Human group II 14 kDa phospholipase A2 activates human platelets.
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ABSTRACT:
SUBMITTER: Polgar J
PROVIDER: S-EPMC1218789 | biostudies-other | 1997 Oct
REPOSITORIES: biostudies-other
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Project description:Several structurally related plant lipids were isolated and their effect was assessed on the enzyme activity of group I (pancreatic and Naja mocambique venom) and group II (Crotalus atrox venom) phospholipase A2 (PLA2) enzymes, with labelled Escherichia coli as an enzyme substrate. The neutral monogalactosyldiacylglycerol (MGDG) and negatively charged diacylglyceryl alpha-D-glucuronide (DGGA) did not influence the enzyme activity of either group. Digalactosyldiacylglycerol (DGDG), another uncharged glycolipid, inhibited PLA2 activity in a dose-dependent manner to 60-70% of the control. Sulphoquinovosyldiacylglycerol (SQDG), which is also anionic, activated both groups of PLA2 enzyme. A similar activation was observed with the zwitterionic diacylglyceryl-O-(N,N,N-trimethylhomoserine) (DGTS) and diacylglyceryl-O-(hydroxymethyl)(N,N, N-trimethyl)-beta-alanine (DGTA). DGDG, SQDG and DGTS are dispersed homogeneously with low critical micelle concentrations (CMCs). The hydrodynamic radius of neutral DGDG is an order of magnitude larger than the charged lipids SQDG and DGTS. The inhibition of pig pancreatic PLA2 by DGDG was dependent on substrate concentration. The intrinsic fluorescence spectra of the enzyme was not changed in the presence of native or hydrogenated DGDG. Thus the inhibition is most probably due to a non-specific interaction of plant lipids with the substrate. Different lengths and saturations of the fatty acyl chains of DGDG did not alter the inhibition of PLA2, whereas deacylation abrogated the inhibitory effect. Both SQDG and DGTS activated pig pancreatic PLA2 in a dose-dependent manner. Saturation of the double bonds of these lipids decreased the activating effect. The fluorescence of pig pancreatic PLA2 incubated with SQDG and DGTS was enhanced by 2-fold and 3-fold respectively, suggesting the formation of a complex between enzyme and lipids. In conclusion, the effect of different plant lipids on PLA2 activity depends on different structural elements of the polar head group and their charge as well as the degree of unsaturation of the fatty acyl chains.
| S-EPMC1217902 | biostudies-other
Project description:1. Vascular endothelial growth factor (VEGF) is a potent inducer of inflammation, and we have shown that this latter effect is mediated through endothelial cell (EC) PAF synthesis. Since the phospholipid remodelling pathway enzymes (CoA-independent transacylase, CoA-IT; phospholipase A2, PLA2; and lyso-PAF acetyltransferase, lyso-PAF-AT) may participate in PAF synthesis, we assessed their contribution to VEGF-induced PAF synthesis in bovine aortic EC (BAEC) and human umbilical vein EC (HUVEC). 2. VEGF enhanced BAEC and HUVEC PAF synthesis by up to 28 and 4 fold above basal levels respectively. 3. A pretreatment with a CoA-IT and lyso-PAF-AT inhibitor (Sanguinarin; 500 nM) blocked VEGF-induced PAF synthesis by 95%, a specific CoA-IT inhibitor (SKF45905; 10 - 50 microM) was without effect, confirming the crucial role of the PLA2 and lyso-PAF-AT. 4. Treatment with secreted PLA2 (sPLA2) inhibitors which have been shown to inhibit both groups IIA and V sPLA2 (SB203347; 10 microM and LY311727; 100 microM) blocked EC PAF synthesis by up to 90%, whereas selective inhibition of group IIA sPLA2 (LY311727; 1 microM) had no significant effect. 5. RT - PCR and Western blot analyses demonstrated the presence of group V sPLA2 whereas group IIA sPLA2 was undetected in EC. 6. Treatment with cytosolic and calcium-independent PLA2 inhibitors (Arachidonyl trifluoromethyl ketone, Bromoenol lactone, Methyl arachydonyl fluorophosphate, up to 50 microM) did not prevent but rather potentiated the VEGF effect on EC PAF synthesis. 7. These results provide evidence that with VEGF activation of EC cells, the group V sPLA2 provides substrate for EC PAF formation.
