANXA7) GTPase. Based on these findings, we hypothesize that ANXA7 is an endogenous regulator of PC-PLC, and targeting ANXA7 by ABO may inhibit atherosclerosis in apoE⁻/⁻ mice. In this study, we tested our hypothesis. The results showed that ABO suppressed oxLDL-induced increase of PC-PLC level and activity and promoted the co-localization of ANXA7 and PC-PLC in VECs. The experiments of ANXA7 knockdown and overexpression demonstrated that the action of ABO was ANXA7-dependent in cultured VECs. To investigate the relation of ANXA7 with PC-PLC in atherosclerosis, apoE⁻/⁻ mice fed with a western diet were treated with 50 or 100 mg/kg/day ABO. The results showed that ABO decreased PC-PLC levels in the mouse aortic endothelium and PC-PLC activity in serum, and enhanced the protein levels of ANXA7 in the mouse aortic endothelium. Furthermore, both dosages of ABO significantly enhanced autophagy and reduced apoptosis in the mouse aortic endothelium. As a result, ABO significantly reduced atherosclerotic plaque area and effectively preserved a stable plaques phenotype, including reduced lipid deposition and pro-inflammatory macrophages, increased anti-inflammatory macrophages, collagen content and smooth muscle cells, and less cell death in the plaques. In conclusion, ANXA7 was an endogenous regulator of PC-PLC, and targeting ANXA7 by ABO inhibited atherosclerosis in apoE⁻/⁻ mice.


Subject(s)
Annexin A7/antagonists & inhibitors , Apolipoproteins E/deficiency , Atherosclerosis/enzymology , Benzoxazines/pharmacology , Human Umbilical Vein Endothelial Cells/enzymology , Small Molecule Libraries/pharmacology , Type C Phospholipases/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/metabolism , Animals , Annexin A7/metabolism , Aorta/enzymology , Aorta/pathology , Apolipoproteins E/metabolism , Atherosclerosis/blood , Atherosclerosis/pathology , Atherosclerosis/prevention & control , Autophagy/drug effects , Body Weight/drug effects , Cells, Cultured , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Endothelium, Vascular/ultrastructure , Heat-Shock Proteins/metabolism , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/pathology , Humans , Interleukin-6/metabolism , Interleukin-8/metabolism , Lipids/blood , Lipoproteins, LDL/pharmacology , Mice , Phagosomes/drug effects , Phagosomes/metabolism , Phagosomes/ultrastructure , Phenotype , Sequestosome-1 Protein , Type C Phospholipases/blood , Type C Phospholipases/metabolism
5.
Br J Nutr ; 110(5): 840-7, 2013 Sep 14.
Article in English | MEDLINE | ID: mdl-23566550

ABSTRACT

The Clostridium-related poultry disease, necrotic enteritis (NE), causes substantial economic losses on a global scale. In the present study, a mixture of two plant-derived phytonutrients, Capsicum oleoresin and turmeric oleoresin (XT), was evaluated for its effects on local and systemic immune responses using a co-infection model of experimental NE in commercial broilers. Chickens were fed from hatch with a diet supplemented with XT, or with a non-supplemented control diet, and either uninfected or orally challenged with virulent Eimeria maxima oocysts at 14 d and Clostridium perfringens at 18 d of age. Parameters of protective immunity were as follows: (1) body weight; (2) gut lesions; (3) serum levels of C. perfringens α-toxin and NE B-like (NetB) toxin; (4) serum levels of antibodies to α-toxin and NetB toxin; (5) levels of gene transcripts encoding pro-inflammatory cytokines and chemokines in the intestine and spleen. Infected chickens fed the XT-supplemented diet had increased body weight and reduced gut lesion scores compared with infected birds given the non-supplemented diet. The XT-fed group also displayed decreased serum α-toxin levels and reduced intestinal IL-8, lipopolysaccharide-induced TNF-α factor (LITAF), IL-17A and IL-17F mRNA levels, while cytokine/chemokine levels in splenocytes increased in the XT-fed group, compared with the animals fed the control diet. In conclusion, the present study documents the molecular and cellular immune changes following dietary supplementation with extracts of Capsicum and turmeric that may be relevant to protective immunity against avian NE.


