Platelet-activating factor acetylhydrolase (PAF-AH)

Platelet-activating factor (PAF) is one of the most potent lipid messengers involved in a variety of physiological events. The acetyl group at the sn-2 position of its glycerol backbone is essential for its biological activity, and its deacetylation induces loss of activity. The deacetylation reaction is catalyzed by PAF-acetylhydrolase (PAF-AH). A series of biochemical and enzymological evaluations revealed that at least three types of PAF-AH exist in mammals, namely the intracellular types I and II and a plasma type. Type I PAF-AH is a G-protein-like complex consisting of two catalytic subunits (alpha1 and alpha2) and a regulatory beta subunit. The beta subunit is a product of the LIS1 gene, mutations of which cause type I lissencephaly. Recent studies indicate that LIS1/beta is important in cellular functions such as induction of nuclear movement and control of microtubule organization. Although substantial evidence is accumulating supporting the idea that the catalytic subunits are also involved in microtubule function, it is still unknown what role PAF plays in the process and whether PAF is an endogenous substrate of this enzyme. Type II PAF-AH is a single polypeptide and shows significant sequence homology with plasma PAF-AH. Type II PAF-AH is myristoylated at the N-terminus and like other N-myristoylated proteins is distributed in both the cytosol and membranes. Plasma PAF-AH is also a single polypeptide and exists in association with plasma lipoproteins. Type II PAF-AH as well as plasma PAF-AH may play a role as a scavenger of oxidized phospholipids which are thought to be involved in diverse pathological processes, including disorganization of membrane structure and PAF-like proinflammatory action. In this review, we will focus on the structures and possible biological functions of intracellular PAF-AHs.     

Molecular and mechanistic characterization of platelet-activating factor-like bioactivity produced upon LDL oxidation

Oxidation of LDL is thought to be involved in both initiating and sustaining atherogenesis through the formation of proinflammatory lipids and the covalent modification of LDL particles. Platelet-activating factor (PAF; 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent phospholipid mediator involved in inflammation. Upon oxidation of LDL, oxidized phospholipids with PAF-like structure are generated, and some of them may act via the PAF receptor. We evaluated the contribution of 1-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) and of other PAF analogs on the PAF-like bioactivity formed upon Cu2+-initiated oxidation of LDL. Reverse-phase HPLC purification and electrospray ionization-MS analyses showed that upon oxidation of LDL with inactivated PAF-acetylhydrolase (PAF-AH), C16:0 PAF accounted for >30% of PAF-like biological activity and its sn-2 butenoyl analog accounted for >50%. However, upon LDL oxidation in the presence of exogenous 1-0-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) without PAF-AH inactivation, C16:0 PAF formation accounted for >90% of the biological activity recovered. We suggest that the C16:0 PAF, despite being a minor constituent of the LDL peroxidation products, may contribute substantially to the bioactivity formed in oxidized LDL. The higher bioactivity of C16:0 PAF, and the higher selectivity of the LDL-attached lyso-PAF transacetylase toward very short acyl chains [acetate (C2) vs. butanate (C4)], may explain the contribution described above.     

Lysophosphatidylcholine and lyso-PAF display PAF-like activity derived from contaminating phospholipids

Lysophosphatidylcholine is an abundant component of plasma and oxidized LDL that displays several biological activities, some of which may occur through the platelet-activating factor (PAF) receptor. We find that commercial lysophosphatidylcholine, its alkyl homolog (lyso-PAF), and PAF all induce inflammation in a murine model of pleurisy. Hydrolysis of PAF to lyso-PAF by recombinant PAF acetylhydrolase abolished this eosinophilic infiltration, implying that lyso-PAF should not have displayed inflammatory activity. Saponification of lyso-PAF or PAF acetylhydrolase treatment of lyso-PAF or lysophosphatidylcholine abolished activity; neither lysolipid should contain susceptible sn-2 residues, suggesting contaminants account for the bioactivity. Lyso-PAF and to a lesser extent lysophosphatidylcholine stimulated Ca(2+) accumulation in 293 cells stably transfected with the human PAF receptor, and this was inhibited by specific PAF receptor antagonists. Again, treatment of lyso-PAF or lysophosphatidylcholine with recombinant PAF acetylhydrolase, a nonselective phospholipase A(2), or saponification of lyso-PAF destroyed the PAF-like activity, a result incompatible with lyso-PAF or lysophosphatidylcholine being the actual agonist.We conclude that neither lyso-PAF nor lysophosphatidylcholine is a PAF receptor agonist, nor are they inflammatory by themselves. We suggest that PAF or a PAF-like mimetic accounts for inflammatory effects of lysophosphatidylcholine and lyso-PAF.     

