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Thomas M Mcintyre - One of the best experts on this subject based on the ideXlab platform.
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determination of phospholipase activity of PAF Acetylhydrolase
Free Radical Biology and Medicine, 2013Co-Authors: Diana M Stafforini, Thomas M McintyreAbstract:This article presents a radiometric assay to determine the enzymatic activity of platelet-activating factor (PAF) Acetylhydrolase (PAF-AH), also known as lipoprotein-associated phospholipase A2 and phospholipase A2 group 7A. The method is based on the release of radioactively labeled acetate from sn-2-labeled PAF and separation of substrate and product using reversed-phase column chromatography on octadecyl silica gel cartridges. The assay is fast, convenient, reproducible, sensitive, and inexpensive. The instrumentation required includes standard laboratory equipment and a liquid scintillation counter. The assay is also useful to determine the activity of intracellular PAF-AH (PAF-AH II), provided that a few modifications are included. The enzymatic activity determined using PAF as the substrate is a direct indication of the ability of plasma samples, purified preparations, and cellular and tissue lysates to hydrolyze short- and medium-chain phospholipids that may or may not harbor oxidized functionalities. In addition, the assay can be used to test the suitability of other phospholipids, including species containing oxidized, long-chain sn-2 fatty acyl groups, as PAF-AH substrates. This versatile assay can be used to accurately determine PAF-AH activity in biological samples and preliminarily assess affinity and efficiency of the hydrolysis of potential substrates present in complex mixtures.
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circulating platelet activating factor is primarily cleared by transport not intravascular hydrolysis by lipoprotein associated phospholipase a2 PAF Acetylhydrolase
Circulation Research, 2011Co-Authors: Rui Chen, Gopal K Marathe, Maria Febbraio, Thomas M McintyreAbstract:Rationale:The phospholipid platelet-activating factor (PAF) stimulates all cells of the innate immune system and numerous cardiovascular cells. A single enzyme (plasma PAF Acetylhydrolase [PAF-AH] or lipoprotein-associated phospholipase [Lp-PL]A2) in plasma hydrolyzes PAF, but significant controversy exists whether its action is pro- or antiinflammatory and accordingly whether its inhibition will slow cardiovascular disease. Objective:We sought to define how PAF and related short-chain oxidized phospholipids turnover in vivo and the role of PAF Acetylhydrolase/Lp-PLA2 in this process. Methods and Results:[3H-acetyl]PAF was hydrolyzed by murine or human plasma (t1/2, 3 and 7 minutes, respectively), but injected [3H-acetyl]PAF disappeared from murine circulation more quickly (t1/2, <30 seconds). [3H]PAF clearance was unchanged in PAF receptor−/− animals, or over the first 2 half-lives in PAF-AH−/− animals. [3H]PAF turnover was reduced by coinjecting excess unlabeled PAF or an oxidatively truncated phospholi...
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Circulating Platelet-Activating Factor Is Primarily Cleared by Transport, Not Intravascular Hydrolysis, by Lipoprotein-Associated Phospholipase A2/PAF Acetylhydrolase
Circulation Research, 2010Co-Authors: Rui Chen, Gopal K Marathe, Maria Febbraio, Thomas M McintyreAbstract:Rationale:The phospholipid platelet-activating factor (PAF) stimulates all cells of the innate immune system and numerous cardiovascular cells. A single enzyme (plasma PAF Acetylhydrolase [PAF-AH] or lipoprotein-associated phospholipase [Lp-PL]A2) in plasma hydrolyzes PAF, but significant controversy exists whether its action is pro- or antiinflammatory and accordingly whether its inhibition will slow cardiovascular disease. Objective:We sought to define how PAF and related short-chain oxidized phospholipids turnover in vivo and the role of PAF Acetylhydrolase/Lp-PLA2 in this process. Methods and Results:[3H-acetyl]PAF was hydrolyzed by murine or human plasma (t1/2, 3 and 7 minutes, respectively), but injected [3H-acetyl]PAF disappeared from murine circulation more quickly (t1/2,
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PAF Acetylhydrolase expressed during megakaryocyte differentiation inactivates PAF like lipids
Blood, 2009Co-Authors: Jason M Foulks, Diana M Stafforini, Gopal K Marathe, Thomas M Mcintyre, Noemi Michetti, Guy A Zimmerman, Andrew S WeyrichAbstract:Platelet activating factor (PAF) and PAF-like lipids induce inflammatory responses in target cells. These lipid mediators are inactivated by PAF-Acetylhydrolase (PAF-AH). The PAF signaling system affects the growth of hematopoietic CD34+ cells, but roles for PAF-AH in this process are unknown. Here, we investigated PAF-AH function during megakaryopoiesis and found that human CD34+ cells accumulate this enzymatic activity as they differentiate toward megakaryocytes, consistent with the expression of mRNA and protein for the plasma PAF-AH isoform. Inhibition of endogenous PAF-AH activity in differentiated megakaryocytes increased formation of lipid mediators that signaled the PAF receptor (PAFR) in fully differentiated human cells such as neutrophils, as well as megakaryocytes themselves. PAF-AH also controlled megakaryocyte αIIbβ3-dependent adhesion, cell spreading, and mobility that relied on signaling through the PAFR. Together these data suggest that megakaryocytes generate PAF-AH to modulate the accumulation of intracellular phospholipid mediators that may detrimentally affect megakaryocyte development and function.