Subject(s)
Capsicum/chemistry , Curcuma/chemistry , Dietary Supplements , Enteritis/veterinary , Plant Extracts/administration & dosage , Poultry Diseases/prevention & control , Animal Feed/analysis , Animals , Antibodies, Bacterial/blood , Bacterial Toxins/blood , Bacterial Toxins/immunology , Calcium-Binding Proteins/blood , Calcium-Binding Proteins/immunology , Clostridium Infections/immunology , Clostridium Infections/prevention & control , Clostridium Infections/veterinary , Clostridium perfringens/immunology , Clostridium perfringens/pathogenicity , Coccidiosis/immunology , Coccidiosis/prevention & control , Coccidiosis/veterinary , Coinfection/prevention & control , Coinfection/veterinary , Cytokines/metabolism , Diet/veterinary , Eimeria/pathogenicity , Enteritis/microbiology , Enteritis/parasitology , Enteritis/prevention & control , Necrosis/veterinary , Plant Extracts/chemistry , Plant Extracts/therapeutic use , Poultry Diseases/immunology , Poultry Diseases/microbiology , Poultry Diseases/parasitology , Type C Phospholipases/blood , Type C Phospholipases/immunology
6.
Nanomedicine ; 8(8): 1329-36, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22542822

ABSTRACT

Nanomaterials are attracting growing interest for their potential use in several applications as nanomedicine; therefore, the analysis of their potential toxic effects on various cellular models, including circulating blood cells, is mandatory. This study aimed to investigate the effect of three unrelated nanomaterials, namely nanoscale silica, multiwalled carbon nanotubes, and carbon black, on platelet activation and aggregation. We found that these nanomaterials stimulate some of the typical biochemical pathways involved in canonical platelet activation, such as the stimulation of phospholipase C and Rap1b, resulting in the integrin α(IIb)ß3-mediated platelet aggregation, through a mechanism largely dependent on the release of the extracellular second messengers ADP and thromboxane A2. Importantly, we found that doses of nanoparticles unable to trigger appreciable responses can synergize with subthreshold amounts of physiological agonists to mediate platelet aggregation, indicating that even small amounts of nanomaterials in the bloodstream might contribute to the development of thrombosis. FROM THE CLINICAL EDITOR: In this study, nanosized particles of three virtually unrelated materials (silica, multi-walled carbon nanotubes and carbon black) were investigated regarding their effects on platelet activation and aggregation. All were found to stimulate some of the typical biochemical pathways involved in canonical platelet activation, and were found to have synergistic effects with physiologic platelet activator agonists.


Subject(s)
Nanoparticles , Nanotubes, Carbon , Platelet Activation/drug effects , Soot , Blood Proteins/metabolism , Humans , In Vitro Techniques , Integrin alpha2/blood , Nanoparticles/toxicity , Nanotubes, Carbon/adverse effects , Phosphoproteins/metabolism , Platelet Aggregation/drug effects , Signal Transduction/drug effects , Silicon Dioxide/pharmacology , Soot/adverse effects , Soot/pharmacology , Type C Phospholipases/blood , rap GTP-Binding Proteins/blood
7.
J Cell Physiol ; 226(6): 1694-701, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21413027

ABSTRACT

Secretion of proinflammatory cytokines by lipopolysaccharide (LPS) activated vascular endothelial cells (VECs) contributes substantially to the pathogenesis of several inflammatory diseases such as atherosclerosis and septic shock. However, the mechanisms involved in this process are not well understood. Here, we investigated the role of phosphatidylcholine-specific phospholipase C (PC-PLC) in LPS-induced IL-8 and MCP-1 production in VECs. The results showed that LPS elevated the level of PC-PLC and the production of IL-8 and MCP-1 in Human umbilical vein vascular endothelial cells (HUVECs). Blocking the function of PC-PLC by exploiting the neutralization antibody of PC-PLC or tricyclodecan-9-yl-xanthogenate (D609), an inhibitor of PC-PLC, significantly inhibited LPS-induced production of IL-8 and MCP-1 in HUVECs. Furthermore, the in vivo experimental results showed that the levels of PC-PLC, IL-8, and MCP-1 in the aortic endothelium and serum were increased in mice injected with LPS. The increased levels of these molecules were also inhibited by the treatment with D609. The data suggested that blocking PC-PLC function significantly inhibited LPS-induced IL-8 and MCP-1 production in cultured HUVECs and in vivo. PC-PLC might be a potential target for therapy in inflammation associated-diseases such as atherosclerosis.


