Platelet-Activating Factor and Related Lipids
The term 'platelet-activating factor' was first
introduced in 1972 to define the activity of a then unknown metabolite, which induced the aggregation of blood platelets released from
basophils stimulated with immunoglobulin E.
In 1979, independently in the laboratories of D.J. Hanahan, J. Benveniste and F. Snyder,
a phospholipid identified as 1‑O‑alkyl-2-acetyl-sn-glycero-3-phosphocholine, an ether analogue of
phosphatidylcholine, was shown to be responsible for this activity
and for the activity of a compound that had been termed ‘antihypertensive polar renal lipid’.
It was thus one of the first complex lipids known to have signalling properties and not simply to have a structural function in
membranes or to act via its hydrolysis products.
In the light of what is now known of the manifold biological activities of this lipid, platelet-activating factor or PAF is not an appropriate name, as it is present in many cell types, including those involved in host defence such as macrophages and mast cells, but it has stuck. It has vital functions in key physiological processes, but in contrast, it is a potent pro-inflammatory mediator that is implicated in innumerable disease states involving inflammation and cell damage or death.
PAF is an unusual lipid in many ways, although it can be considered to be a special case of the more abundant ether lipids with shared biosynthetic pathways. In general, the alkyl groups tend to be mainly saturated and C16 or C18 in chain-length, although vinyl ether (plasmalogen) forms have been detected. There are few other examples of acetic acid esterified directly to glycerol amongst natural lipids in animal tissues, and short-chain fatty acids other than acetate (e.g., propionyl, butyryl) are only occasionally found in position sn-2 of PAF. However, oxidatively truncated phospholipids, i.e., with a short-chain, ω-aldehydo-fatty acid in position sn⏹2 and formed by spontaneous scission of long-chain hydroperoxides, can have PAF-like activity. PAF is present at very low levels in unstimulated animal tissues, and it can be hard to detect experimentally, but it does not appear to be produced by plants.
1. Biosynthesis of Platelet-Activating Factor
PAF is synthesised by a variety of cells, but mainly those involved in host defence, such as platelets, endothelial cells, neutrophils, monocytes and macrophages, from alkylacyl-phospholipids synthesised in the endoplasmic reticulum after the process is begun in peroxisomes as with other ether lipids.
In the main two-step (so-called 'remodelling') pathway, which is always activated in both acute and chronic inflammation, distinct membrane-bound acetyl-CoA:lyso-PAF acetyltransferases (LPLAT8/9 or LPCAT1/2) in contact with the cytoplasm catalyse the transfer of an acetyl residue from acetyl-CoA to 1-O-alkyl-sn-glycerol-3-phosphocholine (lyso-PAF), generated by the action of a bifunctional phospholipase A2 on 1-O-alkyl-2-acyl-phosphatidylcholine, with a high specificity for those molecular species with arachidonic acid in position sn-2. There is thus a strong link between the metabolism of PAF and subsequent eicosanoid production. The acetyltransferase also acts as CoA-independent transacylase to relocate the cleaved arachidonate to position sn-2 of either lysophosphatidylcholine or lysophosphatidylethanolamine, although the lysoplasmalogen form of phosphatidylethanolamine is now believed to be the main acceptor before the vinyl bond is reduced and the head group is exchanged. In addition, lyso-PAF can be generated from 1-O-alkyl-2-acyl-phosphatidylcholine by the action of CoA-independent or CoA-dependent transacylases (reversal of an acyl-CoA acyltransferase reaction), a sequence of reactions that can be compared to the Lands' cycle.
PAF is synthesised continuously by cells but at low levels by LPLAT8, which is believed to be constitutively expressed in the lungs, where it produces PAF and dipalmitoyl-phosphatidylcholine essential for respiration under non-inflammatory conditions. The second isoform of the acetyltransferase (LPLAT9) is inducible and is considered to be pro-inflammatory, as it catalyses a very rapid synthesis of PAF in macrophages following phosphorylation of the enzyme by protein kinase Cα upon stimulation by lipopolysaccharides during bacterial infection or by endogenous G-protein-coupled receptor ligands. Only when the cells are subjected to acute inflammatory stimulation does the activated enzyme produce PAF in appreciable amounts as a possible critical step at the onset or in the early stages of inflammatory responses, with arachidonate released simultaneously for eicosanoid production.