Extracellular phospholipases in atherosclerosis

Phospholipases A2 (PLA2) are a family of enzymes that catalyze the hydrolysis of the sn-2 ester bond of glycerophospholipids liberating lysophospholipids and free fatty acids; important second messengers involved in atherogenesis. Plasma PAF-acetylhydrolase (PAF-AH) or Lp-PLA2 is a Ca²⁺-independent PLA2 which is produced by monocyte-derived macrophages and by activated platelets, and circulates in plasma associated with lipoproteins. PAF-AH catalyzes the removal of the acetyl/short acyl group at the sn-2 position of PAF and oxidized phospholipids produced during inflammation and oxidative stress. In humans, PAF-AH is mainly associated with small dense LDL and to a lesser extent with HDL and with lipoprotein(a). PAF-AH is N-glycosylated prior to secretion which diminishes its association with HDL raising the question of its distribution between the proatherogenic LDL vs the antiatherogenic HDL. Hypercholesterolemic patients have higher plasma PAF-AH activity which is reduced upon hypolipidemic therapy. PAF-AH specific inhibitor darapladib stabilizes human and swine plaques, therefore challenging the antiatherogenic potential of PAF-AH shown in small animal models.     

Inflammatory platelet-activating factor-like phospholipids in oxidized low density lipoproteins are fragmented alkyl phosphatidylcholines.

Oxidation of human low density lipoprotein (LDL) generates proinflammatory mediators and underlies early events in atherogenesis. We identified mediators in oxidized LDL that induced an inflammatory reaction in vivo, and activated polymorphonuclear leukocytes and cells ectopically expressing human platelet-activating factor (PAF) receptors. Oxidation of a synthetic phosphatidylcholine showed that an sn-1 ether bond confers an 800-fold increase in potency. This suggests that rare ether-linked phospholipids in LDL are the likely source of PAF-like activity in oxidized LDL. Accordingly, treatment of oxidized LDL with phospholipase A(1) greatly reduced phospholipid mass, but did not decrease its PAF-like activity. Tandem mass spectrometry identified traces of PAF, and more abundant levels of 1-O-hexadecyl-2-(butanoyl or butenoyl)-sn-glycero-3-phosphocholines (C(4)-PAF analogs) in oxidized LDL that comigrated with PAF-like activity. Synthesis showed that either C(4)-PAF was just 10-fold less potent than PAF as a PAF receptor ligand and agonist. Quantitation by gas chromatography-mass spectrometry of pentafluorobenzoyl derivatives shows the C(4)-PAF analogs were 100-fold more abundant in oxidized LDL than PAF. Oxidation of synthetic alkyl arachidonoyl phosphatidylcholine generated these C(4)-PAFs in abundance. These results show that quite minor constituents of the LDL phosphatidylcholine pool are the exclusive precursors for PAF-like bioactivity in oxidized LDL.     