Subject(s)
Chemokine CCL2/biosynthesis , Endothelial Cells/drug effects , Endothelial Cells/enzymology , Interleukin-8/biosynthesis , Lipopolysaccharides/pharmacology , Type C Phospholipases/metabolism , Animals , Aorta/cytology , Bridged-Ring Compounds/pharmacology , Cytoplasm/drug effects , Cytoplasm/enzymology , Endothelial Cells/cytology , Enzyme Activation/drug effects , Enzyme-Linked Immunosorbent Assay , Humans , Male , Mice , Mice, Inbred C57BL , Norbornanes , Protein Transport/drug effects , Thiocarbamates , Thiones/pharmacology , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/blood , Umbilical Veins/cytology
8.
Cephalalgia ; 31(4): 456-62, 2011 Mar.
Article in English | MEDLINE | ID: mdl-20937607

ABSTRACT

BACKGROUND: Adrenaline, serotonin, cannabinoid and estrogen receptors are involved in migraine pathophysiology. The signaling of these receptors change phosphatidylcholine-specific phospholipase C (PC-PLC) activity, but there have been no reported PC-PLC studies in migraine. METHODS: We identified PC-PLC activity in blood and cerebrospinal fluid (CSF), and quantified it in samples from ictal and interictal migraineurs without aura and healthy controls. RESULTS: Pre-incubation with a specific PC-PLC inhibitor, D609, inhibited enzyme activity (p < .0001) and confirms its presence in CSF. PC-PLC activity was higher in the CSF from ictal migraineurs compared to controls (mean relative fluorescence unit [RFU]/µg/min [standard deviation, SD] 13.1 [3.07] vs. 9.3 [1.97]; p = .002) and, in a paired analysis, in migraineurs during ictal compared to interictal states (11.7 [1.6] vs. 7.9 [1.5]; p = .02). CSF PC-PLC activity in the ictal state correlated negatively with migraine frequency (r = -0.82). Plasma PC-PLC activity was 250-300 times less than in CSF and did not increase in migraine, implicating the brain as the source of the CSF enzyme changes. CONCLUSION: This is the first report of PC-PLC activity in CSF and of its alteration in migraine. We propose that these PC-PLC changes in CSF reflect the overall receptor fluctuations in migraine.


Subject(s)
Migraine Disorders/cerebrospinal fluid , Migraine Disorders/enzymology , Type C Phospholipases/cerebrospinal fluid , Adult , Aged , Aged, 80 and over , Biomarkers/blood , Biomarkers/cerebrospinal fluid , Bridged-Ring Compounds/pharmacology , Enzyme Activation/drug effects , Female , Humans , Male , Middle Aged , Migraine Disorders/blood , Norbornanes , Phosphatidylcholines/cerebrospinal fluid , Thiocarbamates , Thiones/pharmacology , Type C Phospholipases/blood
9.
Blood ; 107(7): 2728-35, 2006 Apr 01.
Article in English | MEDLINE | ID: mdl-16357324

ABSTRACT

The involvement of the small GTPase Rap1b in platelet integrin alpha2beta1-dependent outside-in signaling was investigated. Platelet adhesion to 4 different specific ligands for integrin alpha2beta1, monomeric collagen, decorin, and collagen-derived peptides CB8(II) and CB11(II), induced a robust and rapid activation of Rap1b. This process did not require secreted ADP or thromboxane A2 production but was critically regulated by phospholipase C (PLC)-derived second messengers. Both Ca2+ and protein kinase C were found to organize independent but additive pathways for Rap1b activation downstream of integrin-alpha2beta1, which were completely blocked by inhibition of PLC with U73122. Moreover, integrin alpha2beta1 engagement failed to trigger Rap1b activation in murine platelets lacking CalDAG-GEFI, a guanine nucleotide exchange factor regulated by Ca2+ and diacylglycerol, despite normal phosphorylation and activation of PLCgamma2. In addition, CalDAG-GEFI-deficient platelets showed defective integrin alpha2beta1-dependent adhesion and spreading. We found that outside-in signaling through integrin alpha2beta1 triggered inside-out activation of integrin alphaIIbbeta3 and promoted fibrinogen binding. Similarly to Rap1b stimulation, this process occurred downstream of PLC activation and was dramatically impaired in murine platelets lacking the Rap1 exchange factor CalDAG-GEFI. These results demonstrate that Rap1b is an important element in integrin-dependent outside-in signaling during platelet adhesion and regulates the cross talk between adhesive receptors.


