review To hydrolyze or not to hydrolyze: the dilemma of plateletactivating factor acetylhydrolase Gopal Kedihitlu Marathe,1 Chaitanya Pandit,2 Chikkamenahalli Lakshminarayana Lakshmikanth, Vyala Hanumanthareddy Chaithra, Shancy Petsel Jacob, and Cletus Joseph Michael D’Souza Department of Studies in Biochemistry, University of Mysore, Manasagangothri, Mysore 570006, India

Supplementary key words oxidized phospholipids • platelet-activating factor mimetics • inflammation • platelet-activating factor receptor • cardiovascular disease

PLATELET-ACTIVATING FACTOR ACETYLHYDROLASE Regulated inflammatory responses are defensive and homeostatic, while dysregulated responses are deleterious and impair homeostasis. A key molecule involved in both This work was supported by the University Grants Commission, New Delhi, India [MRP No.F: 41-1284/2012(SR)], Special Assistance Program (SAP) [No. F3-14/2012 (SAP II)], and Vision Group in Science and Technology [VGST/ K-FIST (2010-11)/ GRD-36/2013-14], Government of Karnataka, India. Manuscript received 8 November 2013 and in revised form 22 May 2014. Published, JLR Papers in Press, May 23, 2014 DOI 10.1194/jlr.R045492

regulated and dysregulated inflammation is platelet-activating factor (PAF) (1, 2), structurally identified as 1-alkyl-2acetyl-sn-glycero-3-phosphocholine (Fig. 1A), and its less pharmacologically active acyl analog, 1-acyl-2-acetyl-sn-glycero3-phosphocholine (Fig. 1B). Several structural analogs of PAF, called PAF mimetics, are also documented and are implicated in inflammation. However, all of them are hydrolyzed to the biologically inactive lysoPAF/lysophosphatidylcholine (lysoPC) metabolites by a family of enzymes called PAF acetylhydrolase (PAF-AH) (1–4). The abundant and thoroughly characterized enzyme in this family is the plasma form of PAF-AH [also called lipoprotein-associated phospholipase A2 (PLA2) and group VII PLA2]. The majority of the plasma enzyme (two-thirds) circulates in the plasma bound to LDLs, while the remaining plasma enzyme associates with HDLs and other lipoproteins (5). The plasma PAFAH catalyzes the hydrolysis of acetate (in the case of PAF and acyl PAF) or other substituents at the sn-2 position that exist in oxidized phospholipids, including PAF mimetics. The plasma PAF-AH is a 440 amino acid long protein, with an apparent molecular mass of 45 kDa (5, 6) that varies with the extent of glycosylation. Three PAF-AH isoenzymes have been described to date. One of the enzymes belongs to the subclass PLA2s classified under group VII that includes the plasma form, while the other two enzymes belonging to group VIII are intracellular (3, 7, 8). This localization of the enzymes is not strict, as plasma PAF-AH enzyme is also found intracellularly, and intracellular enzymes are also detected as circulating PAF-AH (9).

SPECTRUM OF SUBSTRATES FOR PAF-AH A salient feature of PAF-AH, unlike other PLA2s, is the absence of a calcium requirement for its full enzymatic

Abbreviations: APC, activated protein C; GPCR, G protein-coupled receptor; lysoPC, lysophosphatidylcholine; PAF, platelet-activating factor; PAF-AH, platelet-activating factor acetylhydrolase; PAF-R, plateletactivating factor receptor; PC, phosphatidylcholine; PLA2, phospholipase A2; PON-1, paraoxonase-1. 1 To whom correspondence should be addressed. e-mail: [email protected] 2 Present address of C. Pandit: Applied Nutrition Division, Defence Food Research Laboratory, Siddharth Nagar, Mysore 570011, India.

Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at

Journal of Lipid Research Volume 55, 2014


Downloaded from at Iowa State Univ Library, on October 18, 2014

Abstract 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 enigThis review seeks to address the above matic. concerns.—Marathe, G. K., C. Pandit, C. L. Lakshmikanth, V. H. Chaithra, S. P. Jacob, and C. J. M. D’Souza. To hydrolyze or not to hydrolyze: the dilemma of platelet-activating factor acetylhydrolase. J. Lipid Res. 2014. 55: 1847–1854.

varying affinities to activate PAF-R, hence popularly referred as PAF mimetics/PAF-like lipids (11, 19–21). However, these oxidized phospholipids accumulate during oxidative insult, as they are formed nonenzymatically, leading to exaggerated inflammatory responses that are also curtailed by the action of PAF-AH (6, 7, 11). Interestingly, noncanonical PAF-R ligands, such as lipoteichoic acid (a component of gram positive bacteria) and endotoxin lipopolysaccharide (a component of gram negative bacteria), may also bind to PAF- R, although with much lower affinity when compared with PAF (22–24).


activity, which allows it to be constitutively active. Another feature of PAF-AH is its inability to hydrolyze intact long chain fatty acids at the sn-2 position of the phospholipids. This allows the plasma PAF-AH to circulate without causing any damage to the cellular components or to lipoproteins (2, 7). With a better understanding of PAF biology and the process of inflammation, it is now clear that the substrate specificity of PAF-AH is vastly relaxed beyond PAF to an array of related molecules. An extreme example includes complex phospholipids such as long chain phospholipid hydroperoxides and isoprostane-containing PCs (Fig. 2) (10). Such modified phospholipids have been identified in oxidized LDLs (11–13); in models of oxidative insult such as alcoholic blood (14), smokers blood (15), and electronegative LDLs (16); and in models of cutaneous inflammation (17). Notable among these lipids are the oxidatively truncated phospholipids butanoyl/butenoyl PAF/PC (11), azelaoyl PAF/PC (18, 19), palmitoyl glutaroyl PC, palmitoyl oxovaleryl PC (12), kodia PC (13), and many more (19) (Table 1). Although PAF is recognized by PAF receptor (PAF-R) at subnanomolar concentrations (11, 20), some of the above mentioned molecules also bind to PAF-R with

BETWEEN THE SUBSTRATE AND PRODUCT OF PAF-AH Fig. 2. Chemical structure of esterified F2 isoprostane PC. A bulky sn-2 group derived from arachidonic acid is present in this phospholipid. PAF-AH can also recognize this as a substrate, but is a poor ligand to PAF-R.