| S-EPMC1572915 | biostudies-other
Project description:The rate-limiting step in the biosynthesis of thromboxane A(2) (TxA(2)) and 12-hydroxyeicosatetraenoic acid (12-HETE) by platelets is activation of cytosolic phospholipase A(2?) (cPLA(2?)), which releases arachidonic acid, which is the substrate for cyclooxygenase-1 (COX-1) and 12-lipoxygenase. We evaluated signaling via the human platelet thrombin receptors, protease-activated receptor (PAR) 1 and PAR4, to the activation of cPLA(2?), which provides a substrate for the biosynthesis of TxA(2) and 12-HETE.Stimulating washed human platelets resulted in delayed biosynthesis of 12-HETE, which continues after maximal formation of TxA(2) is completed, suggesting that 12-HETE is not formed by the same pool of arachidonic acid that provides a substrate to COX-1. PAR1-induced formation of TxA(2) was inhibited by the phosphatidylinositol kinase inhibitor LY294002, whereas this inhibitor did not block 12-HETE biosynthesis. Both 1-butanol and propranolol also blocked TxA(2) biosynthesis but did not inhibit 12-HETE formation.The concerted evidence indicates that the platelet thrombin receptors signal activation of cPLA(2?) coupled to COX-1 by a pathway different from that signaling activation of the cPLA(2?) coupled to 12-lipoxygenase.
| S-EPMC3035574 | biostudies-literature
Project description:Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) catalyzes release of arachidonic acid from glycerophospholipids, leading to thromboxane A(2) (TxA(2)) production. Some platelet agonists stimulate cPLA(2)alpha, but others require fibrinogen binding to alphaIIbbeta3 to elicit TxA(2). Therefore, relationships between cPLA(2)alpha and alphaIIbbeta3 were examined. cPLA(2)alpha and a cPLA(2)alpha binding partner, vimentin, coimmunoprecipitated with alphaIIbbeta3 from platelets, independent of fibrinogen binding. Studies with purified proteins and with recombinant proteins expressed in CHO cells determined that the interaction between cPLA(2)alpha and alphaIIbbeta3 was indirect and was dependent on the alphaIIb and beta3 cytoplasmic tails. Fibrinogen binding to alphaIIbbeta3 caused an increase in integrin-associated cPLA(2)alpha activity in normal platelets, but not in cPLA(2)alpha-deficient mouse platelets or in human platelets treated with pyrrophenone, a cPLA(2)alpha inhibitor. cPLA(2)alpha activation downstream of alphaIIbbeta3 had functional consequences for platelets in that it was required for fibrinogen-dependent recruitment of activated protein kinase Cbeta to the alphaIIbbeta3 complex and for platelet spreading. Thus, cPLA(2)alpha and alphaIIbbeta3 interact to reinforce each other's functions during alphaIIbbeta3 signaling. This provides a plausible explanation for the role of alphaIIbbeta3 in TxA(2) formation and in the defective hemostatic function of mouse or human platelets deficient in cPLA(2)alpha.