Subject(s)
Integrin alpha2beta1/physiology , Platelet Adhesiveness/physiology , Platelet Glycoprotein GPIIb-IIIa Complex/physiology , Receptor Cross-Talk/physiology , rap GTP-Binding Proteins/metabolism , Enzyme Activation , Fibrinogen/metabolism , Humans , Integrin alpha2beta1/blood , Kinetics , Second Messenger Systems/physiology , Type C Phospholipases/blood , rap GTP-Binding Proteins/blood
10.
J Hepatol ; 40(5): 749-56, 2004 May.
Article in English | MEDLINE | ID: mdl-15094221

ABSTRACT

BACKGROUND/AIMS: Circulating neutrophils from cirrhotic patients have a reduced capacity to generate superoxide anion (O(2)(-)), which might contribute to frequent bacterial infections in these patients. We studied the signal transduction pathways involved in the generation of O(2)(-) in neutrophils from 98 cirrhotic patients and 46 healthy controls. METHODS: We measured O(2)(-) production in neutrophils induced by fMLP, opsonized zymosan, TNF alpha, NaF, AlF(4)(-), A23187 and phorbol myristate acetate. Furthermore, we measured phospholipase C activity in neutrophils from healthy controls and end-stage cirrhotic patients. RESULTS: O(2)(-) production was decreased in neutrophils from patients in response to fMLP, opsonized zymosan and TNF alpha. Likewise, response of these cells to G-protein stimulation by fluorides was also decreased. These reduced responses correlated significantly with the degree of liver dysfunction. On the contrary, neutrophils from patients responded normally to A23187 and phorbol esters stimulation indicating that Ca(2+)- and PKC-dependent pathways are intact in these cells. Finally, phospholipase C activity was markedly reduced in neutrophils from end-stage liver cirrhosis. CONCLUSIONS: These data confirm that O(2)(-) generation by neutrophils is decreased in patients with cirrhosis, particularly in those with more severe liver dysfunction, and suggest that this defect involves phosphatidylinositol specific phospholipase C activity.


Subject(s)
Liver Cirrhosis/enzymology , Neutrophils/enzymology , Type C Phospholipases/deficiency , Adult , Aged , Case-Control Studies , Female , Fluorides/pharmacology , GTP-Binding Proteins/blood , Humans , In Vitro Techniques , Liver Cirrhosis/blood , Male , Middle Aged , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , NADPH Oxidases/blood , Neutrophils/drug effects , Neutrophils/metabolism , Peroxidase/blood , Signal Transduction , Superoxides/blood , Tumor Necrosis Factor-alpha/pharmacology , Type C Phospholipases/blood , Zymosan/pharmacology
11.
Immunopharmacol Immunotoxicol ; 25(4): 561-71, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14686798

ABSTRACT

We investigated the effects of the traditional Korean prescription, Danggijakyaksan (DJS) on antiplatelet activity in human platelet suspensions. The effect of oriental medicinal prescriptions, Danggijakyaksan consisting of 6 herbes of Paeoniae Radix (2 g), Poria Cocos (1.33 g), Angelicae Sinensis Radix (1 g), Cnidii Rhizoma (1 g), Atractylodis Macrocephalae Rhizoma (1.33 g) and Alismatis Rhizoma (1.66 g), was studied. In this study, the mechanism involved in the antiplatelet activity of DJS in human platelet suspensions was investigated. Danggijakyaksan did not significantly affect the thromboxane synthetase activity of aspirin-treated platelet microsomes and DJS (20 and 40 microg/mL) significantly inhibited [3H]arachidonic acid (AA) released in collagen-activated platelets but not in unactivated-platelets. Nitric oxide (NO) production in human platelets was measured by a chemiluminesence detection method in this study. Danggijakyaksan did not significantly affect nitrate production in collagen (10 microg/mL)-induced human platelet aggregation. On the other hand, various concentrations of DJS (10, 20, and 40 microg/mL) dose-dependently inhibited [3H]inositol monophosphate (IP) formation stimulated by collagen (10 microg/mL) in [3H]myoinositolloaded platelets at different incubation times (1, 2, 3, and 5 min). It is concluded that the antiplatelet activity of DJS may possibly be due to the inhibition of phospholipase C activity, leading to reduced phosphoinositide breakdown, followed by the inhibition of thromboxane A2 formation, and then inhibition of [Ca2+]i mobilization of platelet aggregation stimulated by agonists.