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a yeast PAF Acetylhydrolase ortholog suppresses oxidative death
Free Radical Biology and Medicine, 2008Co-Authors: Jason M Foulks, Andrew S Weyrich, Guy A Zimmerman, Thomas M McintyreAbstract:Abstract Phospholipids containing sn-2 polyunsaturated fatty acyl residues are primary targets of oxidizing radicals, producing proapoptotic and membrane perturbing fragmented phospholipids. The only known phospholipases that specifically select these oxidized and/or short-chained phospholipids as substrates are mammalian group VII phospholipases A2s that were purified and cloned as PAF Acetylhydrolases. Platelet-activating factor (PAF) is a short-chained phospholipid, and whether these enzymes actually are PAF hydrolases or evolved as oxidized phospholipid phospholipases is unknown. The fission yeast Schizosaccharomyces pombe, which does not form or use PAF as a signaling molecule, contains an open-reading frame potentially homologous to mammalian group VII phospholipase A2s. We cloned this SPBC106.11c locus and expressed it in distantly related Saccharomyces cerevisiae that lack homologous sequences. The S. pombe locus encoded a functional phospholipase A2, now renamed plg7+, that hydrolyzed PAF and a synthetic oxidized phospholipid. Expression of human type II PAF Acetylhydrolase or S. pombe Plg7p enhanced the viability of S. cerevisiae subjected to oxidative stress. We conclude that a single-celled organism with an exceedingly spare genome still expresses an unusually discriminating phospholipase A2, and that selective hydrolysis of phospholipid oxidation products is an early, and critical, way to overcome oxidative membrane damage and oxidant-induced cell death.
Diana M Stafforini - One of the best experts on this subject based on the ideXlab platform.
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determination of phospholipase activity of PAF Acetylhydrolase
Free Radical Biology and Medicine, 2013Co-Authors: Diana M Stafforini, Thomas M McintyreAbstract:This article presents a radiometric assay to determine the enzymatic activity of platelet-activating factor (PAF) Acetylhydrolase (PAF-AH), also known as lipoprotein-associated phospholipase A2 and phospholipase A2 group 7A. The method is based on the release of radioactively labeled acetate from sn-2-labeled PAF and separation of substrate and product using reversed-phase column chromatography on octadecyl silica gel cartridges. The assay is fast, convenient, reproducible, sensitive, and inexpensive. The instrumentation required includes standard laboratory equipment and a liquid scintillation counter. The assay is also useful to determine the activity of intracellular PAF-AH (PAF-AH II), provided that a few modifications are included. The enzymatic activity determined using PAF as the substrate is a direct indication of the ability of plasma samples, purified preparations, and cellular and tissue lysates to hydrolyze short- and medium-chain phospholipids that may or may not harbor oxidized functionalities. In addition, the assay can be used to test the suitability of other phospholipids, including species containing oxidized, long-chain sn-2 fatty acyl groups, as PAF-AH substrates. This versatile assay can be used to accurately determine PAF-AH activity in biological samples and preliminarily assess affinity and efficiency of the hydrolysis of potential substrates present in complex mixtures.
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functional consequences of mutations and polymorphisms in the coding region of the PAF Acetylhydrolase PAF ah gene
Pharmaceuticals, 2009Co-Authors: Diana M StafforiniAbstract:Abstract: In the past several years a number of alterations in the PAF-AH/PLA 2 G7/LpPLA 2 gene have been described. These include inactivating mutations, polymorphisms in the coding region, and other genetic changes located in promoter and intronic regions of the gene. The consequences associated with these genetic variations have been evaluated from different perspectives, including in vitro biochemical and molecular studies and clinical analyses in human subjects. This review highlights the current state of the field and suggests new approaches that can be used to evaluate functional consequences associated with mutations and polymorphisms in the PAF-AH gene. Keywords: PAF Acetylhydrolase; PLA 2 G7; single nucleotide polymorphism; mutation; vascular disease 1. Introduction The first step in the metabolism of two classes of inflammatory phospholipid mediators, Platelet-Activating Factor (PAF) and oxidatively-fragmented phospholipids (OxPL), is catalyzed by enzymatic activities known as PAF Acetylhydrolases (PAF-AHs). This family of phospholipases A
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PAF Acetylhydrolase expressed during megakaryocyte differentiation inactivates PAF like lipids
Blood, 2009Co-Authors: Jason M Foulks, Diana M Stafforini, Gopal K Marathe, Thomas M Mcintyre, Noemi Michetti, Guy A Zimmerman, Andrew S WeyrichAbstract:Platelet activating factor (PAF) and PAF-like lipids induce inflammatory responses in target cells. These lipid mediators are inactivated by PAF-Acetylhydrolase (PAF-AH). The PAF signaling system affects the growth of hematopoietic CD34+ cells, but roles for PAF-AH in this process are unknown. Here, we investigated PAF-AH function during megakaryopoiesis and found that human CD34+ cells accumulate this enzymatic activity as they differentiate toward megakaryocytes, consistent with the expression of mRNA and protein for the plasma PAF-AH isoform. Inhibition of endogenous PAF-AH activity in differentiated megakaryocytes increased formation of lipid mediators that signaled the PAF receptor (PAFR) in fully differentiated human cells such as neutrophils, as well as megakaryocytes themselves. PAF-AH also controlled megakaryocyte αIIbβ3-dependent adhesion, cell spreading, and mobility that relied on signaling through the PAFR. Together these data suggest that megakaryocytes generate PAF-AH to modulate the accumulation of intracellular phospholipid mediators that may detrimentally affect megakaryocyte development and function.