There is an alternative biosynthetic mechanism for PAF production (‘de novo’ pathway) that involves first acetylation of 1-O-alkyl-sn-glycero-3-phosphate, i.e., an intermediate in the biosynthesis of ether lipids and a lysophosphatidic acid analogue, to form 1‑O‑alkyl-2-acetyl-sn-glycero-3-phosphate by means of a quite distinct acetyltransferase from that using lyso-PAF as substrate, i.e., an acetyl-CoA:alkyl-lysoglycerophosphate acetyltransferase. As with other ether lipids, the product is dephosphorylated by an alkylacetylglycerophosphate phosphohydrolase with formation of 1‑O‑alkyl-2-acetyl-sn-glycerol, which can be converted to PAF by a mechanism analogous to that for the biosynthesis of phosphatidylcholine but utilizing a CDP-choline alkylacetylglycerol cholinephosphotransferase distinct from that using diacylglycerols as substrate. This pathway occurs mainly in the brain and kidney and is believed to be important during continuous activation of inflammatory cascades as in the development of inflammation-related disorders. It does not generate free arachidonic acid for eicosanoid synthesis.
Production of PAF is limited by the activity of acetyl hydrolases (see below), and with the involvement of so many synthetic and catabolic mechanisms, regulation of PAF levels is a highly complicated process.
2. Biochemical Functions of Platelet-Activating Factor
After synthesis, PAF is transported to the plasma membrane, where it remains on the cell surface. PAF was one of the first intact phospholipids known to have messenger functions, i.e., in which the signalling results from the molecule binding to specific receptors, rather than from physico-chemical effects on the plasma membrane or other membranes of the cell. There is a strict structural requirement for binding to its unique trans-membrane G‑protein coupled receptor (PAF-R), which is expressed by numerous cells, including all those of the innate immune system, to promote downstream signalling on target cells. Thus, there is a specificity of nearly three orders of magnitude for the ether bond in position sn-1 of PAF in comparison to the 1-acyl analogue, together with considerable specificity for a short acyl chain in position sn-2 and for the phosphocholine head group. In endothelial cells, the receptor is found in both cell surface and large endosomal membranes and is coupled to intracellular Gαq and Gαi heterotrimeric G-proteins, which send distinct yet synergistic signals into target cells such as leukocytes and platelets. On the other hand, some of its activities appear to be independent of the receptor. In primitive marine animals such as corals, which do not possess platelets, PAF and lyso-PAF are produced in response to external stresses.
Initially, PAF was found to effect aggregation of platelets at concentrations as low as 10-11M following its release from immunoglobulin E-stimulated basophils, and it induced a hypertensive response at similarly low levels. Indeed, it is almost always active by 10-9M as an intercellular messenger, and more generally, it is now recognized that its primary role is to mediate intercellular interactions. For example, when PAF binds to its specific receptor (PAF-R), the Gq protein component of this combines with phospholipase Cβ to effect the hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol trisphosphate and diacylglycerol that causes an increase in intracellular Ca2+ downstream of the cell and activation of protein kinase C. In addition, the rise in Ca2+ activates phospholipase A2 (cPLA2α), leading to the release of arachidonic acid for synthesis of eicosanoids and of lysophosphatides, which can serve as substrates for further PAF synthesis. Signalling through PAF-R inhibits the conversion of ATP to cAMP by adenylate cyclase and prevents the activation of protein kinase A and its associated signalling events. In this manner, PAF is now known to exert effects on many different types of biological events and functions, including glycogen degradation, reproduction, brain and retinal function, and blood circulation. Although its inflammatory reactions have received most study, PAF has functions in the central system in relation to neuronal development and to long-term potentiation, a process that is essential for memory formation.
Much
recent work has been concerned with the function of PAF as a mediator of inflammation and in the mechanism of the immune response;
it can activate human inflammatory cells at concentrations as low as 10-14M.
Binding to its receptor on inflammatory cells induces very rapid (within 30 seconds) production of further PAF via enhanced activity
of LPLAT9 mediated by phosphorylation by protein kinase C, and in turn, the increased PAF levels stimulate subsequent inflammatory cascades.
The amount of PAF produced by cellular stimuli of various kinds is dependent on the nature of the cell and specific agonists.
Although it was once thought to be a hormone that acted locally, as it was found initially only on the surface of activated cells so restricting
the inflammatory response, it is now known to be transported in bioactive extracellular vesicles to other tissues to exert its effects.
As PAF can activate inflammasomes directly, i.e., independently of its receptor PAF‑R, to release the pro-inflammatory cytokines
IL-1β and IL-18, this may explain why some PAF-R antagonists are ineffective in blocking PAF-mediated inflammation in clinical trials.