Journal of Lipid Research Volume 55, 2014

PAF and truncated oxidized phospholipids, including PAF mimetics, are among the commonly identified substrates of the plasma form of PAF-AH (2–4, 7, 9, 32, 33).

Downloaded from at Iowa State Univ Library, on October 18, 2014

Fig. 1. Chemical structure of PAF. A: Alkyl analog of PAF. B: Acyl analog of PAF. Alkyl PAF has a nonhydrolyzable ether link at the sn-1 position with hexadecyl or octadecyl moiety and a short chain acetyl group at the sn-2 position. The acyl analog of PAF has a hydrolyzable (sensitive to PLA1) moiety at the sn-1 position. The remaining structural features are common to both. The acyl analog of PAF is several folds less active than the alkyl analog of PAF in a variety of bioassays.

The incidence of CVD in individuals without hypercholesterolemia in the recent past provides a compelling reason to look beyond the traditional risk factors of atherosclerosis. Also, the failure of lipid lowering agents to effectively reduce the risk associated with CVD universally has prompted scientists across the globe to investigate atherosclerosis in a novel dimension, namely inflammation (25–27). A common feature of the various factors causing atherosclerosis is oxidative stress (28). Lipid molecules bearing PUFAs esterified to phospholipids are energetically favored targets for oxidation. Thus, LDL particles generally implied in CVD are oxidized and are no longer native to the body. It is the nature of the human body to effectively clear molecules of foreign nature. The innate immune system polices this task by mounting an inflammatory response (25, 26). In order to effectively eliminate oxidized LDL, monocytes/macrophages are recruited. It is during this phase that the trapped LDL undergoes further oxidation leading to endothelial dysfunction, formation of foam cells (26), and setting into motion a cascade of events leading to atherosclerotic streak formation. Macrophages, a prolific source of PAF-AH, may have a vital role in curtailing the PAF-R-mediated responses by hydrolyzing PAF-R ligands and other oxidized phospholipids during these events (29). In this regard, it is worth mentioning that paraoxonase-1 (PON-1), an esterase associated exclusively with HDL, was once claimed to hydrolyze oxidized phospholipids including PAF (30, 31). Later studies conclusively demonstrated that neither PAF nor oxidized phospholipids are hydrolyzed by PON-1, but trace amounts of the plasma form of PAF-AH copurifying with PON-1 during isolation was responsible for the observed hydrolysis (32). Moreover, recombinant PON-1, though containing significant esterase activities toward synthetic esters, is totally devoid of the claimed PAF or oxidized phospholipid hydrolyzing ability (33). What PON-1 does in HDL remains elusive.


Chemical structures of oxidatively truncated phospholipids

Downloaded from at Iowa State Univ Library, on October 18, 2014

Free radical-mediated oxidative attack generates a host of truncated phospholipids. Oxidatively modified LDL, apoptotic cells are rich sources of these phospholipids. All of them are sensitive to PAF-AH and also recognize PAF-R with varying potency.

PAF is an early mediator of inflammation that is produced rapidly upon appropriate proinflammatory stimulus (1, 2). PAF activates a variety of cells of the innate immune system where it enhances the migratory and adhesive behavior of these cells, enabling them to transmigrate the endothelial

barrier through juxtacrine signaling (34, 35). Being potent, PAF exerts its effects at subnanomolar concentrations through a single cell surface G protein-coupled receptor (GPCR) (11, 20). Thus, not only PAF synthesis but also its subsequent hydrolysis to the biologically inactive To hydrolyze or not to hydrolyze: the dilemma of PAF-AH



Journal of Lipid Research Volume 55, 2014

to oxidation of phospholipids or may reach to millimolar levels in the case of hyperlipidemic subjects (53, 54). Most of this lysoPC is bound to albumin and to lipoprotein particles. However, it is important to note that the optimal concentration of lysoPC required to elicit reported biological responses is in the range of 10–50 ␮M. Thus, the concentration of lysoPC in the plasma is already higher than its action range and further addition of lysoPC should logically be ineffective. As mentioned, lysoPC binds to albumin, other plasma proteins, and even to cells; the real free lysoPC concentration in vivo will always be lower than the measured concentration. Second, the proatherogenic properties of lysoPC probably stem from the experiments that utilized commercial preparations of the molecule that are likely to be contaminated with PAF or PAF mimetics (55). Given the high potency of PAF and trace amount of PAF contaminants, lysoPC may manifest many of the PAF actions through PAF-R. In fact, lysoPC responses are blocked by PAF-R antagonists (55, 56). In a carefully performed study (55), it was proved that PAF present in trace amounts as contaminants in these commercial preparations was the reason for the inflammatory properties of lysoPC/lysoPAF. Moreover, the credibility of lysoPC receptor is seriously questioned (57, 58).

RISK FACTOR OR A RISK MARKER? An important observation that came from WOSCOPS (West of Scotland Coronary Prevention Study) caused increased ambiguity concerning the anti-inflammatory nature of the plasma PAF-AH. This study reported a correlation between elevated levels of PAF-AH and the severity of CVDs (59). On the contrary, additional trials failed to reproduce these findings [reviewed in (28)]. We believe that the increased level of the enzyme serves a protective function based on both in vitro and in vivo experiments and observation from PAF-AH-deficient subjects. For example, retroviral introduction of the plasma form of PAF-AH reduces atherogenesis in a murine model (60). Endothelial cells exposed to electronegative LDL pretreated with PAF-AH were protected from undergoing apoptosis, suggesting again the protective role of PAF-AH (16). Elevating the circulating levels of PAF-AH by exogenous administration was also found to be beneficial (61). Conversely, transgenic mice overexpressing the PAF-R exhibited increased bronchoconstriction (62) and susceptibility to develop spontaneous melanoma (63), while the PAF-R-null mice were less susceptible to systemic inflammation and acid inspiration-induced lung injury (64, 65). More importantly, in a recently concluded phase III STABILITY (Stabilisation of Atherosclerotic Plaque by Initiation of DarapladibTherapy) trial of 16,000 patients by GlaxoSmithKline involving a tightly controlled multi-center study with chronic coronary heart diseases, darapladib, a specific PAF-AH inhibitor, did not yield promising results. The drug failed to produce a statistically significant improvement in the risk of heart attack, stroke, or death, though it added greater reductions for some of the secondary