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The fate of platelet-activating factor: PAF Acetylhydrolases from plasma and tissues
Advances in Lipobiology, 2007Co-Authors: T Imaizumi, Diana M Stafforini, Thomas M Mcintyre, Guy A Zimmerman, Yoshiji Yamada, Stephen M. PrescottAbstract:Abstract Platelet-activating factor (PAF) likely mediates a variety of physiological and pathological events. There is abundant evidence that the concentration of PAF in blood or tissues is influenced by its rate of degradation. Two forms of the degradative enzyme, PAF Acetylhydrolase, have been purified and studied in detail. Changes in the activity of the plasma enzyme have been observed in human diseases, physiological responses, and in animal models, suggesting that it may be a key step. The plasma PAF Acetylhydrolase has several interesting properties including marked substrate specificity and association with lipoproteins. Studies that define the molecular basis for these properties and elucidate the role of the enzyme in physiological processes should be forthcoming, and will provide insight into the function of PAF.
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release of free f2 isoprostanes from esterified phospholipids is catalyzed by intracellular and plasma platelet activating factor Acetylhydrolases
Journal of Biological Chemistry, 2006Co-Authors: Diana M Stafforini, Thomas M Mcintyre, Stephen M. Prescott, James R Sheller, Timothy S Blackwell, Adam Sapirstein, Fiona E Yull, Joseph V Bonventre, Jackson L RobertsAbstract:Abstract F2-isoprostanes are produced in vivo by nonenzymatic peroxidation of arachidonic acid esterified in phospholipids. Increased urinary and plasma F2-isoprostane levels are associated with a number of human diseases. These metabolites are regarded as excellent markers of oxidant stress in vivo. Isoprostanes are initially generated in situ, i.e. when the arachidonate precursor is esterified in phospholipids, and they are subsequently released in free form. Although the mechanism(s) responsible for the release of free isoprostanes after in situ generation in membrane phospholipids is, for the most part, unknown, this process is likely mediated by phospholipase A2 activity(ies). Here we reported that human plasma contains an enzymatic activity that catalyzes this reaction. The activity associates with high density and low density lipoprotein and comigrates with platelet-activating factor (PAF) Acetylhydrolase on KBr density gradients. Plasma samples from subjects deficient in PAF Acetylhydrolase do not release F2-isoprostanes from esterified precursors. The intracellular PAF Acetylhydrolase II, which shares homology to the plasma enzyme, also catalyzes this reaction. We found that both the intracellular and plasma PAF Acetylhydrolases have high affinity for esterified F2-isoprostanes. However, the rate of esterified F2-isoprostane hydrolysis is much slower compared with the rate of hydrolysis of other substrates utilized by these enzymes. Studies using PAF Acetylhydrolase transgenic mice indicated that these animals have a higher capacity to release F2-isoprostanes compared with nontransgenic littermates. Our results suggested that PAF Acetylhydrolases play key roles in the hydrolysis of F2-isoprostanes esterified on phospholipids in vivo.
Masao Miwa - One of the best experts on this subject based on the ideXlab platform.
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Platelet-activating factor Acetylhydrolase gene mutation in Japanese nephrotic children
Kidney International, 2015Co-Authors: Hong Xu, Masao Miwa, Kazumoto Iijima, Yuji Inoue, Taku Shirakawa, Kaoru Nishiyama, Hajime Nakamura, Shunichi Shiozawa Ryojiro Tanaka, Norishige YoshikawaAbstract:Platelet-activating factor Acetylhydrolase gene mutation in Japanese nephrotic children. Background Platelet-activating factor (PAF) may be involved in the pathogenesis of steroid-responsive nephrotic syndrome (SRNS). PAF is degraded to inactive products by PAF Acetylhydrolase. We have investigated whether PAF Acetylhydrolase gene mutation is involved in SRNS in Japanese children. Methods We identified a point mutation in the PAF Acetylhydrolase gene (G994T) using the polymerase chain reaction in 101 Japanese children with SRNS and 100 healthy Japanese. Results There was no difference in the genotype and allele frequencies between patients with SRNS and normal controls. The mean number of relapses during the first year after onset was significantly higher in the 26 patients who were heterozygous for the mutant allele (GT) than in 75 wild-type homozygotes (GG) (2.61 ± 1.98 vs. 1.33 ± 1.35; P = 0.0019). Conclusions We conclude that analysis of the PAF Acetylhydrolase gene mutation at position 994 in Japanese children with SRNS allows the identification of patients who are more likely to have a disease relapse.
