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Platelet-Activating Factor and Related Lipids

Formula of platelet-activating factorThe term 'platelet-activating factor' was introduced 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’. In the light of what is now known of the manifold biological activities of this lipid, platelet-activating factor or PAF is not an especially appropriate name, as it is present in many cell types, especially those involved in host defense such as macrophages, mast cells, neutrophils and so forth, but it has stuck. PAF can be considered a special case of the more abundant ether lipids. It is present at very low levels in unstimulated animal tissues and can be hard to detect experimentally.

It is an unusual lipid in many ways, as lipids with alkyl groups only in position sn-1 are not common in animals, and there are few other examples of acetic acid esterified directly to glycerol amongst natural lipids in animal tissues. In general, the alkyl groups tend to be mainly saturated and C16 or C18 in chain-length, although vinyl ether (plasmalogen) forms have also been detected. Short-chain fatty acids other than acetate (e.g. propionyl, butyryl) in position sn-2 are only occasionally found. While both PAF and lyso-PAF were reported to be present in plants in one study, others have not been able to detect ether lipids. In addition, oxidatively truncated phospholipids can have PAF-like activity.

Biosynthesis of Platelet-Activating Factor

PAF is synthesised by a variety of cells, but especially those involved in host defence, such as platelets, endothelial cells, neutrophils, monocytes, and macrophages. In the main (so-called 'remodelling') pathway, a distinct membrane-bound acetyl-CoA:lyso-PAF acetyltransferase (lyso-PAFAT) in contact with the cytoplasm catalyses 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; this enzyme also acts as CoA-independent transacylase to relocate the cleaved arachidonate to position sn-2 of either lyso-phosphatidylcholine or lyso-phosphatidylethanolamine, regardless of the nature of the radyl moiety on position sn-1.

Biosynthesis of platelet-activating factor

A second lyso-PAFAT, lysophosphatidylcholine acyltransferase 2 (LPCAT2) catalyses a very rapid synthesis of PAF in macrophages following phosphorylation of the enzyme by protein kinase Cα after stimulation by bacterial infection or by endogenous G protein-coupled receptor ligands. This may be a critical step at the onset or in the early stages of inflammatory responses. In addition, lyso-PAF can also be generated by the action of CoA-independent or CoA-dependent (reversal of an acyl-CoA acyltransferase reaction) transacylases.

PAF is synthesised continuously by cells but at low levels by these pathways, and the reaction is regulated by the activity of acetyl hydrolases (see below). However, it is produced in much greater quantities by inflammatory cells when required in response to cell-specific stimuli. Studies with the purified acetyltransferase have shown that with cells in the resting state, the enzyme can utilize arachidonoyl-CoA to produce the membrane-bound PAF precursor 1-O-alkyl-2-arachidonoylglycerol-phosphocholine with even greater facility than the generation of PAF per se. Only when the cells are subjected to acute inflammatory stimulation does the activated enzyme produce PAF in appreciable amounts, probably after phosphorylation by a protein kinase, while arachidonate is released simultaneously for eicosanoid production.

A further lyso-PAF acetyltransferase (LPCAT1) is expressed in the lungs mainly, where it produces PAF and dipalmitoyl-phosphatidylcholine essential for respiration under non-inflammatory conditions. This is a constitutively expressed enzyme, while LPCAT2 is inducible.

There is an alternative second biosynthetic mechanism for PAF production that involves first acetylation of 1-O-alkyl-sn-glycero-3-phosphate to form 1-O-alkyl-2-acetyl-glycero-3-phosphate by means of a quite distinct acetyltransferase from that using lyso-PAF as substrate; the product is dephosphorylated with formation of 1-O-alkyl-2-acetyl-glycerol, which can be converted to PAF by a mechanism analogous to the biosynthesis of phosphatidylcholine. This ‘de novo’ pathway occurs mainly in the brain and kidney and does not generate free arachidonic acid for eicosanoid synthesis; it is believed to be non-inflammatory.

Biochemical Functions of Platelet-Activating Factor

PAF was the first intact phospholipid 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 single trans-membrane G‑protein coupled receptor (PAF-R), which is expressed by numerous cells including all those of the innate immune system, and for recognition as a substrate by enzymes. 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. On the other hand, some of its activities appear to be independent of the receptor.

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 very low levels also. More generally, it is now recognized that its primary role is to mediate intercellular interactions. For example, when PAF binds to its specific receptor, the Gq protein component of this combines with phospholipase Cβ to effect the hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol trisphosphate and diacylglycerol. The result of the latter is an increase in intracellular Ca2+ downstream of the cell and activation of protein kinase C. In this manner, it is now known to exert effects on many different types of biological events and functions, including glycogen degradation, reproduction, brain function and blood circulation.