Downloaded from at Iowa State Univ Library, on October 18, 2014

product, lysoPAF/lysoPC, by the action of PAF-AH is an important mechanism employed to prevent an exaggerated inflammatory response, and hence PAF-AH is aptly termed as “signal terminator” (36, 37). Oxidative stress leads to an uncontrolled nonenzymatic chemical attack of the PUFAs in the phospholipids generating a pool of PAF mimetics and other oxidized phospholipids (11, 20, 38). Amid a sea of various other oxidation products produced during LDL oxidation, butanoyl and butenoyl species (Table 1) account for the bulk of the PAF activity, and these PAF mimetics were identified to be up to 1/10th as potent as PAF (11). In a case control study, mean plasma PAF levels of 23.8 pg/ml were reported in healthy subjects while CVD patients had elevated PAF levels of 49.7 pg/ml (39). Studies by Ninio and her coworkers detected the presence of PAF preferentially in intermediate LDL during LDL oxidation, where it reached up to 8.6 ± 5.7 pmol/mg in 3 h of oxidation (40). In another report, serum PAF levels were directly correlated with severity of anaphylaxis, where the PAF levels rose up to 805 ± 595 pg/ml in patients while control subjects had a basal levels of 127 ± 104 pg/ml (41). The oxidized phospholipids could be a part of the cellular membrane or the lipoprotein particle. In either case, monitoring its effective removal by the action of PAF-AH is important due to the damage they cause by forming whiskers in membranes (42). Considering the proinflammatory properties of the substrates of PAF-AH, it appears important that the enzyme be maximally active to protect cells from uncontrolled oxidative/inflammatory damage. In this regard, it is not surprising that the enzyme is constitutively active. Moreover, overexpression of the enzyme confers cell survival, which otherwise would undergo cell death in response to an oxidative insult (43). Consistent with the concept that this enzyme serves a protective role, some forms of PAF-AH even change their subcellular location to potentially allow the removal of PAF/PAF mimetics at the very site of their origin. For example, measurable translocation of the type 2b enzyme from cytosol to plasma membrane in models of oxidative stress has been shown (44, 45). LysoPC, the product of PAF-AH action when acyl PAF/ diacyl phospholipid oxidation products are the substrates, has gained much attention in the past (46). Although acyl PAF is a moderate PAF-R ligand (it is just several fold less active than its alkyl counterpart), its hydrolytic product, lysoPC, is not an effective PAF-R agonist. However, lysoPC is believed to possess a variety of activities such as induction of cytokine synthesis (47), augmented migration of monocytes (48), chemoattraction in smooth muscle cells (49), etc.; all processes are potentially proatherogenic. Presently, a proinflammatory (hence proatherogenic) property is also ascribed to lysoPC/lysoPAF that not only questions the very anti-inflammatory nature of the PAFAH, but also the proinflammatory nature of PAF/PAF mimetics (50, 51). However, a “proinflammatory” status cannot be assigned to lysoPC for two simple reasons. First, the normal serum concentration of lysoPC is between 140 and 150 ␮M (52); this value might increase by 40–50% owing

inflammation (77), for example the promoter of the PAFAH gene is positively regulated by PAF and negatively regulated by interferon ␥ and lipopolysaccharide [reviewed in (28)]. Because the final levels of PAF-AH activity are due to the result of both positive and negative modulators, it is difficult to assign precisely whether PAF-AH is a risk factor or a risk marker. The elevated levels of the enzyme probably help in curtailing the ill effects of the increased PAF and oxidized phospholipids during atherosclerosis by hydrolyzing it to a less harmful lysoPC/lysoPAF. Therefore, the increased serum concentration of the enzyme is most likely to be a potential risk marker.

MAKING AND BREAKING PAF AND PAF MIMETICS: BI-CYCLE OF PAF AND PAF MIMETICS Despite the fact that lysoPC is not proatherogenic and PAF-AH is not a true risk factor, whether PAF-AH is a friend or a foe is still a debated issue (78, 79). It may be possible that overwhelming levels of acyl PAF and its mimetics may have a role in curtailing PAF-R activation. In fact, endothelial cells predominantly make acyl PAF when stimulated with proinflammatory agonists (80, 81). In any cell engaged in PAF biosynthesis, acyl PAF constitutes a major part of the PAF pool under normal conditions and alkyl PAF accounts for a very minor fraction. This is also true in cell free systems undergoing oxidation, such as oxidation of LDL (11). The acyl PAF can also bind and activate PAF-R at high concentration and is hydrolyzed by the same PAF-AH that also hydrolyzes alkyl PAF (1, 2, 4). The difference in the relative abundance of the two species of PAF is subjected to the availability of the precursor molecules. It is now believed that PAF (acyl and alkyl) is continuously produced at basal levels irrespective of a stimulus due to membrane remodeling (82). But the continuous PAF synthesis is counteracted by constitutive PAF-AHmediated degradation (82). Also noteworthy is that acyl PAF is far less potent than alkyl PAF, and upon binding to PAF-R, presumably silences the cells. This is evident from the observation that PLA1 treatment of a lipid extract possessing PAF-like activity increases its activity several fold after the removal of diacyl PCs including acyl PAF (11). These two features are among a variety of remarkable mechanisms that the nature employs to ensure homeostasis. Thus, acyl PAF and acyl PAF mimetics may act as biological antagonists of PAF-R under normal conditions to keep PAF-sensitive cells quiescent. Making and breaking alkyl and acyl PAF can be thought of as a bi-cycle of PAF (Fig. 3) involving two different cycles each producing the respective species of PAF. The rate of the reactions producing acyl PAF is higher than that of the alkyl PAF. Thus, it is apparent that under normal conditions, the two wheels of the PAF bi-cycle operate under different rates. Considering the potency of alkyl PAF acting at subnanomolar concentrations upon a suitable proinflammatory stimulus, it is obvious that the rate of production of alkyl PAF increases only by a small degree while the increased rate of acyl PAF production is To hydrolyze or not to hydrolyze: the dilemma of PAF-AH