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a single nucleotide polymorphism in the plasma PAF Acetylhydrolase gene and risk of atherosclerosis in japanese patients with peripheral artery occlusive disease
Journal of Surgical Research, 2006Co-Authors: Naoki Unno, Masao Miwa, Junko Sugatani, Takanori Sakaguchi, Toshio Nakamura, Naoto Yamamoto, Hiroyuki KonnoAbstract:Background Plasma PAF-Acetylhydrolase (PAF-AH) gene polymorphisms (G994 → T in exon 9) and the resulting deficiency of enzyme activity were identified in the Japanese population. The objective of this study was to assess the joint effect of the polymorphism and hypercholesterolemia on risk of atherosclerosis. Methods and Results We performed a case-control study including 150 patients who underwent operation for peripheral arterial occlusive disease (PAOD) and 158 controls matched for age and sex. Genomic DNA was analyzed for the mutant allele by a specific polymerase-chain reaction. Plasma PAF-AH activity was measured in both groups. The patients with multiple atherosclerotic diseases showed higher levels of PAF-AH activities than the patients with only peripheral artery occlusive disease among normal genotypes. PAOD patients were assessed either with or without polymorphism or hypercholesterolemia in regard to accompanying coronary artery disease or stroke. The prevalence of the polymorphism was significantly more frequent in the patients with PAOD. The plasma PAF-AH activity was correlated with total cholesterol and LDL level, and inversely related with HDL in normal genotype (GG) PAOD patients. However, neither the correlation nor the inverse relation was found in patients with the polymorphism. Patients with both hypercholesterolemia and the polymorphisms revealed a relative risk for other atherosclerotic disease of 11.5 (6.0-40.3) compared with normal genotype and normal lipid level. Conclusion The plasma PAF-AH gene polymorphism and hypercholesterolemia may interact and increase the risk of atherosclerosis.
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Platelet-activating factor Acetylhydrolase gene mutation in Japanese children with Escherichia coli O157-associated hemolytic uremic syndrome.
American Journal of Kidney Diseases, 2000Co-Authors: Hong Xu, Masao Miwa, Kazumoto Iijima, Taku Shirakawa, Shunichi Shiozawa, Hajime Nakamura, Kanji Yamaoka, Naohisa Kawamura, Norishige YoshikawaAbstract:Abstract Platelet-activating factor (PAF) may be involved in the pathogenesis of Escherichia coli O157–associated hemolytic uremic syndrome (HUS). PAF is degraded to inactive products by PAF Acetylhydrolase. In this study, we investigated whether a PAF Acetylhydrolase gene mutation (G→T transversion at position 994) is involved in HUS in Japanese children. A point mutation in the PAF Acetylhydrolase gene (G994T) was identified using polymerase chain reaction in 50 Japanese children with E coli O157–associated HUS and 100 healthy Japanese. We then determined the relationship between the PAF Acetylhydrolase G994T gene mutation and clinical features of HUS. There was no difference in genotype and allele frequencies between patients with HUS and healthy controls. The mean duration of oligoanuria was significantly longer in patients with the GT genotype than in those with the GG genotype ( P = 0.012). Although 11 of 15 patients (73%) heterozygous for the mutant allele (GT) required dialysis, only 13 of the 35 wild-type homozygotes (GG; 37%) required dialysis ( P = 0.030). Mean plasma PAF Acetylhydrolase activity was significantly less in patients with the GT genotype than in those with the GG genotype ( P E coli O157–associated HUS. Our study suggests that analysis of the PAF Acetylhydrolase gene mutation in Japanese children with E coli O157–associated HUS may allow the prediction of the severity of HUS.
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Role of platelet-activating factor Acetylhydrolase gene mutation in Japanese childhood IgA nephropathy
American Journal of Kidney Diseases, 1999Co-Authors: Ryojiro Tanaka, Masao Miwa, Kazumoto Iijima, Hong Xu, Yuji Inoue, Ryusuke Murakami, Taku Shirakawa, Kaoru Nishiyama, Shunichi Shiozawa, Hajime NakamuraAbstract:Abstract Platelet-activating factor (PAF) is a potent mediator of inflammatory injury in renal diseases. PAF is degraded to inactive products by PAF Acetylhydrolase. Recently, a point mutation (G to T transversion) of the PAF Acetylhydrolase gene was observed at position 994, and this mutation was found to contribute to the variability in plasma PAF levels, with undetectable plasma PAF Acetylhydrolase activity occurring in homozygous patients (TT genotype) and reduced levels of activity in heterozygous patients (GT genotype). Therefore, we investigated the effect of the PAF Acetylhydrolase gene mutation on the pathogenesis and progression of immunoglobulin A (IgA) nephropathy. Genomic DNA was obtained from 89 children with IgA nephropathy and 100 controls. We identified the PAF Acetylhydrolase gene mutation (G994T) by polymerase chain reaction. There was no significant difference in genotypic frequency between patients and controls. However, urinary protein excretion at the time of biopsy was significantly greater in patients with the GT/TT genotypes than in those with the GG genotype. The percentage of glomeruli with mesangial cell proliferation was significantly greater in patients with the GT/TT genotypes than in those with the GG genotype. These results indicate the PAF Acetylhydrolase gene mutation may influence the degree of proteinuria and the extent of mesangial proliferation in the early stage of childhood IgA nephropathy.