| S-EPMC2615656 | biostudies-literature
Project description:BACKGROUND:Phospholipase A2s mediate the rate-limiting step in the formation of eicosanoids such as cysteinyl leukotrienes (CysLTs). Group IVA cytosolic PLA2? (cPLA2?) is thought to be the dominant PLA2 in eosinophils; however, eosinophils also have secreted PLA2 (sPLA2) activity that has not been fully defined. OBJECTIVES:To examine the expression of sPLA2 group X (sPLA2-X) in eosinophils, the participation of sPLA2-X in the formation of CysLTs, and the mechanism by which sPLA2-X initiates the synthesis of CysLTs in eosinophils. METHODS:Peripheral blood eosinophils were obtained from volunteers with asthma and/or allergy. A rabbit polyclonal anti-sPLA2-X antibody identified sPLA2-X by Western blot. We used confocal microscopy to colocalize the sPLA2-X to intracellular structures. An inhibitor of sPLA2-X (ROC-0929) that does not inhibit other mammalian sPLA2s, as well as inhibitors of the mitogen-activated kinase cascade (MAPK) and cPLA2?, was used to examine the mechanism of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-mediated formation of CysLT. RESULTS:Eosinophils express the mammalian sPLA2-X gene (PLA2G10). The sPLA2-X protein is located in the endoplasmic reticulum, golgi, and granules of eosinophils and moves to the granules and lipid bodies during fMLP-mediated activation. Selective sPLA2-X inhibition attenuated the fMLP-mediated release of arachidonic acid and CysLT formation by eosinophils. Inhibitors of p38, extracellular-signal-regulated kinases 1/2 (p44/42 MAPK), c-Jun N-terminal kinase, and cPLA2? also attenuated the fMLP-mediated formation of CysLT. The sPLA2-X inhibitor reduced the phosphorylation of p38 and extracellular-signal-regulated kinases 1/2 (p44/42 MAPK) as well as cPLA2? during cellular activation, indicating that sPLA2-X is involved in activating the MAPK cascade leading to the formation of CysLT via cPLA2?. We further demonstrate that sPLA2-X is activated before secretion from the cell during activation. Short-term priming with IL-13 and TNF/IL-1? increased the expression of PLA2G10 by eosinophils. CONCLUSIONS:These results demonstrate that sPLA2-X plays a significant role in the formation of CysLTs by human eosinophils. The predominant role of the enzyme is the regulation of MAPK activation that leads to the phosphorylation of cPLA2?. The sPLA2-X protein is regulated by proteolytic cleavage, suggesting that an inflammatory environment may promote the formation of CysLTs through this mechanism. These results have important implications for the treatment of eosinophilic disorders such as asthma.
| S-EPMC4695325 | biostudies-literature
Project description:Human group IIA secretory phospholipase A2 (hGIIA) promotes the proliferation of cancer cells, making it a compelling therapeutic target, but it is also significant in other inflammatory conditions. Consequently, suitable inhibitors of hGIIA have always been sought. The activation of phospholipases A2 and the catalysis of glycerophospholipid substrates generally leads to the release of fatty acids such as arachidonic acid (AA) and lysophospholipid, which are then converted to mediator compounds, including prostaglandins, leukotrienes, and the platelet-activating factor. However, this ability of hGIIA to provide AA is not a complete explanation of its biological role in inflammation, as it has now been shown that it also exerts proinflammatory effects by a catalysis-independent mechanism. This mechanism is likely to be highly dependent on key specific molecular interactions, and the full mechanistic descriptions of this remain elusive. The current candidates for the protein partners that may mediate this catalysis-independent mechanism are also introduced in this review. A key discovery has been that selective inhibition of the catalysis-independent activity of hGIIA is achieved with cyclised derivatives of a pentapeptide, FLSYK, derived from the primary sequence of hGIIA. The effects of hGIIA on cell function appear to vary depending on the pathology studied, and so its mechanism of action is complex and context-dependent. This review is comprehensive and covers the most recent developments in the understanding of the many facets of hGIIA function and inhibition and the insight they provide into their clinical application for disease treatment. A cyclic analogue of FLSYK, c2, the most potent analogue known, has now been taken into clinical trials targeting advanced prostate cancer.
| S-EPMC7583969 | biostudies-literature
Project description:Lipid mediators play pivotal roles in colorectal cancer and colitis, but only a limited member of the phospholipase A2 (PLA2) subtypes, which lie upstream of various lipid mediators, have been implicated in the positive or negative regulation of these diseases. Clinical and biochemical evidence suggests that secreted PLA2 group III (sPLA2-III) is associated with colorectal cancer, although its precise role remains obscure. Here we have found that sPLA2-III-null (Pla2g3 -/-) mice are highly resistant to colon carcinogenesis. Furthermore, Pla2g3 -/- mice are less susceptible to dextran sulfate-induced colitis, implying that the amelioration of colonic inflammation by sPLA2-III ablation may underlie the protective effect against colon cancer. Lipidomics analysis of the colon revealed significant reduction of pro-inflammatory/pro-tumorigenic lysophosholipids as well as unusual steady-state elevation of colon-protective fatty acids and their oxygenated metabolites in Pla2g3 -/- mice. Overall, our results establish a role of sPLA2-III in the promotion of colorectal inflammation and cancer, expand our understanding of the divergent roles of multiple PLA2 enzymes in the gastrointestinal tract, and point to sPLA2-III as a novel druggable target for colorectal diseases.