Downloaded from at Iowa State Univ Library, on October 18, 2014

endpoints. Currently, GlaxoSmithKline is thoroughly investigating this data for subgroup differences and an additional SOLID-TIMI 52 (Stabilization of Plaques using Darapladib-Thrombolysis in Myocardial Infarction 52) trial of 13,000 patients is ongoing. Better understanding of the complex inflammatory disorders in general, especially those that involve the PAF signaling system, is a prerequisite prior to developing novel drugs targeting PAF-AH (66). The relevance of PAF and PAF-AH in health and disease is observed not just in CVDs, but in a host of diseases affecting the respiratory, dermal, gastrointestinal, pancreatic, and other systems. Both elevated and decreased activities of PAF-AH have been reported in a variety of diseased conditions. For example, dynamic variations in endogenous levels of PAF-AH occur over time in both experimental sepsis and in critically ill sepsis patients (67). In one study involving genetically deficient plasma PAFAH mice, initially mice were protected from mortality when exposed to bacteria, but later developed significant necrotizing enterocolitis when compared with wild-type mice (68). On the other hand, decreased levels of the enzyme are associated with a number of diseases such as asthma, systemic Lupus erythematosus, and Crohn’s disease [reviewed in (4)]. More than the murine examples, humans from Asian populations, who are deficient in circulating plasma PAF-AH due to a common mutation at position 994 of the PLA2G7 gene resulting in valine to phenylalanine substitution (V279F), are at an increased risk to develop a range of inflammatory disorders (69). Unexpectedly, using recombinant PAF-AH in sepsis patients did not decrease the mortality rate, as reported by Opal et al. (70) in their phase III clinical trial. This study was carried out in comparison with activated protein C (APC), the only critical care drug that was available for sepsis. However, the population that Opal et al. (70) studied was at a low mortality risk when compared with the previous APC study. Unfortunately, the recombinant form of APC (commercially sold as Xigris) that was in use until now, has recently been withdrawn, leaving the critical care physicians without a drug to treat sepsis (71). Outcomes from previous clinical sepsis trials using anti-PAF agents also were not of much promise. This has raised the question of whether the PAF signaling pathway should be targeted in sepsis (72). More recently other targets have also been identified, for example GPCRs coupled to endothelial G␣q/ G␣11 signaling and spingosine-1-phosphate-dependent Gi signaling may play a critical role in mediating lethal responses to anaphylactic mediators such as PAF (73, 74). The plasma PAF-AH is susceptible to oxidant attack and suffers inactivation from modification of the residues that contribute to enzymatic activity (75). Whether highly variable levels of circulating PAF-AH arise from oxidation in the general population is not currently known. In an elegant study (76), variation in PAF-AH levels is not due to variations in the efficiency of transcription, translation, and mRNA stability, but due to the presence or absence of the N-terminal domain. Additionally, expression of plasma PAF-AH is transcriptionally modulated by the mediators of

unlikely to make a significant difference as far as PAF-R activation is concerned (Fig. 3). Thus, the sheer abundance of acyl PAF enables it as the obvious target of PAFAH action. Under normal conditions, though PAF-AH is engaged in hydrolyzing acyl PAF, low levels of alkyl PAF may transiently bind to the PAF-R. But, these low levels are unlikely to elicit an inflammatory response. Upon proinflammatory stimulation, the rate of acyl PAF synthesis goes up as does the synthesis of alkyl PAF. Now PAFAH is busy in hydrolyzing overwhelming levels of acyl PAF, leaving alkyl PAF to effectively bind to PAF-R, resulting in a transient inflammatory response. Under oxidative insult conditions, free radical-mediated oxidative attack generates a plethora of oxidized phospholipids, all of which are PAF-AH substrates and some of which are also excellent PAF-R ligands (PAF mimetics). These oxidized phospholipids amplify the inflammatory responses. Failure of the PAF-AH to efficiently hydrolyze alkyl PAF and PAF mimetics due to their low abundance amid a pool of abundant acyl PAF and acyl PAF mimetics might lead to an exaggerated but unnecessary inflammatory response. This is evident in murine models of inflammation wherein massive amounts of recombinant PAF-AH (approximately 10-fold over the endogenous PAF-AH levels) had to be administered in order to generate a noninflammatory phenotype (36, 60). A dedicated PAF synthase is yet to be described (83). Thus we propose that the ambiguity is not concerning the nature of the products formed from PAF-AH hydrolysis or the elevated levels of PAF-AH during CVD, but rather the heterogeneous group of molecules that can act as agonists/antagonists for the PAF-R and as substrates for PAF-AH. We remain 1852

Journal of Lipid Research Volume 55, 2014

incompletely informed of the biology of this enigmatic molecule. The authors are thankful to Drs. Stephen M. Prescott (Oklahoma Medical Research Foundation, OK), Jeffrey B. Travers (Indiana University, Indianapolis, IN), Thomas M. McIntyre (Lerner Research Institute, Cleveland, OH), and K. Madhusudan (Department of Biochemistry, University of Mysore, Manasagangotri, Mysore) for their critical comments.

REFERENCES 1. Prescott, S. M., and G. A. Zimmerman. 1990. Platelet-activating factor. J. Biol. Chem. 265: 17381–17384. 2. Prescott, S. M., G. A. Zimmerman, D. M. Stafforini, and T. M. McIntyre. 2000. Platelet-activating factor and related lipid mediators. Annu. Rev. Biochem. 69: 419–445. 3. Stafforini, D. M. 2009. Biology of platelet-activating factor acetylhydrolase (PAF-AH, lipoprotein associated phospholipase A2). Cardiovasc. Drugs Ther. 23: 73–83. 4. Tjoelker, L. W., and D. M. Stafforini. 2000. Platelet-activating factor acetylhydrolases in health and disease. Biochim. Biophys. Acta. 1488: 102–123. 5. Stafforini, D. M., T. M. McIntyre, M. E. Carter, and S. M. Prescott. 1987. Human plasma platelet-activating factor acetylhydrolase. Association with lipoprotein particles and role in the degradation of platelet-activating factor. J. Biol. Chem. 262: 4215–4222. 6. Tjoelker, L. W., C. Eberhardt, J. Unger, H. L. Trong, G. A. Zimmerman, T. M. McIntyre, D. M. Stafforini, S. M. Prescott, and P. W. Gray. 1995. Plasma platelet-activating factor acetylhydrolase is a secreted phospholipase A2 with a catalytic triad. J. Biol. Chem. 270: 25481–25487. 7. McIntyre, T. M., S. M. Prescott, and D. M. Stafforini. 2009. The emerging roles of PAF acetylhydrolase. J. Lipid Res. 50: S255–S259. 8. Dennis, E. A. 1997. The growing phospholipase A2 superfamily of signal transduction enzymes. Trends Biochem. Sci. 22: 1–2.