Subject(s)
Blood Platelets/drug effects , Blood Platelets/enzymology , Plant Extracts/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Type C Phospholipases/antagonists & inhibitors , Arachidonic Acid/blood , Collagen/pharmacology , Humans , In Vitro Techniques , Nitric Oxide/blood , Phosphatidylinositols/blood , Thromboxane-A Synthase/blood , Type C Phospholipases/blood
12.
J Biol Chem ; 278(37): 34812-22, 2003 Sep 12.
Article in English | MEDLINE | ID: mdl-12832405

ABSTRACT

Platelet adhesion to fibrinogen is important for platelet aggregation and thrombus growth. In this study we have examined the mechanisms regulating platelet adhesion on immobilized fibrinogen under static and shear conditions. We demonstrate that integrin alpha IIb beta 3 engagement of immobilized fibrinogen is sufficient to induce an oscillatory calcium response, necessary for lamellipodial formation and platelet spreading. Released ADP increases the proportion of platelets exhibiting a cytosolic calcium response but is not essential for calcium signaling or lamellipodial extension. Pretreating platelets with the Src kinase inhibitor PP2, the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (APB-2), or the phospholipase C (PLC) inhibitor U73122 abolished calcium signaling and platelet spreading, suggesting a major role for Src kinase-regulated PLC isoforms in these processes. Analysis of PLC gamma 2-/- mouse platelets revealed a major role for this isoform in regulating cytosolic calcium flux and platelet spreading on fibrinogen. Under flow conditions, platelets derived from PLC gamma 2-/- mice formed less stable adhesive interactions with fibrinogen, particularly in the presence of ADP antagonists. Our studies define an important role for PLC gamma 2 in integrin alpha IIb beta 3-dependent calcium flux, necessary for stable platelet adhesion and spreading on fibrinogen. Furthermore, they establish an important cooperative signaling role for PLC gamma 2 and ADP in regulating platelet adhesion efficiency on fibrinogen.


Subject(s)
Blood Platelets/physiology , Calcium Signaling/physiology , Fibrinogen/physiology , Platelet Adhesiveness/physiology , Platelet Glycoprotein GPIIb-IIIa Complex/physiology , Type C Phospholipases/blood , Animals , Apyrase/pharmacology , Aspirin/pharmacology , Calcium Signaling/drug effects , Humans , Kinetics , Mice , Mice, Knockout , Phospholipase C gamma , Platelet Aggregation Inhibitors/pharmacology , Platelet Glycoprotein GPIIb-IIIa Complex/drug effects , Type C Phospholipases/deficiency
13.
Blood ; 102(4): 1367-73, 2003 Aug 15.
Article in English | MEDLINE | ID: mdl-12730118

ABSTRACT

Collagen stimulates platelet activation through a tyrosine kinase-based pathway downstream of the glycoprotein VI (GPVI)-Fc receptor (FcR) gamma-chain complex. Genetic ablation of FcR gamma-chain results in a complete inhibition of aggregation to collagen. In contrast, a steady increase in light transmission is induced by collagen in phospholipase Cgamma2-deficient (PLCgamma2-/-) platelets in a Born aggregometer, indicating a weak level of activation. This increase is inhibited partially in the presence of an alpha2beta1-blocking antibody or an alphaIIbbeta3 antagonist and completely by a combination of the 2 inhibitors. It is also abolished by the Src kinase inhibitor PP1 and reduced in the presence of the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin. The GPVI-specific agonists convulxin and collagen-related peptide (CRP) also stimulate weak aggregation in PLCgamma2-/- platelets, which is inhibited by wortmannin and PP1. Collagen and CRP stimulate tyrosine phosphorylation of PLCgamma1 at its regulatory site, Tyr 783, in murine but not in human platelets through a Src kinase-dependent pathway. Adhesion of PLCgamma2-/- platelets to a collagen monolayer is severely reduced at a shear rate of 800 s-1, relative to controls, whereas it is abolished in FcR gamma-chain-/- platelets. These results provide strong evidence that engagement of GPVI stimulates limited integrin activation in PLCgamma2-/- platelets via PLCgamma1 and PI3-kinase.