Plasma platelet activating factor-acetylhydrolase (PAF-AH)

The platelet-activating factor-acetylhydrolase (PAF-AH) is an enzyme which catalyzes the hydrolysis of acetyl ester at the sn-2 position of PAF. The family of PAF-AHs consists of two intracellular isoforms (Ib and II), and one secreted isoform (plasma). These PAF-AHs show different biochemical characteristics and molecular structures. Plasma PAF-AH and intracellular isoform, II degrade not only PAF but also oxidatively fragmented phospholipids with potent biological activities. Among these PAF-AHs, plasma PAF-AH has been the target of many clinical studies in inflammatory diseases, such as asthma, sepsis, and vascular diseases, because the plasma PAF-AH activity in the patients with these diseases is altered when compared with normal individuals. Finding a genetic deficiency in the plasma PAF-AH opened the gate in elucidating the protecting role of this enzyme in inflammatory diseases. The most common loss-of-function mutation, V279F, is found in more than 30% of Japanese subjects (4% homozygous, 27% heterozygous). This single nucleotide polymorphism in plasma PAF-AH and the resulting enzymatic deficiency is thought to be a genetic risk factor in various inflammatory diseases in Japanese subjects. Administration of recombinant plasma PAF-AH or transfer of the plasma PAF-AH gene improves pathology in animal models. Therefore, substitution of plasma PAF-AH would be an effective in the treatment of the patients with the inflammatory diseases and a novel clinical approach. In addition, the detection of polymorphisms in the plasma PAF-AH gene and abnormalities in enzyme activity would be beneficial in the diagnosis of the inflammatory diseases.     

To hydrolyze or not to hydrolyze: the dilemma of platelet-activating factor acetylhydrolase

Mounting ambiguity persists around the functional role of the plasma form of platelet-activating factor acetylhydrolase (PAF-AH). Because PAF-AH hydrolyzes PAF and related oxidized phospholipids, it is widely accepted as an anti-inflammatory enzyme. On the other hand, its actions can also generate lysophosphatidylcholine (lysoPC), a component of bioactive atherogenic oxidized LDL, thus allowing the enzyme to have proinflammatory capabilities. Presence of a canonical lysoPC receptor has been seriously questioned for a multitude of reasons. Animal models of inflammation show that elevating PAF-AH levels is beneficial and not deleterious and overexpression of PAF receptor (PAF-R) also augments inflammatory responses. Further, many Asian populations have a catalytically inert PAF-AH that appears to be a severity factor in a range of inflammatory disorders. Correlation found with elevated levels of PAF-AH and CVDs has led to the design of a specific PAF-AH inhibitor, darapladib. However, in a recently concluded phase III STABILITY clinical trial, use of darapladib did not yield promising results. Presence of structurally related multiple ligands for PAF-R with varied potency, existence of multi-molecular forms of PAF-AH, broad substrate specificity of the enzyme and continuous PAF production by the so called bi-cycle of PAF makes PAF more enigmatic. This review seeks to address the above concerns.     

The proinflammatory mediator Platelet Activating Factor is an effective substrate for human group X secreted phospholipase A2

Platelet Activating Factor (PAF) is a potent mediator of inflammation whose biological activity depends on the acetyl group esterified at the sn-2 position of the molecule. PAF-acetylhydrolase (PAF-AH), a secreted calcium-independent phospholipase A(2), is known to inactivate PAF by formation of lyso-PAF and acetate. However, PAF-AH deficient patients are not susceptible to the biological effects of inhaled PAF in airway inflammation, suggesting that other enzymes may regulate extracellular levels of PAF. We therefore examined the hydrolytic activity of the recently described human group X secreted phospholipase A(2) (hGX sPLA(2)) towards PAF. Among different sPLA(2)s, hGX sPLA(2) has the highest affinity towards phosphatidylcholine (PC), the major phospholipid of cellular membranes and plasma lipoproteins. Our results show that unlike group IIA, group V, and the pancreatic group IB sPLA(2), recombinant hGX sPLA(2) can efficiently hydrolyze PAF. The hydrolysis of PAF by hGX sPLA(2) rises abruptly when the concentration of PAF passes through its critical micelle concentration suggesting that the enzyme undergoes interfacial binding and activation to PAF. In conclusion, our study shows that hGX sPLA(2) may be a novel player in PAF regulation during inflammatory processes.     