Downloaded from at Iowa State Univ Library, on October 18, 2014

Fig. 3. Bi-cycle of PAF. Resting cells make minute amounts of alkyl PAF (smaller circle) and relatively more acyl PAF (bigger circle) from alkyl/acyl PC and diacyl PC, respectively, via PLA2 action. A nonspecific PAF synthase/acetyltransferase catalyzes the formation of acyl PAF and alkyl PAF. Continuous hydrolysis of both analogs by PAF-AH makes PAF undetectable in resting cells. Upon proinflammatory stimulus, relative abundance of alkyl/acyl PAF goes up. This elevated amount of PAF may not be susceptible for effective hydrolysis by PAF-AH leading to the accumulation of PAF. Although acyl PAF may act as PAF-R antagonist, transient increased accumulation of alkyl PAF over basal levels may evoke an inflammatory response. Under oxidative insult conditions, oxidant attack (dashed lines) may inactivate PAF-AH, leading to generation of truncated oxidized phospholipids all of which are PAF-AH substrates and some of which are also excellent PAF-R ligands (PAF mimetics). Hence, PAF-AH is a real signal terminator and unlikely to be a risk factor.

28. Rosenson, R. S., and D. M. Stafforini. 2012. Modulation of oxidative stress, inflammation, and atherosclerosis by lipoprotein- associated phospholipase A2. J. Lipid Res. 53: 1767–1782. 29. Elstad, M. R., D. M. Stafforini, T. M. McIntyre, S. M. Prescott, and G. A. Zimmerman. 1989. Platelet-activating factor acetylhydrolase increases during macrophage differentiation. A novel mechanism that regulates accumulation of platelet-activating factor. J. Biol. Chem. 264: 8467–8470. 30. Watson, A. D., J. A. Berliner, S. Y. Hama, B. N. La Du, K. F. Faull, A. M. Fogelman, and M. Navab. 1995. Protective effect of high density lipoprotein associated paraoxonase. Inhibition of the biological activity of minimally oxidized low density lipoprotein. J. Clin. Invest. 96: 2882–2891. 31. Rodrigo, L., B. Mackness, P. N. Durrington, A. Hernandez, and M. I. Mackness. 2001. Hydrolysis of platelet-activating factor by human serum paraoxonase. Biochem. J. 354: 1–7. 32. Marathe, G. K., G. A. Zimmerman, and T. M. McIntyre. 2003. Platelet-activating factor acetylhydrolase, and not paraoxonase-1, is the oxidized phospholipid hydrolase of high density lipoprotein particles. J. Biol. Chem. 278: 3937–3947. 33. Kriska, T., G. K. Marathe, J. C. Schmidt, T. M. McIntyre, and A. W. Girotti. 2007. Phospholipase action of platelet-activating factor acetylhydrolase, but notparaoxonase-1, on long fatty acyl chain phospholipid hydroperoxides. J. Biol. Chem. 282: 100–108. 34. Zimmerman, G. A., S. M. Prescott, and T. M. McIntyre. 1992. Endothelial cell interactions with granulocytes: tethering and signalling molecules. Immunol. Today. 13: 93–100. 35. Alon, R., M. Aker, S. Feigelson, M. Sokolovsky-Eisenberg, D. E. Staunton, G. Cinamon, V. Grabovsky, R. Shamri, and A. Etzioni. 2003. A novel genetic leukocyte adhesion deficiency in sub second triggering of integrin avidity by endothelial chemokines results in impaired leukocyte arrest on vascular endothelium under shear flow. Blood. 101: 4437–4445. 36. Tjoelker, L. W., C. Wilder, C. Eberhardt, D. M. Stafforini, G. Dietsch, B. Schimpf, S. Hooper, H. Le Trong, L. S. Cousens, G. A. Zimmerman, et al. 1995. Anti-inflammatory properties of a plateletactivating factor acetylhydrolase. Nature. 374: 549–553. 37. Bazan, N. G. 1995. Inflammation. A signal terminator. Nature. 374: 501–502. 38. Marathe, G. K., K. A. Harrison, R. C. Murphy, S. M. Prescott, G. A. Zimmerman, and T. M. McIntyre. 2000. Bioactive phospholipid oxidation products. Free Radic. Biol. Med. 28: 1762–1770. 39. Zheng, G. H., S. Q. Xiong, L. J. Mei, H. Y. Chen, T. Wang, and J. F. Chu. 2012. Elevated plasma platelet activating factor, platelet activating factor acetylhydrolase levels and risk of coronary heart disease or blood stasis syndrome of coronary heart disease in Chinese: a case control study. Inflammation. 35: 1419–1428. 40. Tsoukatos, D. C., M. Arborati, T. Liapikos, K. L. Clay, R. C. Murphy, M. J. Chapman, and E. Ninio. 1997. Copper-catalyzed oxidation mediates PAF formation in human LDL subspecies. Protective role of PAF: acetylhydrolase in dense LDL. Arterioscler. Thromb. Vasc. Biol. 17: 3505–3512. 41. Vadas, P., M. Gold, B. Perelman, G. M. Liss, G. Lack, T. Blyth, F. E. Simons, K. J. Simons, D. Cass, and J. Yeung. 2008. Platelet-activating factor, PAF acetylhydrolase, and severe anaphylaxis. N. Engl. J. Med. 358: 28–35. 42. Greenberg, M. E., X. M. Li, B. G. Gugiu, X. Gu, J. Qin, R. G. Salomon, and S. L. Hazen. 2008. The lipid whisker model of the structure of oxidized cell membranes. J. Biol. Chem. 283: 2385–2396. 43. Latchoumycandane, C., G. K. Marathe, R. Zhanq, and T. M. McIntyre. 2012. Oxidatively truncated phospholipids are required agents of tumor necrosis factor ␣ (TNF␣)-induced apoptosis. J. Biol. Chem. 287: 17693–17705. 44. Matsuzawa, A., K. Hattori, J. Aoki, H. Arai, and K. Inoue. 1997. Protection against oxidative stress-induced cell death by intracellular platelet-activating factor-acetylhydrolase II. J. Biol. Chem. 272: 32315–32320. 45. Marques, M., Y. Pei, M. D. Southall, J. M. Johnston, H. Arai, J. Aoki, T. Inoue, H. Seltmann, C. C. Zouboulis, and J. B. Travers. 2002. Identification of platelet-activating factor acetylhydrolase II in human skin. J. Invest. Dermatol. 119: 913–919. 46. Huang, Y. H., L. Schäfer-Elinder, R. Wu, H. E. Claesson, and J. Frostegård. 1999. Lysophosphatidylcholine (LPC) induces proinflammatory cytokines by a platelet-activating factor (PAF) receptordependent mechanism. Clin. Exp. Immunol. 116: 326–331. 47. Nishi, E., N. Kume, H. Ochi, H. Moriwaki, S. Higashiyama, N. Taniguchi, and T. Kita. 1997. Lysophosphatidylcholine induces