Subject(s)
Blood Platelets/enzymology , Integrin alpha2beta1/metabolism , Lectins, C-Type , Phosphatidylinositol 3-Kinases/blood , Platelet Activation/physiology , Platelet Membrane Glycoproteins/agonists , Type C Phospholipases/blood , Animals , Blood Platelets/drug effects , Collagen/metabolism , Collagen/pharmacology , Crotalid Venoms/pharmacology , Enzyme Inhibitors/pharmacology , Fibrinogen/metabolism , Humans , Integrin alpha2beta1/antagonists & inhibitors , Mice , Phosphoinositide-3 Kinase Inhibitors , Phospholipase C gamma , Phosphorylation , Platelet Activation/drug effects , Platelet Aggregation Inhibitors/pharmacology , Platelet Glycoprotein GPIIb-IIIa Complex/antagonists & inhibitors , Platelet Membrane Glycoproteins/pharmacology , Receptors, Fc/metabolism , Type C Phospholipases/deficiency , Tyrosine/metabolism , src-Family Kinases/antagonists & inhibitors , src-Family Kinases/metabolism
14.
Biochem Biophys Res Commun ; 303(2): 446-51, 2003 Apr 04.
Article in English | MEDLINE | ID: mdl-12659837

ABSTRACT

Normal cellular prion protein (PrP(C)) and decay-accelerating factor (DAF) are glycoproteins linked to the cell surface by glycosylphosphatidylinositol (GPI) anchors. Both PrP(C) and DAF reside in detergent insoluble complex that can be isolated from human peripheral blood mononuclear cells. However, these two GPI-anchored proteins possess different cell biological properties. The GPI anchor of DAF is markedly more sensitive to cleavage by phosphatidylinositol-specific phospholipase C (PI-PLC) than that of PrP(C). Conversely, PrP(C) has a shorter cell surface half-life than DAF, possibly due to the fact that PrP(C) but not DAF is shed from the cell surface. This is the first demonstration that on the surface of the same cell type two GPI-anchored proteins differ in their cell biological properties.


Subject(s)
CD55 Antigens/physiology , Glycosylphosphatidylinositols/blood , Prions/physiology , Type C Phospholipases/blood , Cell Membrane/physiology , Clone Cells , Humans , Jurkat Cells , Kinetics , Neutrophils/physiology , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoinositide Phospholipase C , T-Lymphocytes
15.
Life Sci ; 72(2): 113-24, 2002 Nov 29.
Article in English | MEDLINE | ID: mdl-12417245

ABSTRACT

We previously showed that a dissociated form of a low-molecular-weight heat shock-related protein 20 (HSP20) but not an aggregated form of HSP20 suppresses platelet aggregation. In the present study, we investigated the behavior of HSP20 in response to endothelial injury and the possible mechanism of HSP20 in platelet functions. The levels of HSP20 in vessel wall after endothelial injury were markedly reduced. This observation was supported by the results of Western blotting analysis and immunohistochemical analysis. Additionally, the plasma levels of HSP20 in cardiomyopathic hamsters were markedly elevated. Centrifugation on sucrose density gradients allowed detection mainly of the dissociated form of plasma HSP20 in these hamsters. Human platelets showed specific binding sites for HSP20. Moreover, HSP20 markedly reduced thrombin-induced phosphoinositide hydrolysis by phospholipase C in human platelets. Taken together, our results strongly suggest that HSP20, which immediately responds to pathological events, acts extracellularly as a regulator of platelet functions.


Subject(s)
Blood Platelets/physiology , Heat-Shock Proteins/physiology , Phosphoproteins/physiology , Animals , Binding Sites , Blood Platelets/enzymology , Blotting, Western , Cardiomyopathies/metabolism , Cardiomyopathies/pathology , Carotid Arteries/pathology , Cricetinae , Endothelium, Vascular/injuries , Endothelium, Vascular/pathology , Extracellular Space/physiology , HSP20 Heat-Shock Proteins , Heat-Shock Proteins/metabolism , Humans , Immunohistochemistry , In Vitro Techniques , Inositol/metabolism , Male , Myocardium/chemistry , Myocardium/pathology , Phosphatidylinositols/metabolism , Phosphoproteins/metabolism , Platelet Function Tests , Thrombin/antagonists & inhibitors , Thrombin/chemistry , Type C Phospholipases/blood
16.
Biochem J ; 364(Pt 3): 755-65, 2002 Jun 15.
Article in English | MEDLINE | ID: mdl-12049640

