Biochemistry of Platelet Activating Factor

Fundamentals

Structure

All phospholipids, including PAF, have a glycerol backbone. Three carbons of glycerol are named sn-1, sn-2, and sn-3. PAF has an alkyl group at the sn-1 position, acetyl group at the sn-2 position (middle carbon), phosphocholine at the sn-3 position. Alkyl groups at the sn-1 position of PAF produced enzymatically by leukocytes have a mixture of hexadecyl or octadecyl groups (i.e., 1-O-alkyl-2-acetyl sn-glycero-3-phosphocholines). One of the most critical aspects of PAF is its strict structural requirement for receptor binding and recognition by degradative enzymes. The specificity by degradative enzymes is a highly practical requirement because if the degradative enzymes were not specific, they would also hydrolyze structural phospholipids.[3]

Biosynthesis

There are two pathways to synthesize PAF: Remodeling and de-novo synthesis.

The remodeling pathway starts with a phospholipid called phosphatidylcholine. It involves substituting an acetyl residue for the long-chain fatty acyl residue a the sn-2 of phosphatidylcholine. A trace amount of phosphatidylcholine is present in all cells as a part of the normal plasma membrane. However, in PAF-producing cells such as endothelial cells and neutrophils, phosphatidylcholine content ranges from 10% to 40%.

Biosynthesis through the remodeling pathway has two steps, and it contributes to the bulk of the synthesis of PAF during inflammation. In the first step, phospholipase A2 (PLA2) acts on phosphatidylcholine producing eicosanoid (arachidonic acid) and lysophosphatidylcholine (LPC). In the second and final step, acetyl residue is transferred to LPC by LPC acetyltransferase to produce PAF.

Synthesis of PAF with the remodeling pathway occurs inside the cell on the nuclear membrane or the endoplasmic reticulum. The mechanism of the translocation of PAF from the interior to the exterior is not known. The fate of synthesized PAF depends on the cell type. Endothelial cells and leukocytes display PAF on their cell surface for juxtracrine signaling, whereas monocytes release most of the PAF they produce. Leukocytes can also be triggered to release some of the PAF they produced.

The alternative pathway of PAF synthesis, de novo pathway, mainly contributes to the physiologic levels of PAF needed for normal cellular function and has three steps:

1. The first step is to transfer acetyl residue onto a phospholipid, alkyl-lyso-glycerophosphate, by the acetyltransferase to form alkyl-acetyl-glycerophosphate. Acetyltransferase in this pathway is different from acetyltransferase in the remodeling pathway.
2. The second step is the hydrolysis of alkyl-lyso-glycerophosphate to alkyl-acetyl-glycerol by phosphohydrolase.
3. The final step involves transferring CDP-choline to alkyl-acetyl-glycerol by choline phosphotransferase to form PAF (alkyl-acetyl-glycerophosphocholine).

Under normal circumstances, the synthesis of PAF is maintained at a very low concentration by de novo synthesis, and its synthesis peaks during an inflammatory response through the remodeling pathway. Various agents can stimulate PAF syntheses, including antigen-antibody interactions, collagen, thrombin, and other inflammatory mediators involved in inflammation. 

Metabolism

PAF acetylhydrolases degrades PAF by removal of acetyl group at the sn-2 position to generate lyso-PAF and acetate. There are various isoforms of PAF acetylhydrolase with variable specificity to PAF distributed widely in various tissues, including plasma and blood cells. PAF acetylhydrolases are unique enzymes because they hydrolyze phospholipids with short chains at the sn-2 position, and they can act in the absence of calcium.[4] Their specificity allows them to work independently of calcium and prevents them from hydrolyzing phospholipid components of plasma membranes and lipoproteins.[5]

Issues of Concern

There are three PAF features that the PAF receptor recognizes: ether bond at sn-1, short residue at sn-2, and phosphocholine head group at sn-3. There is a polyunsaturated arachidonoyl residue on the sn-2 position in a precursor molecule of PAF, phosphatidylcholine. It is replaced by alkyl residue during the controlled synthesis of PAF. However, if there is uncontrolled oxidation of this alkyl residue at the sn-2 position, an oxidized phospholipid is generated, and it can act as a potent ligand and agonist of the PAF receptor. These phospholipids are called “PAF-like lipids.”[6]

Any alteration in the structure of PAF significantly reduces its binding to the PAF receptor. If two carbons elongate the acetyl group at the sn-2, the activity of PAF decreases by a factor of 10-100-fold. Any further elongation results in loss of function unless the alkyl group is oxidized. The PAF receptor can accept oxidized PAF even if it has an elongated alkyl group at sn-2 residue. In vivo, uncontrolled free-radical reactions generate large amounts of PAF-like oxidized phospholipids, especially if the injury is prolonged and vigorous.[7] It is especially crucial in syndromes of inflammatory tissue damage such as ischemic and reperfusion injury because these injuries release significant quantities of oxidants. Effects of PAF-like lipids generated locally in large amounts may be difficult to counteract with PAF receptor antagonists without achieving the high concentration of the drug in that area.