Platelet-activating factor (PAF)-like oxidized phospholipids: relevance to atherosclerosis

Lipid peroxidation is involved in the pathogenesis of chronic diseases including atherosclerosis. Oxidized lipoprotein has diverse biological activities and is believed to initiate atheroma formation and maturate fatty plaque. The active components of oxidized lipoproteins still remain to be clarified, but a likely candidate is the phosphatidylcholine (PC) having an sn-2-short-chain acyl group with a methyl, hydroxyl, aldehydic or carboxylic terminal. These unique PCs, formed by oxidative fragmentation of the polyunsaturated acyl group of the parent PC in liposomes, low density lipoproteins and blood plasma, induce platelet aggregation through the activation of the receptor for platelet-activating factor (PAF), due to their resemblance in structure with PAF. We have found that PAF-like lipids regulate DNA synthesis and production of nitric oxide independently of the activation of the PAF receptor in vascular smooth muscle cells. Regulation of vascular cell function through two distinct signaling pathways mediated by PAF-like lipids provides new insight into the mechanism of induction of atherosclerosis.     

Lung production of platelet-activating factor acetylhydrolase in oleic acid-induced acute lung injury

Platelet-activating factor (PAF) is a proinflammatory mediator that plays a central role in acute lung injury (ALI). PAF- acetylhydrolases (PAF-AHs) terminate PAF's signals and regulate inflammation. In this study, we describe the kinetics of plasma and bronchoalveolar lavage (BAL) PAF-AH in the early phase of ALI. Six pigs with oleic acid induced ALI and two healthy controls were studied. Plasma and BAL samples were collected every 2h and immunohistochemical analysis of PAF-AH was performed in lung tissues. PAF-AH activity in BAL was increased at the end of the experiment (BAL PAF-AH Time 0=0.001+/-0.001 nmol/ml/min/g vs Time 6=0.031+/-0.018 nmol/ml/min/g, p=0.04) while plasma activity was not altered. We observed increased PAF-AH staining of macrophages and epithelial cells in the lungs of animals with ALI but not in healthy controls. Our data suggest that increases in PAF-AH levels are, in part, a result of alveolar production. PAF-AH may represent a modulatory strategy to counteract the excessive pro-inflammatory effects of PAF and PAF-like lipids in lung inflammation.     

An oxidized derivative of phosphatidylcholine is a substrate for the platelet-activating factor acetylhydrolase from human plasma

Platelet-activating factor (PAF) is a glycerophospholipid that has diverse potent biological actions. A plasma enzyme catalyzes the hydrolysis of the sn-2 acetoyl group of PAF and thereby abolishes its bioactivity. This PAF acetylhydrolase is specific for phospholipids, such as PAF, with a short acyl group at the sn-2 position. The majority of it (60-70%) is associated with low density lipoprotein (LDL), and the remainder is with high density lipoprotein (HDL). LDL also has a phospholipase A2 activity that is specific for oxidized polyunsaturated fatty acids, which may be important in determining how LDL is recognized by cellular receptors. We previously have purified and characterized the PAF acetylhydrolase from human plasma. We now have found that the purified PAF acetylhydrolase catalyzes the hydrolysis of the oxidized fragments of arachidonic acid from the sn-2 position of phosphatidylcholine. One of the preferred substrates appeared by mass spectrometry to have 5-oxovalerate at the sn-2 position. We synthesized 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine and found that the PAF acetylhydrolase had the same apparent Km for it (11.3 microM) as for PAF (12.5 microM), with Vmax values of 100 and 167 mumol/h/mg of protein, respectively. We also conclude that the PAF acetylhydrolase is the sole activity in LDL that degrades oxidized phospholipids since we found co-localization of the activity against both substrates to LDL and HDL, and precipitation of enzyme activity with an antibody to the PAF acetylhydrolase. Thus, the PAF acetylhydrolase in human plasma degrades oxidized phospholipids, which may be involved in the modification of apolipoprotein B100 and other pathological processes.     