ABSTRACT

The platelet collagen receptor glycoprotein VI (GPVI) and the fibrinogen receptor integrin alphaIIbbeta3 trigger intracellular signalling cascades involving the tyrosine kinase Syk, the adapter SLP-76 and phospholipase Cgamma2 (PLCgamma2). Similar pathways are activated downstream of immune receptors in lymphocytes, where they have been localized in part to glycolipid-enriched membrane domains (GEMs). Here we provide several lines of evidence that GPVI-mediated tyrosine phosphorylation of PLCgamma2 in platelets is dependent on GEM-organized signalling and utilizes the GEM resident adapter protein LAT (linker for activation of T cells). In sharp contrast, although fibrinogen binding to platelets stimulates alphaIIbbeta3-dependent activation of Syk and tyrosine phosphorylation of SLP-76 and PLCgamma2, it does not utilize GEMs to promote these responses or to support platelet aggregation. These results establish that GPVI and alphaIIbbeta3 trigger distinct patterns of receptor signalling in platelets, leading to tyrosine phosphorylation of PLCgamma2, and they highlight the role of GEMs in compartmentalizing signalling reactions involved in haemostasis.


Subject(s)
Blood Platelets/enzymology , Cell Membrane/enzymology , Isoenzymes/blood , Platelet Glycoprotein GPIIb-IIIa Complex/pharmacology , Platelet Membrane Glycoproteins/pharmacology , Type C Phospholipases/blood , Binding Sites , Blood Platelets/drug effects , Blood Platelets/physiology , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Membrane/chemistry , Fibrinogen/pharmacology , Glycolipids/blood , Humans , Membrane Lipids/blood , Phospholipase C gamma , Phosphorylation , Phosphotyrosine/metabolism , Signal Transduction
17.
Platelets ; 13(1): 31-5, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11918834

ABSTRACT

Platelets were labelled separately with six different, radioactive unsaturated fatty acids. The cells were isolated from the radioactive precursors and treated with and without 2 U/ml of thrombin. The formation of radioactive free fatty acid+oxygenated fatty acids and of radioactive radioactive phosphatidic acid+diacylglycerol was taken as a measure of the PLA(2) and PLC reactions, respectively. We found that that in intact platelets PLA(2) prefers phospholipid molecular species containing unsaturated acyls, most likely in the sn-2 position, in the priority order: 20:4>20:5>18:2 = 18:3 = 22:6>>18:1, while PLC prefers its substrates in the priority order 20:5>20:4>18:2>18:3 = 22:6>18:1.


Subject(s)
Blood Platelets/enzymology , Phospholipases A/blood , Thrombin/pharmacology , Type C Phospholipases/blood , Fatty Acids, Nonesterified/blood , Humans , Substrate Specificity
18.
Neuropsychopharmacology ; 26(2): 216-28, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11790517

ABSTRACT

Phospholipase C (PLC) and protein kinase C (PKC) are important components of the phosphoinositide (PI) signaling system. To examine if the abnormalities observed in the PI signaling system of patients with affective disorders, reported in previous studies, are related to abnormalities in one or more of its components, we studied PKC, PI-PLC activity, the expression of their specific isozymes, and expression of myristoylated alanine-rich C-kinase substrate (MARCKS) in platelets obtained from 15 drug-free hospitalized patients with bipolar disorder and 15 with major depressive disorder (unipolar) and from 15 nonhospitalized normal control subjects. We observed a significant decrease in PI-PLC and PKC activity and the expression of selective PKC alpha, betaI, betaII, and PLC delta(1) isozymes in membrane and cytosol fraction of platelets from bipolar but not unipolar patients. On the other hand, the level of MARCKS was significantly increased in membrane and cytosol fraction of platelets from patients with bipolar but not unipolar disorders. These results suggest that alterations in PKC, PLC, and MARCKS may be involved in the pathophysiology of bipolar illness.


Subject(s)
Bipolar Disorder/enzymology , Blood Platelets/enzymology , Gene Expression Regulation, Enzymologic/physiology , Intracellular Signaling Peptides and Proteins , Membrane Proteins , Mood Disorders/enzymology , Phosphoproteins/metabolism , Protein Kinase C/metabolism , Type C Phospholipases/metabolism , Adult , Aging/metabolism , Blotting, Western , Brain/enzymology , Calcium-Binding Proteins , Cell Membrane/enzymology , Cytosol/enzymology , Female , Glucosidases , Humans , Isoenzymes/biosynthesis , Isoenzymes/metabolism , Male , Myristoylated Alanine-Rich C Kinase Substrate , Protein Kinase C/blood , Psychiatric Status Rating Scales , Sex Characteristics , Type C Phospholipases/blood
19.
J Biol Chem ; 276(24): 20882-9, 2001 Jun 15.
Article in English | MEDLINE | ID: mdl-11287424