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identification of a major PAF Acetylhydrolase in human serum plasma as a 43 kda glycoprotein containing about 9 kda asparagine conjugated sugar chain s
Journal of Biochemistry, 1998Co-Authors: Masaki Akiyama, Junko Sugatani, Takashi Suzuki, Yasuo Suzuki, Masao MiwaAbstract:: Platelet-activating factor (PAF) Acetylhydrolase from human serum/plasma was identified on a polyvinylidene difluoride (PVDF) membrane by electroblotting proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme activity was detected in the 43 kDa region on the membrane as a decrease in the beta-radioluminescence of [3H]acetyl-PAF or by the convenient method for determining PAF Acetylhydrolase activity (the TCA precipitation method). The enzyme activity on treatment with N-glycosidase F shifted to the 34 kDa region on the PVDF membrane. On the other hand, only one band was observed, corresponding to a molecular mass of 53 kDa, on analysis by SDS-PAGE with silver staining. Treatment of the 53 kDa protein with N-glycosidase F changed its molecular mass to 43 kDa (protein A). The NH2-terminal 32 amino acid sequence of protein A completely corresponds to that of the heterogenous enzyme with 54 amino acids deleted from the NH2 terminus reported by Tjoelker et al. (Nature 374, 549-553, 1995). Even after trypsin treatment of the N-glycosidase F-digested enzyme, its PAF-AH activity remained in the 34 kDa region, but the contaminating protein A disappeared, on the PVDF membrane. In addition, the majority of serum PAF-AH was retained on a Sambucus sieboldiana agglutinin (SSA)-agarose column and was eluted with the hapten sugar, lactose. These results indicate that PAF Acetylhydrolase consisting of a 34 kDa protein and about 9 kDa asparagine-conjugated sugar chain(s) is a major enzyme in human serum/plasma.
Alexandros D Tselepis - One of the best experts on this subject based on the ideXlab platform.
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the low density lipoprotein ldl associated PAF Acetylhydrolase activity and the extent of ldl oxidation are important determinants of the autoantibody titers against oxidized ldl in patients with coronary artery disease
Prostaglandins Leukotrienes and Essential Fatty Acids, 2006Co-Authors: Evangelia S Lourida, John A Goudevenos, Athanasios Papathanasiou, Alexandros D TselepisAbstract:Abstract The oxidation of low-density lipoprotein (LDL) induces immunogenic epitopes, many of which are due to oxidatively modified phospholipids (oxPL). Lysophosphatidylcholine (lyso-PC) which is generated during LDL oxidation through the hydrolysis of oxPL by LDL-associated PAF-Acetylhydrolase (PAF-AH) is also immunogenic. We investigated whether the LDL-associated PAF-AH and the extent of LDL oxidation influence the autoantibody titers against oxidized LDL (oxLDL) in patients with stable angina as well as in apparently healthy volunteers. Three types of copper-oxidized LDL, were prepared at the end of the lag, propagation or decomposition phase (oxLDL L , oxLDL P and oxLDL D , respectively). Similar types of oxidized LDL were prepared after previous inactivation of endogenous PAF-AH [oxLDL(−)]. All these types of oxLDL as well as malondialdehyde-modified LDL (MDA-LDL) were used as antigens. Antibody titers against the above antigens were measured with an ELISA method in the serum of 65 patients with stable angina and 47 apparently healthy volunteers. Both groups exhibited higher autoantibody titers against each type of oxLDL(−) compared to the respective type of oxLDL ( P P and oxLDL D or oxLDL(-) P and oxLDL(−) D were used as antigens compared to oxLDL L ( P L , respectively ( P P are associated with a significantly higher risk for coronary artery disease. LDL-associated PAF-AH activity may play an important role in decreasing the overall immunogenicity of oxLDL, whereas the extent of LDL oxidation seems to modulate the epitopes formed on oxLDL. Lyso-PC, a major component of oxLDL P , could be mainly responsible for the elevated autoantibody titers against oxLDL in patients with stable angina.
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lipoprotein associated PAF Acetylhydrolase activity in subjects with the metabolic syndrome
Prostaglandins Leukotrienes and Essential Fatty Acids, 2005Co-Authors: Evangelos C Rizos, Alexandros D Tselepis, Afroditi P Tambaki, Irene F Gazi, Moses ElisafAbstract:Abstract Plasma- and lipoprotein-associated activity of the platelet activating factor Acetylhydrolase (PAF-Acetylhydrolase, PAF-AH) plays an important role in inflammation and in atherosclerotic process, which are present in the metabolic syndrome (MS). Paraoxonase 1 (PON1) is an esterase associated with high-density lipoprotein (HDL) which contributes to the anti-atherogenic effects of this lipoprotein. We investigated the activities of both enzymes in 60 patients with MS and 110 age- and sex-matched subjects without it (non-MS group). Plasma PAF-AH activity was higher in the MS compared to the non-MS group, while HDL–PAF-AH and serum PON1 activities were lower in the MS compared to the non-MS group. Univariate regression analysis in the MS group showed that plasma PAF-AH activity was positively associated with systolic blood pressure, whereas HDL–PAF-AH activity was inversely associated with the homeostasis model assessments (HOMA) index. Both associations remained significant in the multivariate regression analysis, suggesting that insulin resistance and systolic hypertension are major determinants for the alterations in plasma and HDL-associated PAF-AH activity among those observed in MS patients.