Clinical aspects of plasma platelet-activating factor-acetylhydrolase

Plasma platelet-activating factor (PAF)-acetylhydrolase (PAF-AH), which is characterized by tight association with plasma lipoproteins, degrades not only PAF but also phospholipids with oxidatively modified short fatty acyl chain esterified at the sn-2 position. Production and accumulation of these phospholipids are associated with the onset of inflammatory diseases and preventive role of this enzyme has been evidenced by many recent studies including prevalence of the genetic deficiency of the enzyme in the patients and therapeutic effects of treatment with recombinant protein or gene transfer. With respect to the atherosclerosis, however, it is not fully cleared whether this enzyme plays an anti-atherogenic role or pro-atherogenic role because plasma PAF-AH also might produce lysophosphatidylcholine (LysoPC) and oxidatively modified nonesterified fatty acids with potent pro-inflammatory and pro-atherogenic bioactivities. These dual roles of plasma PAF-AH might be regulated by the altered distribution of the enzyme between low density lipoprotein (LDL) and high density lipoprotein (HDL) particles because HDL-associated enzymes are considered to contribute to the protection of LDL from oxidative modification. This review focuses on the recent findings which address the role of this enzyme in the human diseases especially including asthma, septic shock and atherosclerosis.     

Platelet-activating factor acetylhydrolases: broad substrate specificity and lipoprotein binding does not modulate the catalytic properties of the plasma enzyme

Platelet-activating factor acetylhydrolases (PAF-AHs) are a group of enzymes that hydrolyze the sn-2 acetyl ester of PAF (phospholipase A(2) activity) but not phospholipids with two long fatty acyl groups. Our previous studies showed that membrane-bound human plasma PAF-AH (pPAF-AH) accesses its substrate only from the aqueous phase, which raises the possibility that this enzyme can hydrolyze a variety of lipid esters that are partially soluble in the aqueous phase. Here we show that pPAF-AH has broad substrate specificity in that it hydrolyzes short-chain diacylglycerols, triacylglycerols, and acetylated alkanols, and displays phospholipase A(1) activity. On the basis of all of the substrate specificity results, it appears that the minimal structural requirement for a good pPAF-AH substrate is the portion of a glyceride derivative that includes an sn-2 ester and a reasonably hydrophobic chain in the position occupied by the sn-1 chain. In vivo, pPAF-AH is bound to high and low density lipoproteins, and we show that the apparent maximal velocity for this enzyme is not influenced by lipoprotein binding and that the enzyme hydrolyzes tributyroylglycerol as well as the recombinant pPAF-AH does. Broad substrate specificity is also observed for the structurally homologous PAF-AH which occurs intracellularly [PAF-AH(II)] as well as for the PAF-AH from the lower eukaryote Physarum polycephalum although pPAF-AH and PAF-AH(II) tolerate the removal of the sn-3 headgroup better than the PAF-AH from P. polycephalum does. In contrast, the intracellular PAF-AH found in mammalian brain [PAF-AH(Ib) alpha 1/alpha 1 and alpha 2/alpha 2 homodimers] is more selectively operative on compounds with a short acetyl chain although this enzyme also displays significant phospholipase A(1) activity.     

Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for platelet-activating factor.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) activates neutrophils (polymorphonuclear leukocytes, PMN) through a receptor that specifically recognizes short sn-2 residues. We oxidized synthetic [2-arachidonoyl]phosphatidylcholine to fragment and shorten the sn-2 residue, and then examined the phospholipid products for the ability to stimulate PMN. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine was fragmented by ozonolysis to 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine. This phospholipid activated human neutrophils at submicromolar concentrations, and is effects were inhibited by specific PAF receptor antagonists WEB2086, L659,989, and CV3988. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine next was fragmented by an uncontrolled free radical-catalyzed reaction: it was treated with soybean lipoxygenase to form its sn-2 15-hydroperoxy derivative (which did not activate neutrophils) and then allowed to oxidize under air. The secondary oxidation resulted in the formation of numerous fragmented phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103), some of which activated PMN. Hydrolysis of sn-2 residues with phospholipase A2 destroyed biologic activity, as did hydrolysis with PAF acetylhydrolase. PAF acetylhydrolase is specific for short or intermediate length sn-2 residues and does not hydrolyze the starting material (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103). Neutrophil activation was completely blocked by L659,989, a specific PAF receptor antagonist. We conclude that diacylphosphatidylcholines containing an sn-2 polyunsaturated fatty acyl residue can be oxidatively fragmented to species with sn-2 residues short enough to activate the PAF receptor of neutrophils. This suggests a new mechanism for the appearance of biologically active phospholipids, and shows that PAF receptor antagonists block the action of both PAF and these PAF-like lipids.     