To hydrolyze or not to hydrolyze: the dilemma of PAF-AH


Downloaded from at Iowa State Univ Library, on October 18, 2014

9. Zhou, G., G. K. Marathe, B. Willard, and T. M. McIntyre. 2011. Intracellular erythrocyte platelet-activating factor acetylhydrolase I inactivates aspirin in blood. J. Biol. Chem. 286: 34820–34829. 10. Stafforini, D. M., J. R. Sheller, T. S. Blackwell, A. Sapirstein, F. E. Yull, T. M. McIntyre, J. V. Bonventre, S. M. Prescott, and L. J. Roberts. 2006. Release of free F2-isoprostanes from esterified phospholipids is catalyzed by intracellular and plasma platelet-activating factor acetylhydrolases. J. Biol. Chem. 281: 4616–4623. 11. Marathe, G. K., S. S. Davies, K. A. Harrison, A. R. Silva, R. C. Murphy, H. Castro-Faria-Neto, S. M. Prescott, G. A. Zimmerman, and T. M. McIntyre. 1999. Inflammatory platelet-activating factor-like phospholipids in oxidized low density lipoproteins are fragmented alkyl phosphatidylcholines. J. Biol. Chem. 274: 28395–28404. 12. Watson, A. D., N. Leitinger, M. Navab, K. F. Faull, S. Hörkkö, J. L. Witztum, W. Palinski, D. Schwenke, R. G. Salomon, W. Sha, et al. 1997. Structural identification by mass spectrometry of oxidized phospholipids in minimally oxidized low density lipoprotein that induce monocyte/endothelial interactions and evidence for their presence in vivo. J. Biol. Chem. 272: 13597–13607. 13. Podrez, E. A., E. Poliakov, Z. Shen, R. Zhang, Y. Deng, M. Sun, P. J. Finton, L. Shan, M. Febbraio, D. P. Hajjar, et al. 2002. A novel family of atherogenic oxidized phospholipids promotes macrophage foam cell formation via the scavenger receptor CD36 and is enriched in atherosclerotic lesions. J. Biol. Chem. 277: 38517–38523. 14. Yang, L., C. Latchoumycandane, M. R. McMullen, B. T. Pratt, R. Zhang, B. G. Papouchado, L. E. Nagy, A. E. Feldstein, and T. M. McIntyre. 2010. Chronic alcohol exposure increases circulating bioactive oxidized phospholipids. J. Biol. Chem. 285: 22211–22220. 15. Lehr, H. A., A. S. Weyrich, R. K. Saetzler, A. Jurek, K. E. Arfors, G. A. Zimmerman, S. M. Prescott, and T. M. McIntyre. 1997. Vitamin C blocks inflammatory platelet-activating factor mimetics created by cigarette smoking. J. Clin. Invest. 99: 2358–2364. 16. Chen, C. H., T. Jiang, J. H. Yang, W. Jiang, J. Lu, G. K. Marathe, H. J. Pownall, C. M. Ballantyne, T. M. McIntyre, P. D. Henry, et al. 2003. Low-density lipoprotein in hypercholesterolemic human plasma induces vascular endothelial cell apoptosis by inhibiting fibroblast growth factor 2 transcription. Circulation. 107: 2102–2108. 17. Marathe, G. K., C. Johnson, S. D. Billings, M. D. Southall, Y. Pei, D. Spandau, R. C. Murphy, G. A. Zimmerman, T. M. McIntyre, and J. B. Travers. 2005. Ultraviolet B radiation generates platelet-activating factor-like phospholipids underlying cutaneous damage. J. Biol. Chem. 280: 35448–35457. 18. Davies, S. S., A. V. Pontsler, G. K. Marathe, K. A. Harrison, R. C. Murphy, J. C. Hinshaw, G. D. Prestwich, A. S. Hilaire, S. M. Prescott, G. A. Zimmerman, et al. 2001. Oxidized alkyl phospholipids are specific, high affinity peroxisome proliferator-activated receptor gamma ligands and agonists. J. Biol. Chem. 276: 16015–16023. 19. Chen, R., X. Chen, R. G. Salomon, and T. M. McIntyre. 2009. Platelet activation by low concentrations of intact oxidized LDL particles involves the PAF receptor. Arterioscler. Thromb. Vasc. Biol. 29: 363–371. 20. Marathe, G. K., G. A. Zimmerman, S. M. Prescott, and T. M. McIntyre. 2002. Activation of vascular cells by PAF-like lipids in oxidized LDL. Vascul. Pharmacol. 38: 193–200. 21. Smiley, P. L., K. E. Stremler, S. M. Prescott, G. A. Zimmerman, and T. M. McIntyre. 1991. Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for plateletactivating factor. J. Biol. Chem. 266: 11104–11110. 22. Zhang, Q., N. Mousdicas, Q. Yi, M. Al-Hassani, S. D. Billings, S. M. Perkins, K. M. Howard, S. Ishii, T. Shimizu, and J. B. Travers. 2005. Staphylococcal lipoteichoic acid inhibits delayed-type hypersensitivity reactions via the platelet-activating factor receptor. J. Clin. Invest. 115: 2855–2861. 23. Lemjabbar, H., and C. Basbaum. 2002. Platelet-activating factor receptor and ADAM10 mediate responses to Staphylococcus aureus in epithelial cells. Nat. Med. 8: 41–46. 24. Nakamura, M., Z. Honda, I. Waga, T. Matsumoto, M. Noma, and T. Shimizu. 1992. Endotoxin transduces Ca2+ signaling via plateletactivating factor receptor. FEBS Lett. 314: 125–129. 25. Ross, R. 1999. Atherosclerosis - an inflammatory disease. N. Engl. J. Med. 340: 115–126. 26. Choudhury, R. P., J. M. Lee, and D. R. Greaves. 2005. Mechanisms of disease: macrophage-derived foam cells emerging as therapeutic targets in atherosclerosis. Nat. Clin. Pract. Cardiovasc. Med. 2: 309–315. 27. Brewer, H. B., Jr. 2000. The lipid-laden foam cell: an elusive target for therapeutic intervention. J. Clin. Invest. 105: 703–705.