PAF is presumed to have evolved as part of a protective mechanism in the innate host defence system, and its pro-inflammatory properties are necessary for the day-to-day protection of tissues from pathogenic insults. When produced in an uncontrolled manner, it can have harmful effects, and it has been implicated in the pathogenesis of several disease states, ranging from allergic reactions to stroke, sepsis, myocardial infarction, cancer, colitis, HIV infection, the effects of ultra-violet radiation, and in the central nervous system, multiple sclerosis and Alzheimer's disease amongst others. It has a key role in the destabilizing and rupture of atherosclerotic plaques that leads to acute cardiovascular events, and an increase in the concentration of PAF has been observed in the blood of patients with acute myocardial infarction and arrhythmia. In relation to asthma, platelet-activating factor can act directly as a chemotactic factor and indirectly by stimulating the release of other inflammatory agents. Administration of PAF can produce many of the symptoms observed in asthma, including bronchoconstriction, mucus hypersecretion and inflammation of bronchi, probably via the formation of leukotrienes as secondary mediators. Recently, PAF has been shown to be an anti-obesity factor, functioning through stimulation of its receptor in brown but not white adipose tissue, and reduction in this activity may be responsible for increasing adiposity with age. Of course, the eicosanoids produced as a by-product of PAF biosynthesis are also mediators of inflammation and may act synergistically with PAF.
As a pro-inflammatory mediator, PAF has been implicated in the development of cancer, especially that of the skin, where it is involved in transmitting the immunosuppressive signal of UV irradiation from the skin surface to the immune system in keratinocytes and thence in activating mast cell migration in vivo. Elevated levels of the PAF receptor are present in tumour cells and cells that infiltrate tumours with negative impacts upon the efficacies of chemotherapy and radiation therapy, resulting in promotion of tumour cell proliferation, production of survival signals, migration of vascular cells and formation of new vessels. In experimental models, it has been shown that blocking of the PAF receptor reduces tumour growth and increases animal survival. A synthetic analogue of PAF, 1‑O‑octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (‘edelfosine’) is a potent anticancer agent in animal models by causing selective apoptosis of cancer cells while sparing healthy cells, but it appears to be too toxic for use with humans. On the other hand, this may simply be the first step in the development of new ether-linked drugs, and analogues in which the phosphocholine moiety is replaced by a carbohydrate are showing promise for their therapeutic potential. Nutraceuticals containing natural PAF agonists are under development.
PAF is reported to be an important regulator of membrane channels and transporters (collectively named the transportome). By binding to its receptor, it enhances calcium entry into cells and drives neuronal development. It is a factor in ocular disorders, including age-related macular degeneration and diabetic retinopathy, and when in deficit, it can influence neurodegeneration. By increasing blood-brain barrier permeability, it facilitates inflammation.
The nature of the alkyl group in position sn-1 may be important to such processes, and it has been established that C16- and C18-PAF cause death to cerebellar granule neurons, although they signal through different pathways. In addition, PAF receptor signalling can be either pro- or anti-apoptotic, depending upon the nature of the sn-1 alkyl moiety, probably because of differential binding of each homologue to the receptor. Phosphatidylethanolamine analogues of PAF have been studied, but they are much less potent biologically. A further comparable signalling molecule, N‑acetylsphingosine, is produced by a CoA-independent transacetylase, which transfers the acetyl group of PAF to sphingosine (see our web page on ceramides). Although lyso-PAF can cause neurotoxicity and an inflammatory response, it may have opposing effects to PAF in relation to neutrophil superoxide production and platelet aggregation.
Alkylacetylglycerols, analogues of 1,2-diacyl-sn-glycerols and PAF precursors, have biological activities, some of which are independent of subsequent conversion to PAF. They have been shown to promote differentiation of cultured leukaemia cells and to affect the biological activity of many other cell types in vitro at least, although some of this activity may be due to formation of metabolites. Further, they can be phosphorylated to generate 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphate, a phosphatidic acid analogue, which is thought to compete with diacylglycerols to regulate protein kinase C signalling and/or its membrane location.
1-O-Acyl analogues of PAF, i.e., with a saturated fatty acid rather than an alkyl moiety in position sn-1, are produced in tissues in amounts that surpass that of PAF, and while they may have mild proinflammatory properties, their main function appears to be to attenuate and possibly regulate PAF signalling by acting as sacrificial substrates for PAF acetylhydrolases and perhaps as endogenous PAF-receptor antagonist/partial agonists. For example, administration of alkyl-PAF causes sudden death in Swiss albino mice, but this effect is suppressed by administering boluses of acyl-PAF at the same time. When assayed directly, depending on the bioassay system used, the difference in potency of the acyl analogue ranged from a 100-fold to a 2000-fold decrease relative to PAF per se.