Bovine platelet-activating factor acetylhydrolase (PAF-AH) activity related to fertility

Plasma platelet-activating factor acetylhydrolase (PAF-AH), the enzyme characterized by the association with plasma lipoproteins, degrades platelet-activating factor (PAF) as well as PAF-like oxidatively fragmented phospholipids produced during oxidative stress. Apart from pro-inflammatory properties, PAF is also related to reproductive processes and successful fertility. In order to get a better insight into the involvement of PAF-AH in the fertility of cows, the aim of the study was to determine the PAF-AH activity as well as the C-reactive protein, cholesterol and high density lipoprotein-cholesterol (HDL-C) in the serum of dairy cows throughout the pregnancy and lactation, as well as in infertile cows. The results showed that serum PAF-AH activity changes throughout pregnancy and lactation with a lower level during periparturient period. It is also found higher PAF-AH activity in lactating cows with reproductive disorders compared to high lactating cows without reproductive disorders. Strong correlation between PAF-AH activity and HDL-C concentration indicates that HDL could have considerable influence on PAF-AH activity in bovine plasma. CRP concentration was also lower during transition period suggesting that lactation might stimulate CRP synthesis in bovine. A higher CRP concentration in cows with reproductive disorders compared to fertile cows at the peak of lactation, demonstrates that milk production is not the only factor influencing CRP in cows. A significant correlation between PAF-AH activity and CRP level shows that both parameters could be influenced by reproductive status of dairy cows.     

Structural identification of phosphatidylcholines having an oxidatively shortened linoleate residue generated through its oxygenation with soybean or rabbit reticulocyte lipoxygenase

Phosphatidylcholines (PCs) with platelet-activating factor (PAF)-like biological activities are known to be generated by fragmentation of the sn-2-esterified polyunsaturated fatty acyl group. The reaction is free radical-mediated and triggered by oxidants such as metal ions, oxyhemoglobin, and organic hydroperoxides. In this study, we characterized the PAF-like phospholipids produced on reaction of PC having a linoleate group with lipoxygenase enzymes at low oxygen concentrations. When the oxidized PCs were analyzed by gas chromatography-mass spectrometry, two types of oxidatively fragmented PC were detected. One PC had an sn-2-short chain saturated or unsaturated acyl group (C(8)-C(13)) with an aldehydic terminal; the abundant species were PCs with C(9) and C(13). The other PC had a short chain saturated acyl group (C(6)-C(9)) with a methyl terminal, and the most predominant species was PC with C(8). When the extracts of oxidation products were subjected to catalytic hydrogenation, PCs having saturated acyl groups (C(6)-C(14)) were detected; the most abundant was C(12) species. The less regiospecific formation of PAF-like lipids suggests that they were generated by oxidative fragmentation of PC hydroperoxides formed by non-stereoselective oxygenation of the alkyl radical of esterified linoleate that escaped from the active centers of lipoxygenases. One of the PAF-like PC with an aldehydic terminal was found to be bioactive; it inhibited the production of nitric oxide induced by lipopolysaccharide and interferon-gamma in vascular smooth muscle cells from rat aorta.     