48. 49.


51. 52.




57. 58. 59.








Journal of Lipid Research Volume 55, 2014


67. 68.



71. 72. 73.





78. 79. 80.




responses with intact sensitivity to endotoxin in mice lacking a platelet-activating factor receptor. J. Exp. Med. 187: 1779–1788. Serruys, P. W., H. M. García-García, P. Buszman, P. Erne, S. Verheye, M. Aschermann, H. Duckers, O. Bleie, D. Dudek, H. E. Bøtker, et al. 2008. Effects of the direct lipoprotein-associated phospholipase A2 inhibitor darapladib on human coronary atherosclerotic plaque. Circulation. 118: 1172–1182. Yost, C. C., A. S. Weyrich, and G. A. Zimmerman. 2010. The platelet activating factor (PAF) signalling cascade in systemic inflammatory responses. Biochimie. 92: 692–697. Lu, J., M. Pierce, A. Franklin, T. Jilling, D. M. Stafforini, and M. Caplan. 2010. Dual roles of endogenous platelet-activating factor acetylhydrolase in a murine model of necrotizing enterocolitis. Pediatr. Res. 68: 225–230. Stafforini, D. M., K. Satoh, D. L. Atkinson, L. W. Tjoelker, C. Eberhardt, H. Yoshida, T. Imaizumi, S. Takamatsu, G. A. Zimmerman, T. M. McIntyre, et al. 1996. Platelet-activating factor acetylhydrolase deficiency. A missense mutation near the active site of an anti-inflammatory phospholipase. J. Clin. Invest. 97: 2784–2791. Opal, S., P. F. Laterre, E. Abraham, B. Francois, X. Wittebole, S. Lowry, J. F. Dhainaut, B. Warren, T. Dugernier, A. Lopez, et al. 2004. Recombinant human platelet-activating factor acetylhydrolase for treatment of severe sepsis: results of a phase III, multicenter, randomized, double-blind, placebo-controlled clinical trial. Crit. Care Med. 32: 332–341. Williams, S. C. P. 2012. After Xigris, researchers look to new targets to combat sepsis. Nat. Med. 18: 1001. Minneci, P. C., K. J. Deans, S. M. Banks, P. Q. Eichacker, and C. Natanson. 2004. Should we continue to target the platelet-activating factor pathway in septic patients? Crit. Care Med. 32: 585–588. Camerer, E., J. B. Regard, I. Cornelissen, Y. Srinivasan, D. N. Duong, D. Palmer, T. H. Pham, J. S. Wong, R. Pappu, and S. R. Coughlin. 2009. Sphingosine-1-phosphate in the plasma compartment regulates basal and inflammation-induced vascular leak in mice. J. Clin. Invest. 119: 1871–1879. Korhonen, H., B. Fisslthaler, A. Moers, A. Wirth, D. Habermehl, T. Wieland, G. Schutz, N. Wettschureck, I. Fleming, and S. Offermanns. 2009. Anaphylactic shock depends on endothelial Gq/G11. J. Exp. Med. 206: 411–420. MacRitchie, A. N., A. A. Gardner, S. M. Prescott, and D. M. Stafforini. 2007. Molecular basis for susceptibility of plasma platelet-activating factor acetylhydrolase to oxidative inactivation. FASEB J. 21: 1164–1176. Gardner, A. A., E. C. Reichert, T. S. Alexander, M. K. Topham, and D. M. Stafforini. 2010. Novel mechanism for regulation of plasma platelet-activating factor acetylhydrolase expression in mammalian cells. Biochem. J. 428: 269–279. Cao, Y., D. M. Stafforini, G. A. Zimmerman, T. M. McIntyre, and S. M. Prescott. 1998. Expression of plasma platelet-activating factor acetylhydrolase is transcriptionally regulated by mediators of inflammation. J. Biol. Chem. 273: 4012–4020. Chen, C-H. 2004. Platelet-activating factor acetylhydrolase: is it good or bad for you? Curr. Opin. Lipidol. 15: 337–341. Karabina, S. A., and E. Ninio. 2006. Plasma PAF-acetylhydrolase: an unfulfilled promise? Biochim. Biophys. Acta. 1761: 1351–1358. Whatley, R. E., K. L. Clay, F. H. Chilton, M. Triggiani, G. A. Zimmerman, T. M. McIntyre, and S. M. Prescott. 1992. Relative amounts of 1-O-alkyl- and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in stimulated endothelial cells. Prostaglandins. 43: 21–29. McIntyre, T. M., G. A. Zimmerman, K. Satoh, and S. M. Prescott. 1985. Cultured endothelial cells synthesize both platelet-activating factor and prostacyclin in response to histamine, bradykinin, and adenosine triphosphate. J. Clin. Invest. 76: 271–280. Chen, J., L. Yang, J. M. Foulks, A. S. Weyrich, G. K. Marathe, and T. M. McIntyre. 2007. Intracellular PAF catabolism by PAF acetylhydrolase counteracts continual PAF synthesis. J. Lipid Res. 48: 2365–2376. Shindou, H., D. Hishikawa, H. Nakanishi, T. Harayama, S. Ishii, R. Taguchi, and T. Shimizu. 2007. A single enzyme catalyzes both platelet-activating factor production and membrane biogenesis of inflammatory cells. Cloning and characterization of acetyl-CoA: LYSO-PAF acetyltransferase. J. Biol. Chem. 282: 6532–6539.