3. Catabolism of Platelet-Activating Factor
Control of PAF concentration
and activity is regulated partly by tight control of its synthesis and partly by the action of specific
PAF acetylhydrolases, three of which exist with one in plasma and two intracellular and cytosolic
(all Ca2+-independent); they are classified as part of the large phospholipase A2 family of enzymes.
Their main function is to remove the acetyl group from PAF to eliminate its biological activity,
and they are not active against conventional phospholipids.
One of the intracellular forms (PAF-AH(I)) is enriched in brain and is completely specific for PAF but is required for spermatogenesis
and is increasingly recognized as an oncogenic factor.
The second intracellular isoform (PAF-AH(II)) is expressed in virtually all tissues, but most abundantly in the liver, kidney, intestine and testis, and it has a broader specificity in that, like the plasma form, it will hydrolyse truncated acyl moieties from oxidized phospholipids (see next section). In mast cells and many other cell types, this enzyme releases enzymatically oxidized fatty acids, including eicosanoids such as F2‑isoprostane residues and hydroperoxy- or epoxy-octadecadienoyl/eicosatrienoyl moieties (lipid mediators), that are esterified to phospholipids (oxidized phospholipids), so it may have a role in allergic diseases.
The third most abundant and best characterized PAF-acetylhydrolase is the plasma form, which is associated with both circulating LDL and HDL particles and functions on the lipid-aqueous interface, where it is sometimes termed the ‘lipoprotein-associated phospholipase A2’ (Lp‑PLA2 - group VII family). This is secreted constitutively by blood cellular components and to a lesser extent by liver cells, aorta cells and adipocytes, and it a 45 KDa protein, which circulates in plasma in its active form. All three enzymes hydrolyse unmodified fatty acyl residues up to 5 or 6 carbon atoms long in the sn-2 position also, albeit relatively slowly, although even this restriction is relaxed when the terminal-end of the fatty acyl moiety is oxidized (i.e., aldehydic or carboxylic), such as in the oxidatively truncated phospholipids. By removing the latter within cells, PAF acetylhydrolase protects cells from apoptosis. One further enzyme, alkaline sphingomyelinase, deactivates platelet-activating factor but by removing the phosphocholine moiety.
One effect of the plasma form may be to remove oxidized phospholipids from lipoproteins and atherosclerotic plaques that might otherwise contribute to their inflammatory properties. Thus, while oxysterols accumulate as atherosclerotic lesions mature, formation and destruction of oxidized phosphatidylcholines is a continuous process in both early and advanced lesions. In contrast, it has been suggested that the other products of PAF hydrolases, lysophosphatidylcholines, may be pro-atherogenic, and in support of the latter view, large-scale epidemiological studies have found that elevated plasma PAF-acetylhydrolase/Lp-PLA2 levels are associated with an increased risk of coronary disease, stroke and mortality. The balance between pro- and anti-inflammatory effects is obviously critical.
Expression of these enzymes is up-regulated at the transcriptional level by mediators of inflammation in response to inflammatory stimuli, but they are susceptible to oxidative inactivation. Decreased levels are associated with various diseases, including asthma, systemic Lupus erythematosus and Crohn’s disease. In clinical and preclinical studies, inhibition of Lp-PLA2 has shown promising therapeutic effects in diabetic macular oedema and Alzheimer’s disease.
PAF-acetylhydrolase has trans-acetylase activity and can transfer short-chain fatty acids from PAF to ether/ester-linked lysophospholipids. The ether linkage in the lysophospholipid can be cleaved oxidatively by the microsomal alkylglycerol monooxygenase to yield a fatty aldehyde, which is then further oxidized to the corresponding acid as described in our web page on ether lipids.
4. Oxidatively Truncated Phospholipids
PAF-like molecules with some biological activities are produced in tissues by non-enzymatic oxidation of polyunsaturated fatty acids in phospholipids and in phosphatidylcholine especially, resulting in cleavage near the first double bond to leave a short-chain acid with a terminal aldehyde group in position 2, a so‑called ‘core aldehyde’, together with a volatile aldehyde. This process is discussed in greater detail in our web page dealing with oxidized phospholipids. While the biosynthesis of PAF is tightly regulated, the various non-enzymatic oxidation reactions that produce core aldehydes are essentially uncontrolled. Such compounds are formed in plasma lipoproteins and human atherosclerotic lesions, and indeed, they were first identified as the bioactive components of oxidized LDL that mediate many of the pro-inflammatory and pro-atherogenic effects reported for these lipoproteins.