| S-EPMC5612992 | biostudies-literature
Project description:Secretory phospholipase A(2)s (sPLA(2)) hydrolyze glycerophospholipids to liberate lysophospholipids and free fatty acids. Although group X (GX) sPLA(2) is recognized as the most potent mammalian sPLA(2) in vitro, its precise physiological function(s) remains unclear. We recently reported that GX sPLA(2) suppresses activation of the liver X receptor in macrophages, resulting in reduced expression of liver X receptor-responsive genes including ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), and a consequent decrease in cellular cholesterol efflux and increase in cellular cholesterol content (Shridas et al. 2010. Arterioscler. Thromb. Vasc. Biol. 30: 2014-2021). In this study, we provide evidence that GX sPLA(2) modulates macrophage inflammatory responses by altering cellular cholesterol homeostasis. Transgenic expression or exogenous addition of GX sPLA(2) resulted in a significantly higher induction of TNF-?, IL-6, and cyclooxygenase-2 in J774 macrophage-like cells in response to LPS. This effect required GX sPLA(2) catalytic activity, and was abolished in macrophages that lack either TLR4 or MyD88. The hypersensitivity to LPS in cells overexpressing GX sPLA(2) was reversed when cellular free cholesterol was normalized using cyclodextrin. Consistent with results from gain-of-function studies, peritoneal macrophages from GX sPLA(2)-deficient mice exhibited a significantly dampened response to LPS. Plasma concentrations of inflammatory cytokines were significantly lower in GX sPLA(2)-deficient mice compared with wild-type mice after LPS administration. Thus, GX sPLA(2) amplifies signaling through TLR4 by a mechanism that is dependent on its catalytic activity. Our data indicate this effect is mediated through alterations in plasma membrane free cholesterol and lipid raft content.
| S-EPMC3119755 | biostudies-literature
Project description:Simplified analogs of YM-26734, a known inhibitor of secreted phospholipase A(2) (sPLA(2)) group IIA, were synthesized and found to also display potent inhibition at low nanomolar concentrations. Analogs were based on the didodecanoylphloroglucinol portion of YM-26734 which contains the predicted active site calcium binding group.
| S-EPMC2578825 | biostudies-literature
Project description:Although benign hepatocellular adenomas (HCA) are very rare, recent observations have shown their occurrence in patients with diabetes mellitus. Consequently, most of these cases are treated by resection due to concerns regarding their potential progression to hepatocarcinoma (HCC). This decision is largely driven by the limited number of studies on HCC subtyping and the lack of molecular and biological insights into the carcinogenic potential of benign tumors. This study aimed to comprehensively investigate the subtype classification of HCA and to compare and analyze gene expression profiling between HCA and HCC tissues. One fresh inflammatory HCA (I-HCA), three non-B non-C HCCs, two hepatitis B virus-HCCs, and one normal liver tissue sample were subjected to single-cell RNA sequencing (scRNA-seq). Comparative analysis of scRNA-seq among different tissues showed that phospholipase A2 group IIA (PLA2G2A) mRNA was specifically expressed in I-HCA, following RNA-seq analysis in formalin-fixed paraffin-embedded tissues from other HCAs. Immunohistochemistry using the PLA2G2A antibody in these tissues indicated that the positive reaction was mainly observed in hepatocytes of I-HCAs and stromal cells surrounding the tumor tissue in HCC were also stained. According to a clinical database, PLA2G2A expression in HCC does not correlate with poor prognosis. This finding may potentially help develop a new definition for I-HCA, resulting in a significant clinical contribution, but it requires validation with other fresh HCA samples.
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