Downloaded from at Iowa State Univ Library, on October 18, 2014


heparin-binding epidermal growth factor-like growth factor and interferon-gamma in human T-lymphocytes. Ann. N. Y. Acad. Sci. 811: 519–524. Liu-Wu, Y., E. Hurt-Camejo, and O. Wiklund. 1998. Lysophosphatidylcholine induces the production of IL-1beta by human monocytes. Atherosclerosis. 137: 351–357. Chai, Y. C., P. H. Howe, P. E. DiCorleto, and G. M. Chisolm. 1996. Oxidized low density lipoprotein and lysophosphatidylcholine stimulate cell cycle entry in vascular smooth muscle cells. Evidence for release of fibroblast growth factor-2. J. Biol. Chem. 271: 17791–17797. Jeong, Y. I., I. D. Jung, C. M. Lee, J. H. Chang, S. H. Chun, K. T. Noh, S. K. Jeong, Y. K. Shin, W. S. Lee, M. S. Kang, et al. 2009. The novel role of platelet-activating factor in protecting mice against lipopolysaccharide-induced endotoxic shock. PLoS ONE. 4: e6503–e6512. Bochkov, V. N., A. Kadl, J. Huber, F. Gruber, B. R. Binder, and N. Leitinger. 2002. Protective role of phospholipid oxidation products in endotoxin-induced tissue damage. Nature. 419: 77–81. Rabini, R. A., R. Galassi, P. Fumelli, N. Dousset, M. L. Solera, P. Valdiguie, G. Curatola, G. Ferretti, M. Taus, and L. Mazzanti. 1994. Reduced Na(+)-K(+)-ATPase activity and plasma lysophosphatidylcholine concentrations in diabetic patients. Diabetes. 43: 915–919. Chen, L., B. Liang, D. E. Froese, S. Liu, J. T. Wong, K. Tran, G. M. Hatch, D. Mymin, E. A. Kroeger, R. Y. Man, et al. 1997. Oxidative modification of low density lipoprotein in normal and hyperlipidemic patients: effect of lysophosphatidylcholine composition on vascular relaxation. J. Lipid Res. 38: 546–553. Chisolm, G. M., and Y. Chai. 2000. Regulation of cell growth by oxidized LDL. Free Radic. Biol. Med. 28: 1697–1707. Marathe, G. K., A. R. Silva, H. C. Faria Neto, L. W. Tjoelker, S. M. Prescott, G. A. Zimmerman, and T. M. McIntyre. 2001. Lysophosphatidylcholine and lyso-PAF display PAF-like activity derived from contaminating phospholipids. J. Lipid Res. 42: 1430–1437. Ogita, T., Y. Tanaka, T. Nakaoka, R. Matsuoka, Y. Kira, M. Nakamura, T. Shimizu, and T. Fujita. 1997. Lysophosphatidylcholine transduces Ca2+ signaling via the platelet-activating factor receptor in macrophages. Am. J. Physiol. 272: H17–H24. Kabarowski, J. H., K. Zhu, L. Q. Le, O. N. Witte, and Y. Xu. 2001. Lysophosphatidylcholine as a ligand for the immunoregulatory receptor G2A. Science. 293: 702–705. Witte, O. N., J. H. Kabarowski, Y. Xu, L. Q. Le, and K. Zhu. 2005. Retraction. Science. 307: 206. Packard, C. J., D. S. O’Reilly, M. J. Caslake, A. D. McMahon, I. Ford, J. Cooney, C. H. Macphee, K. E. Suckling, M. Krishna, F. E. Wilkinson, et al. 2000. Lipoprotein-associated phospholipase A2 as an independent predictor of coronary heart disease. N. Engl. J. Med. 343: 1148–1155. Quarck, R., B. De Geest, D. Stengel, A. Mertens, M. Lox, G. Theilmeier, C. Michiels, M. Raes, H. Bult, D. Collen, et al. 2001. Adenovirus-mediated gene transfer of human platelet-activating factor-acetylhydrolase prevents injury-induced neointima formation and reduces spontaneous atherosclerosis in apolipoprotein E-deficient mice. Circulation. 103: 2495–2500. Henderson, W. R., Jr., J. Lu, K. M. Poole, G. N. Dietsch, and E. Y. Chi. 2000. Recombinant human platelet-activating factor acetylhydrolase inhibits airway inflammation and hyperreactivity in mouse asthma model. J. Immunol. 164: 3360–3367. Nagase, T., S. Ishii, H. Shindou, Y. Ouchi, and T. Shimizu. 2002. Airway hyperresponsiveness in transgenic mice overexpressing platelet activating factor receptor is mediated by an atropine-sensitive pathway. Am. J. Respir. Crit. Care Med. 165: 200–205. Sato, S., K. Kume, C. Ito, S. Ishii, and T. Shimizu. 1999. Accelerated proliferation of epidermal keratinocytes by the transgenic expression of the platelet-activating factor receptor. Arch. Dermatol. Res. 291: 614–621. Nagase, T., S. Ishii, K. Kume, N. Uozumi, T. Izumi, Y. Ouchi, and T. Shimizu. 1999. Platelet-activating factor mediates acid-induced lung injury in genetically engineered mice. J. Clin. Invest. 104: 1071–1076. Ishii, S., T. Kuwaki, T. Nagase, K. Maki, F. Tashiro, S. Sunaga, W. H. Cao, K. Kume, Y. Fukuchi, K. Ikuta, et al. 1998. Impaired anaphylactic

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 hydro...
743KB Sizes 1 Downloads 3 Views