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effect of lipoprotein a on platelet activation induced by platelet activating factor role of apolipoprotein a and endogenous PAF Acetylhydrolase
Cardiovascular Research, 2004Co-Authors: Loukas D Tsironis, Haralampos J Milionis, Moses Elisaf, John V Mitsios, Alexandros D TselepisAbstract:OBJECTIVE: Lipoprotein (a) [Lp(a)] is considered an atherogenic lipoprotein, which is also implicated in thrombosis. Lp(a) binds to platelets and modulates the effects of various platelet agonists. Platelet activating factor (PAF) is a potent platelet agonist degraded and inactivated by PAF-Acetylhydrolase (PAF-AH), which in plasma is associated with lipoproteins. Lp(a) is enriched in PAF-AH, thus a functional characteristic of this lipoprotein is its capability to hydrolyze and inactivate PAF. In the present study, we investigated the effect of Lp(a) on PAF-induced platelet activation. The potential roles of the apo(a) moiety and especially of the PAF-AH content of Lp(a) on the above effect were also addressed. METHODS: Lp(a) was isolated by affinity chromatography from plasma of apparently healthy fasting donors with serum Lp(a) concentrations >/=20 mg/dl. Reduced Lp(a) [Lp(a-)] was prepared by incubation of Lp(a) with dithiothreitol (DTT), whereas inactivation of Lp(a)-associated PAF-AH was performed by incubation of Lp(a) with pefabloc [pefa-Lp(a)]. Platelet-rich plasma (PRP) or washed platelets were prepared from peripheral venous blood samples of normolipidemic, apparently healthy fasting donors with their serum Lp(a) levels lower than 0.8 mg/dl. The surface expression of the platelet integrin-receptor alpha(IIb)beta3 and the fibrinogen binding to the activated alpha(IIb)beta3 was studied by flow cytometry. RESULTS: Lp(a), at doses higher than 20 microg/ml, inhibits PAF-induced platelet activation in a dose-dependent manner. Pefa-Lp(a), lacking PAF-AH activity, exhibited a similar to Lp(a) inhibitory effect. Importantly, the Lp(a) inhibitory effect was not influenced by the apo(a) isoform size, whereas Lp(a-) was a more potent inhibitor compared to Lp(a). Similarly to PAF, Lp(a) inhibits platelet aggregation induced by ADP or Calcium ionophore A23187. Lp(a), pefa-Lp(a) or Lp(a-) effectively inhibited PAF- or ADP-induced surface expression of alphaIIbbeta3, the Lp(a-) being more potent compared to Lp(a) or to pefa-Lp(a). Finally, Lp(a) significantly inhibited fibrinogen binding to platelets activated with PAF. CONCLUSIONS: Lp(a) inhibits PAF-induced platelet activation in a non-specific manner. The Lp(a)-associated PAF-AH does not play any important role in this effect, whereas the apo(a) moiety of Lp(a) significantly reduces its inhibitory effect. The inhibition of alpha(IIb)beta3 activation and fibrinogen binding to the activated platelets may represent the major mechanism by which Lp(a) inhibits PAF-induced platelet aggregation.
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urine of patients with nephrotic syndrome contains the plasma type of PAF Acetylhydrolase associated with lipoproteins
Nephron Physiology, 2004Co-Authors: Olga Balafa, Alexandros D Tselepis, Soniaathina Karabina, Moses Elisaf, Charalambos Pappas, Kostas C SiamopoulosAbstract:Background: Platelet-activating factor (PAF) is a proinflammatory phospholipid mediator involved in the pathogenesis of glomerulonephritis (GN). In plasma, PAF is hydrolyzed and inactivated by PAF-Acetylhydrolase (PAF-AH), an enzyme associated with lipoproteins, mainly with the low-density lipoprotein. PAF-AH activity has been found in urine of patients with primary GN, however the source and type of urinary PAF-AH remain unknown. We characterized the type of PAF-AH excreted in the urine of patients with primary GN and studied the possible relationship of this enzyme with the lipiduria and proteinuria observed in these patients. Methods: Eighteen patients with primary GN (8 with nephrotic syndrome (NS) and 10 with non-nephrotic range proteinuria (NNRP)) and 20 normolipidemic age- and sex-matched controls participated in the study. PAF-AH activity in plasma, in urine and in individual lipoprotein particles was determined by the trichloroacetic acid precipitation procedure, whereas the PAF-AH protein was detected by Western blotting analysis. Plasma and urine lipoproteins were fractionated by gradient ultracentrifugation and characterized by Western blotting analysis. Results: Plasma PAF-AH activity was higher in NS patients compared with NNRP patients and controls, whereas the enzyme activity associated with high-density lipoprotein was significantly lower in both patient groups compared with controls. PAF-AH was detected only in the urine of NS patients. It was the plasma type of PAF-AH and was associated with lipoprotein particles. Enzyme activity was also positively correlated with urine cholesterol levels. Conclusion: Urine of NS patients contains the plasma type of PAF-AH, which is related to the extent of lipiduria and is associated with urine lipoproteins.