Oxidatively truncated phospholipids have been reported to possess a wide range of biological activities, many of which correspond to those of PAF. They bring about platelet aggregation at nanomolar concentrations by activating the PAF receptor, and they may be involved in thrombosis and acute coronary events by inducing proliferation of smooth muscle cells. By means of a mechanism that is independent of the PAF receptor, they are pro-apoptotic and have a substantial influence on regulated cell death, while as might be expected, they have a disruptive effect upon cell membranes. In contrast, they can prevent endotoxin shock induced by exposure to bacterial lipopolysaccharides in vivo.
Catabolism: The oxidized fatty acids in position sn-2 are removed by the PAF-acetylhydrolases described above to yield short-chain aldehydo fatty acids (unesterified) and lysophosphatidylcholine, and in plasma, the lecithin-cholesterol acyltransferase (LCAT) functions in a similar way, presumably as a detoxification mechanism for oxidized lipoproteins. Thirdly, the lysosomal phospholipase A2 can remove the fatty acids from position sn-1 of these lipids.
Suggested Reading
- Christie, W.W. and Han, X. Lipid Analysis - Isolation, Separation, Identification and Lipidomic Analysis (4th edition), 446 pages (Oily Press, Woodhead Publishing and now Elsevier) (2010) - see Science Direct.
- Damiani, E. and Ullrich, S.E. Understanding the connection between platelet-activating factor, a UV-induced lipid mediator of inflammation, immune suppression and skin cancer. Prog. Lipid Res., 63, 14-27 (2016); DOI.
- Davies, S.S. and Guo, L.L. Lipid peroxidation generates biologically active phospholipids including oxidatively N-modified phospholipids. Chem. Phys. Lipids, 181, 1-33 (2014); DOI.
- Deng, M., Guo, H., Tam, J.W., Johnson, B.M., Brickey, W.J., New, J.S., Lenox, A., Shi, H., Golenbock, D.T., Koller, B.H., McKinnon, K.P., Beutler, B. and Ting, J.P.-Y. Platelet-activating factor (PAF) mediates NLRP3-NEK7 inflammasome induction independently of PAFR. J. Exp. Med., 216, 2838-2853 (2019); DOI.
- Gill, P., Jindal, N.L., Jagdis, A. and Vadas, P. Platelets in the immune response: Revisiting platelet-activating factor in anaphylaxis. J. Allergy Clin. Immunol., 135, 1424-1432 (2015); DOI.
- Harishkumar, R., Hans, S., Stanton, J.E., Grabrucker, A.M., Lordan, R. and Zabetakis, I. Targeting the platelet-activating factor receptor (PAF-R): antithrombotic and anti-atherosclerotic nutrients. Nutrients, 14, 4414 (2022); DOI.
- Kimura, T., Jennings, W. and Epand, R.M. Roles of specific lipid species in the cell and their molecular mechanism. Prog. Lipid Res., 62, 75-92 (2016); DOI.
- Kono, N. and Arai, H. Platelet-activating factor acetylhydrolases: An overview and update. Biochim. Biophys. Acta, Lipids, 1864, 922-931 (2019); DOI.
- Lordan, R., Tsoupras, A., Zabetakis, I. and Demopoulos, C.A. Forty years since the structural elucidation of platelet-activating factor (PAF): historical, current, and future research perspectives. Molecules, 24, 4414 (2019); DOI.
- Marathe, G.K., Chaithra, V.H., Ke, L.-Y. and Chen, C.-H. Effect of acyl and alkyl analogs of platelet-activating factor on inflammatory signaling. Prostaglandins Other Lipid Mediators, 151, 106478 (2020); DOI.
- Pantazi, D., Tellis, C. and Tselepis, A.D. 'SI: PAF' Oxidized phospholipids and lipoprotein-associated phospholipase A2 (Lp-PLA2) in atherosclerotic cardiovascular disease: An update. Biofactors, 48, 1257-1270 (2022); DOI.
- Ramakrishnan, A.V.K.P., Varghese, T.P., Vanapalli, S., Nair, N.K. and Mingate, M.D. Platelet activating factor: A potential biomarker in acute coronary syndrome? Card. Ther., 35, 64-70 (2017); DOI.
- Rangholia, N., Leisner, T.M. and Holly, S.P. Bioactive ether lipids: primordial modulators of cellular signaling. Metabolites, 11, 41 (2021); DOI.
- Travers, J.B., Rohan, J.G. and Sahu, R.P. New insights into the pathologic roles of the platelet-activating factor system. Front. Endocrinol., 11, 624132 (2021); DOI.
- Upton, J.E.M., Grunebaum, E., Sussman, G. and Vadas, P. Platelet activating factor (PAF): a mediator of inflammation. Biofactors, 48, 1189-1202 (2022); DOI.
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