Scottish thistleMuch 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. While it is presumed to have evolved as part of a protective mechanism in the innate host defence system, there is particular interest in its involvement in uncontrolled pathological conditions where it can have harmful effects. For example, it has a number of pro-inflammatory properties, and in excess it has been implicated in the pathogenesis of a number of disease states, ranging from allergic reactions to stroke, sepsis, myocardial infarction, colitis and HIV infection, and in the central nervous system, multiple sclerosis and Alzheimer's disease amongst others. Thus, it has a key role in the destabilizing and rupture of atherosclerotic plaques that leads to acute cardiovascular events. In relation to asthma, platelet-activating factor is able to 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. Also 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. However, the amount of PAF produced by cellular stimuli of various kinds is dependent on the nature of the cell and specific agonists. In essence it is a hormone that acts locally, as it is found only on the surface of activated cells so restricting the inflammatory response. Of course, the eicosanoids produced as a byproduct of PAF biosynthesis are also mediators of inflammation and may act synergistically with PAF.

Recently, PAF has been shown to be an anti-obesity factor, functioning through stimulation of its receptor in brown but not white adipose tissue. Reduction in this activity may be responsible for increasing adiposity with age.

The nature of the alkyl group in position sn-1 may be important to such processes. For example, It has been established that C16-PAF and C18-PAF cause death to cerebellar granule neurons, but that 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. A synthetic analogue of PAF, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (‘edelfosine’) is a potent anticancer agent in animal models, but appears too toxic for use with humans. However, analogues in which the phosphocholine moiety is replaced by a carbohydrate are showing therapeutic promise. Similarly, PAF receptor antagonists are under study for their therapeutic potential.

Alkylacetylglycerols, analogues of 1,2-diacyl-sn-glycerols, have biological activity also, some of which is independent of subsequent conversion to PAF. 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).

Catabolism of Platelet-Activating Factor

Catabolism of platelet-activating factorControl of PAF concentration and activity is regulated partly by tight control of its synthesis, and partly by the action of specific PAF acetylhydrolases, two small sub-families of which exist (both Ca2+-independent) and are classified as part of the large phospholipase A2 family of enzymes. These are not active against conventional phospholipids, and their main function is to remove the acetyl group from PAF thus eliminating its biological activity. One isoform of the sub-family of group VIII PAF-acetylhydrolases is enriched in brain and erythrocytes, it is cytosolic and it is completely specific for PAF. A second isoform is present mainly in liver and kidney, where it is located in both the cytosol and membranes, and has a broader specificity in that it will also hydrolyse truncated acyl moieties from oxidized phospholipids (see next section). Both isoforms function intracellularly.

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’ (group VII family). This is secreted constitutively by macrophages and is a 45 KDa protein, which circulates in plasma in its active form. These enzymes also hydrolyse unmodified fatty acyl residues up to 5 or 6 carbon atoms long in the sn-2 position, although even this restriction is relaxed when the terminal-end of the fatty acyl moiety is oxidized (i.e. aldehydic or carboxylic), such as the oxidatively truncated phospholipids described above. Indeed, the enzyme will hydrolyse phosphatidylcholine molecules containing hydroperoxy-octadecadienoyl and F2-isoprostane residues. The effect is to remove any oxidized phospholipids from lipoproteins and from 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. Similarly, by removing intracellular truncated phospholipids, PAF acetylhydrolase protects cells from apoptosis. 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.

The plasma PAF-acetylhydrolase is therefore considered an important anti-inflammatory enzyme. Decreased levels are associated with various diseases, including asthma, systemic Lupus erythematosus and Crohn’s disease.

The other products of PAF hydrolases are lysophospholipids, but at concentrations orders of magnitude lower than normal circulating levels, so they are not expected to make a significant inflammatory contribution. 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.

PAF-acetylhydrolase has trans-acetylase activity also, and is able to transfer short-chain fatty acids from PAF to ether/ester-linked lysophospholipids.

Oxidatively Truncated Phospholipids

PAF-like molecules with some biological activity 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 leaving 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 bioactive aldehydes. While the biosynthesis of PAF involves tightly regulated reactions, the various reactions involving chemical oxidation that produce core aldehydes are essentially uncontrolled. Such compounds are formed in plasma lipoproteins and are present in human atherosclerotic lesions. 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.

Formula of a core aldehyde

Oxidatively truncated phospholipids have been reported to possess a wide range of biological activities that correspond to those of PAF. For example, 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. Such lipids (and PAF) are also pro-apoptotic by a mechanism that is independent of the PAF receptor, and they have a substantial influence on regulated cell death. 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 oxidized truncated fatty acids (unesterified) and lysophosphatidylcholine. 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.

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Lipid listings Credits/disclaimer Updated: August 23rd, 2017 Author: William W. Christie LipidWeb icon