ABSTRACT

Aggretin, a potent platelet activator, was isolated from Calloselasma rhodostoma venom, and 30-amino acid N-terminal sequences of both subunits were determined. Aggretin belongs to the heterodimeric snake C-type lectin family and is thought to activate platelets by binding to platelet glycoprotein alpha(2)beta(1). We now show that binding to glycoprotein (GP) Ib is also required. Aggretin-induced platelet activation was inhibited by a monoclonal antibody to GPIb as well as by antibodies to alpha(2)beta(1). Binding of both of these platelet receptors to aggretin was confirmed by affinity chromatography. No binding of other major platelet membrane glycoproteins, in particular GPVI, to aggretin was detected. Aggretin also activates platelets from Fc receptor gamma chain (Fcgamma)-deficient mice to a greater extent than those from normal control mice, showing that it does not use the GPVI/Fcgamma pathway. Platelets from Fcgamma-deficient mice expressed fibrinogen receptors normally in response to collagen, although they did not aggregate, indicating that these platelets may partly compensate via other receptors including alpha(2)beta(1) or GPIb for the lack of the Fcgamma pathway. Signaling by aggretin involves a dose-dependent lag phase followed by rapid tyrosine phosphorylation of a number of proteins. Among these are p72(SYK), p125(FAK), and PLCgamma2, whereas, in comparison with collagen and convulxin, the Fcgamma subunit neither is phosphorylated nor coprecipitates with p72(SYK). This supports an independent, GPIb- and integrin-based pathway for activation of p72(SYK) not involving the Fcgamma receptor.


Subject(s)
Blood Platelets/physiology , Enzyme Precursors/blood , Integrins/blood , Integrins/physiology , Isoenzymes/blood , Lectins, C-Type , Lectins/pharmacology , Platelet Activation/physiology , Platelet Glycoprotein GPIb-IX Complex/physiology , Protein-Tyrosine Kinases/blood , Type C Phospholipases/blood , Viper Venoms/chemistry , Viper Venoms/pharmacology , Agkistrodon , Amino Acid Sequence , Animals , Blood Platelets/drug effects , Chromatography, Affinity , Collagen/pharmacology , Crotalid Venoms/pharmacology , Humans , In Vitro Techniques , Intracellular Signaling Peptides and Proteins , Lectins/chemistry , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Phospholipase C gamma , Phosphorylation , Phosphotyrosine/blood , Platelet Activation/drug effects , Platelet Glycoprotein GPIb-IX Complex/drug effects , Protein Subunits , Receptors, Collagen , Sequence Alignment , Sequence Homology, Amino Acid , Syk Kinase , Viper Venoms/isolation & purification
20.
Platelets ; 12(2): 121-3, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11297033

ABSTRACT

Phospholipase Cgamma2 (PLCgamma2), the predominant isoform of phospholipase C expressed in platelets, plays a major role in activation of platelets by collagen. Although PLCgamma2 has been shown to be tyrosine phosphorylated upon collagen-induced activation, the phosphorylation sites are yet to be determined. We have sequenced the 3' terminal cDNA of human phospholipase C-gamma-2 and found it different from the human PLCgamma2 cDNA sequence previously reported by Ohta et al. (Ohta S, Matsui A, Nazawa Y, Kagawa Y. FEBS Lett 1988; 242: 31-5). There is an extra guanosine at position 3723 which causes a shift in the reading frame. The new carboxyl terminal amino acid (aa) sequence beyond the frame shift is 88% identical to that of rat (21 out of 24 aa residues) which is considerably higher than the identity with published sequence (26% identity). The new deduced aa sequence contains two tyrosine residues at positions 1245 and 1264 which might be phosphorylated upon stimulation and hence might be important for the activation of the PLCgamma2.


Subject(s)
Isoenzymes/chemistry , Type C Phospholipases/chemistry , Amino Acid Sequence , Animals , Blood Platelets/enzymology , DNA, Complementary/genetics , Humans , Isoenzymes/blood , Isoenzymes/genetics , Molecular Sequence Data , Open Reading Frames , Phospholipase C gamma , Phosphorylation , Phosphotyrosine/analysis , Protein Processing, Post-Translational , Rats , Sequence Alignment , Sequence Analysis, Protein , Sequence Homology, Amino Acid , Type C Phospholipases/blood , Type C Phospholipases/genetics
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