Endotoxins stimulate neutrophil adhesion followed by synthesis and release of platelet-activating factor in microparticles

Lipopolysaccharides and triacyl-cysteine-modified proteins of Gram-negative and positive organisms are potent endotoxins. Animal models show that the receptor for platelet-activating factor (PAF) is responsible for many of the deleterious effects of endotoxin, where regulated, localized PAF production localizes the inflammatory response. In contrast, biologically active analogs of PAF (PAF-like lipids) are generated by oxidative attack on phospholipids by chemical reactions that are unregulated and unlocalized. The identity and distribution of the PAF receptor ligand in endotoxemia is unknown. We found human polymorphonuclear leukocytes (PMNs) were a significant source of PAF receptor agonists after stimulation by either class of endotoxin. Production of PAF receptor agonists required that the PMN adhere to a surface, and adhesion (and therefore accumulation of PAF-like bioactivity) in response to endotoxic stimulation was delayed for several minutes. PAF-like oxidized phospholipids were found by mass spectroscopy, but biosynthetic PAF accounted for most of the phospholipid agonists arising from endotoxic stimulation. A significant portion of the PAF made by PMNs was secreted, in contrast to its near complete retention by other inflammatory cells. Endotoxic stimulation induced a respiratory burst with the production of superoxide and the formation and shedding of microparticles. Free and microparticle-bound PAF appeared in the media, and blocking microvesiculation with calpeptin blocked PAF release. The released material activated platelets, and platelets co-aggregated with endotoxin-stimulated PMNs. Adherent PMNs therefore behave differently than suspended cells and are a significant source of free PAF after endotoxin exposure. Leukocytes can couple endotoxic challenge to the widespread circulatory and inflammatory effects of endotoxin.     

Pioglitazone induces plasma platelet activating factor-acetylhydrolase and inhibits platelet activating factor-mediated cytoskeletal reorganization in macrophage

Platelet activating factor (PAF) is a key molecule for inflammation. To examine a role of peroxisome proliferator-activated receptor gamma (PPARgamma) in inflammatory reactions of atherosclerosis, we investigated the effects of 15-deoxy-(Delta12,14)-Prostaglandin J2 (15d-PGJ2) and pioglitazone, PPARgamma ligands, on plasma PAF-acetylhydrolase (PAF-AH) expression in THP-1 macrophages. PAF-AH mRNA and protein were up-regulated by the PPARgamma ligands. Prostaglandin F2alpha (PGF2alpha), a PARgamma inhibitor, abrogated the up-regulation of PAF-AH mRNA by pioglitazone, suggesting that PPARgamma activation is involved in the induction of PAF-AH by pioglitazone. As PAF promotes the cell motility with cytoskeletal reorganization, we investigated the effect of pioglitazone on PAF-mediated morphological changes in THP-1 macrophages. In the absence of pioglitazone, PAF promoted the elongation of actin cytoskeleton, which was inhibited by pretreatment with pioglitazone. In contrast, pioglitazone was not able to inhibit the morphological changes induced by C-PAF, a non-hydrolyzable PAF agonist. Thus, it is suggested that PAF-induced morphological changes could be inhibited by pioglitazone through PAF-AH, which rapidly hydrolyzed PAF. These data propose that PPARgamma/PAF-AH pathway is a clinical target for the prevention against atherosclerosis.     

Platelet-activating factor (PAF)-like activity, localization of PAF receptor (PAF-R) and PAF-acetylhydrolase (PAF-AH) activity in bovine endometrium at different stages of the estrous cycle and early pregnancy

PAF-like activity in the endometrium increased from days 2-4 to day 12 and day 20 in both cyclic and pregnant cows. There was an increase in platelet aggregation induced by PAF-like activity in the endometrium of pregnant animals on day 20 as compared to cyclic animals at the same point in time. Two major bands of PAF-R protein at 67 kDa and 97 kDa were detected by Western blot analysis. PAF-R was localized mainly in luminal and glandular epithelium of the endometrium, but the staining was markedly increased in the endometrium of pregnant cows on day 20 compared to cyclic animals on the same day. The purified PAF-AH from the endometrium is similar to in plasma. In cyclic cattle, no changes in PAF-AH activity of endometrium were observed, whereas a decrease in enzyme activity occurred in pregnant cows on day 20 as compared to cyclic animals on the same day. We suggest that the bovine endometrium produces PAF-like activity, expresses the PAF-R and possesses a PAF-AH activity which varies during pregnancy.