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platelet associated and secreted PAF Acetylhydrolase activity in patients with stable angina sequential changes of the enzyme activity after angioplasty
European Journal of Clinical Investigation, 2001Co-Authors: John A Goudevenos, Alexandros D Tselepis, Demokritos Tsoukatos, Ewa Ninio, Moses Elisaf, M P Vini, L K Michalis, D A SiderisAbstract:Background Platelet-activating factor (PAF), the lipid mediator of inflammation and potent platelet agonist, can be hydrolysed and inactivated by PAF-Acetylhydrolase (PAFAH). We investigated the PAF-AH activity in relation to PAF formation in platelets from patients with stable angina undergoing elective percutaneous transluminal coronary angioplasty (PTCA). Design Twenty-seven patients with stable angina, undergoing PTCA, and 30 age- and sexmatched controls were studied. The platelet-associated and secreted PAF-AH activity was measured, before PTCA, as well as at 4 h, 48 h and 6 months afterwards. PAF formation by thrombin-stimulated platelets and the platelet aggregation responses to PAF and ADP were also determined. Results The PAF-AH activity secreted by thrombin-stimulated platelets before PTCA (in pmol/10 9 cells/h) was significantly higher compared to controls (892 ^ 222 vs. 624 ^ 144, P , 0·001). The enzyme activity was not altered at 4 h after PTCA, but was significantly increased at 48 h (1284 ^ 312, P , 0·005) to return to the levels observed in the control group 6 months afterwards. Detectable levels of PAF in thrombin-stimulated platelets were found only at 6 months after PTCA. Furthermore, the cell-associated enzyme activity in resting platelets before PTCA was significantly lower compared with controls. Unlike in controls, the platelet-associated enzyme activity in the patient group was not increased after stimulation with thrombin and it was associated by a platelet hyperaggregability to PAF. Both the intact cell-associated activity and the platelet hyper-reactivity to PAF were restored at 6 months after PTCA. Conclusions Alterations in the platelet PAF-AH activity, which affect the PAF formation in thrombin-stimulated platelets and are associated by an increased aggregatory response to PAF, are observed in patients with stable angina and are completely restored after PTCA.
Junko Sugatani - One of the best experts on this subject based on the ideXlab platform.
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Serum platelet-activating factor Acetylhydrolase activity in rats with gastric ulcers induced by water-immersion stress.
Scandinavian Journal of Gastroenterology, 2009Co-Authors: Kazuyo Fujimura, Junko Sugatani, M Miwa, Takako Mizuno, Yoshiko Sameshima, Kunihiko SaitoAbstract:Platelet-activating factor (PAF) Acetylhydrolase is an enzyme which hydrolyzes PAF to yield inactive lysoPAF. This study focused on the influence of water-immersion stress on serum PAF Acetylhydrolase activity. The enzyme activity was determined by measurement of [3H]acetate produced from l-O-alkyl-2-[3H]acetyl-sn-glycero-3-phosphocholine upon precipitation of the complex of the radioactive substrate and albumin with trichloroacetic acid. The onset of water-immersion stress caused ‘the devefopment of gastric fesions associated’ with a significant increase in serum PAF Acetylhydrolase activity. Serum PAF Acetylhydrolase may leak into the blood from some tissues in rats with gastric injury induced by water-immersion stress and might control the action of PAF.
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a single nucleotide polymorphism in the plasma PAF Acetylhydrolase gene and risk of atherosclerosis in japanese patients with peripheral artery occlusive disease
Journal of Surgical Research, 2006Co-Authors: Naoki Unno, Masao Miwa, Junko Sugatani, Takanori Sakaguchi, Toshio Nakamura, Naoto Yamamoto, Hiroyuki KonnoAbstract:Background Plasma PAF-Acetylhydrolase (PAF-AH) gene polymorphisms (G994 → T in exon 9) and the resulting deficiency of enzyme activity were identified in the Japanese population. The objective of this study was to assess the joint effect of the polymorphism and hypercholesterolemia on risk of atherosclerosis. Methods and Results We performed a case-control study including 150 patients who underwent operation for peripheral arterial occlusive disease (PAOD) and 158 controls matched for age and sex. Genomic DNA was analyzed for the mutant allele by a specific polymerase-chain reaction. Plasma PAF-AH activity was measured in both groups. The patients with multiple atherosclerotic diseases showed higher levels of PAF-AH activities than the patients with only peripheral artery occlusive disease among normal genotypes. PAOD patients were assessed either with or without polymorphism or hypercholesterolemia in regard to accompanying coronary artery disease or stroke. The prevalence of the polymorphism was significantly more frequent in the patients with PAOD. The plasma PAF-AH activity was correlated with total cholesterol and LDL level, and inversely related with HDL in normal genotype (GG) PAOD patients. However, neither the correlation nor the inverse relation was found in patients with the polymorphism. Patients with both hypercholesterolemia and the polymorphisms revealed a relative risk for other atherosclerotic disease of 11.5 (6.0-40.3) compared with normal genotype and normal lipid level. Conclusion The plasma PAF-AH gene polymorphism and hypercholesterolemia may interact and increase the risk of atherosclerosis.
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Identification of a Major PAF Acetylhydrolase in Human Serum/Plasma as a 43 kDa Glycoprotein Containing About 9 kDa Asparagine-Conjugated Sugar Chain(s)
Journal of Biochemistry, 1998Co-Authors: Masaki Akiyama, Junko Sugatani, Takashi Suzuki, Yasuo Suzuki, Masao MiwaAbstract:: Platelet-activating factor (PAF) Acetylhydrolase from human serum/plasma was identified on a polyvinylidene difluoride (PVDF) membrane by electroblotting proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme activity was detected in the 43 kDa region on the membrane as a decrease in the beta-radioluminescence of [3H]acetyl-PAF or by the convenient method for determining PAF Acetylhydrolase activity (the TCA precipitation method). The enzyme activity on treatment with N-glycosidase F shifted to the 34 kDa region on the PVDF membrane. On the other hand, only one band was observed, corresponding to a molecular mass of 53 kDa, on analysis by SDS-PAGE with silver staining. Treatment of the 53 kDa protein with N-glycosidase F changed its molecular mass to 43 kDa (protein A). The NH2-terminal 32 amino acid sequence of protein A completely corresponds to that of the heterogenous enzyme with 54 amino acids deleted from the NH2 terminus reported by Tjoelker et al. (Nature 374, 549-553, 1995). Even after trypsin treatment of the N-glycosidase F-digested enzyme, its PAF-AH activity remained in the 34 kDa region, but the contaminating protein A disappeared, on the PVDF membrane. In addition, the majority of serum PAF-AH was retained on a Sambucus sieboldiana agglutinin (SSA)-agarose column and was eluted with the hapten sugar, lactose. These results indicate that PAF Acetylhydrolase consisting of a 34 kDa protein and about 9 kDa asparagine-conjugated sugar chain(s) is a major enzyme in human serum/plasma.
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identification of a major PAF Acetylhydrolase in human serum plasma as a 43 kda glycoprotein containing about 9 kda asparagine conjugated sugar chain s
Journal of Biochemistry, 1998Co-Authors: Masaki Akiyama, Junko Sugatani, Takashi Suzuki, Yasuo Suzuki, Masao MiwaAbstract:: Platelet-activating factor (PAF) Acetylhydrolase from human serum/plasma was identified on a polyvinylidene difluoride (PVDF) membrane by electroblotting proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme activity was detected in the 43 kDa region on the membrane as a decrease in the beta-radioluminescence of [3H]acetyl-PAF or by the convenient method for determining PAF Acetylhydrolase activity (the TCA precipitation method). The enzyme activity on treatment with N-glycosidase F shifted to the 34 kDa region on the PVDF membrane. On the other hand, only one band was observed, corresponding to a molecular mass of 53 kDa, on analysis by SDS-PAGE with silver staining. Treatment of the 53 kDa protein with N-glycosidase F changed its molecular mass to 43 kDa (protein A). The NH2-terminal 32 amino acid sequence of protein A completely corresponds to that of the heterogenous enzyme with 54 amino acids deleted from the NH2 terminus reported by Tjoelker et al. (Nature 374, 549-553, 1995). Even after trypsin treatment of the N-glycosidase F-digested enzyme, its PAF-AH activity remained in the 34 kDa region, but the contaminating protein A disappeared, on the PVDF membrane. In addition, the majority of serum PAF-AH was retained on a Sambucus sieboldiana agglutinin (SSA)-agarose column and was eluted with the hapten sugar, lactose. These results indicate that PAF Acetylhydrolase consisting of a 34 kDa protein and about 9 kDa asparagine-conjugated sugar chain(s) is a major enzyme in human serum/plasma.
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corneal PAF Acetylhydrolase increases in anterior segment ischemia in rabbits
Experimental Eye Research, 1993Co-Authors: Junko Sugatani, Hirohiko Miki, M Uyama, Kunihiko SaitoAbstract:Abstract The involvement of platelet-activating factor (PAF) in the development of anterior segment necrosis after occlusion of the bilateral long posterior ciliary arteries was investigated in rabbits. With the progression of anterior segment necrosis, which was characterized by corneal edema and neovascularization, the contents of protein and total phospholipids increased in the cornea and aqueous humor, but not in the iris. PAF Acetylhydrolase activity was significantly increased on the first postoperative day in the peripheral cornea and on the second day in the central cornea prior to the development of marked corneal edema and neovascularization, but it did not increase in the iris. The accumulation of newly synthesized PAF in the cornea reached a plateau level on the third postoperative day, which coincided with the progression of the corneal lesion. In the aqueous humor, PAF Acetylhydrolase activity was increased by the induced ischemia, but no PAF was detected. The increased PAF Acetylhydrolase activity may reflect adaptation to the participation of PAF in the progression of anterior segment necrosis.