LDL particles contain an abundance of PAF-like lipids. These PAF analogs are not synthesized by remodeling or de novo pathways because they have 4-carbon butanoyl residues at sn-2 instead of the acetyl residue found in PAF. Therefore, PAF-like activity by these lipids is due to oxidation. Oxidation of LDL is a well-known trigger for atherosclerosis cascade, and PAF-like biologic activity by the oxidized lipid plays a key role in its pathophysiology.[8] Potential oxidation pathways and products are defined in the lab under a controlled environment. However, it is difficult to define the potential oxidation pathways and products in vivo because intermediates of oxidized lipids are highly unstable and get metabolized quickly.[9]

Isoprostanes are the products of oxidized arachidonate and are currently the best marker of oxidative processes. They are increased in some disease states and can be increased even further by the oxidative stress of cigarette smoking.[10] Isoprostanes are also potent vasoconstrictors and are present in umbilical blood after maternal smoking.[11] Identification of oxidized fragments in vivo is difficult not only because of the numerous isomers but also because PAF-like lipids can induce synthesis of PAF, leading to its accumulation.[12] Isolated oxidized lipids from LDL, which contains a small amount of PAF, when injected into animals can cause edema and activate and recruit inflammatory cells in injected animals. Therefore, PAF and PAF-like lipids may occur individually or in combination, and despite the structural differences between these lipids, functionally, they are equivalent.[13]

Cellular Level

PAF primary role is to mediate intercellular interactions. A variety of cells synthesize it, and it binds to the extracellular receptors of other cells, activating them and causing a change in their phenotypes. There are also intracellular receptors of PAF that are yet to be rigorously characterized.

Intercellular signaling by PAF is best understood in vascular and inflammatory systems. However, PAF also transmits information intercellularly in CNS, endocrine, GI, and other organs. There is tight control over the PAF intercellular-signaling system through several regulatory mechanisms. Some of these mechanisms include controlling synthetic pathways, biologic availability of PAF through spatial regulation of display, expression of PAF receptor on specific cells, desensitization of the receptor, and rapid degradation of PAF by PAF acetylhydrolases found both intracellularly and extracellularly. These mechanisms have evolved to precisely control PAF biologic activities. Therefore, dysregulation or unregulated intercellular signaling can be a mechanism of disease.

PAF has a well-understood role in mediating cell-cell interaction in acute and chronic inflammation in all organs. Endothelial cells and several classes of leukocytes are involved in inflammation. PAF can mediate its effects by acting over short distances in a paracrine fashion or circulating in the blood in an endocrine fashion. It has a short half-life because of Lp-PLA2 activity in the blood, limiting its endocrine mode of action. Acute inflammation experimental models have shown that most of the PAF signaling occurs in a juxtacrine fashion. PAF on the surface of one cell binds to the PAF receptor on the second cell and activates it.[14]

Activation of endothelial cells by histamine or thrombin causes them to express PAF on their surface.[15] P-selectin, a transiently expressed glycoprotein on activated endothelial cells, and PAF have the same kinetics and work in concert for neutrophil activation and adhesion. P-selectin tethers leukocyte and endothelial cells, allowing PAF from endothelial cells to bind with the PAF receptor on leukocytes.[16] The binding of endothelial cells with leukocytes results in a qualitative change in surface adhesive proteins (CD11/CD18 integrins), making them competent to bind to ligands (e.g., ICAM-1) on activated endothelial cells. Also, PAF stimulation induces the development of polarized shape in leukocytes, prime them for enhanced granular secretion, enhances their motility, and causes a redistribution of surface ligands.[17]

Activation of PAF receptors on monocytes leads to increased secretion of monocyte chemotactic protein (MCP-1) and tumor necrosis factor (TNF-alpha). PAF receptor is a G-protein coupled receptor that translates nuclear factor-kappa B (NF-kB), a transcriptional factor, into the nucleus required for expression of MCP-1, TNF-alpha, and other immediate-early genes.[18]

Molecular Level

Because PAF mediates the actions of other signaling molecules, its activity is controlled at the molecular level. One of the ways to control the activity of PAF is the variable expression of the PAF receptor on cell surfaces. PAF receptor gene, located on chromosome 1, has two distinct promotors (PAFR promoters 1 and 2). As a result, there are two PAF receptor transcripts (PAFR transcripts 1 and 2) differentially expressed in human tissues. Peripheral leukocytes and differentiated eosinophilic cell lines express PAFR transcript-1. Heart, lungs, spleen, kidney express PAFR transcript-2.

PAFR transcript-1 is mostly involved in inflammation and pathological processes and is synthesized in response to stimulation by PAF in a positive feedback manner through the activation of NF-kB. PAFR transcript-2 is controlled by various hormones and cytokines such as estrogen, TGF-beta, retinoic acid, or T3. Retinoic acid and thyroid hormone are potent inducers of PAFR transcript-2. Estrogen also increases PAFR transcript-2 levels, and TFG-beta decreases its levels.[19]

Function

PAF is primarily involved in inflammatory reactions and the activation of thrombotic cascades. It exerts its effects through the receptor present on the target cells, resulting in the production of various inflammatory mediators such as prostaglandins, cytokines, and other inflammatory mediators. It also acts in conjunction with these inflammatory mediators in the inflammatory response. It is also known to affect other systems such as cardiovascular, nervous, and respiratory systems.[20] Some of its functions are below:

Liver: PAF actions on the liver results in increased glycogenolysis. The mechanism of induction of breakdown of glycogen is from the hemodynamic effects of PAF rather than enzymatic regulation.[21]

Lungs: PAF induces the production of leukotrienes D4 and C4 in the lungs. As a result, it leads to fluid loss from the microvasculature, a potentially significant event in the outcome of allergic or inflammatory disease. PAF can also cause massive bronchoconstriction. It is also a sensitive regulator of surfactant secretion in type II alveolar cells.[22]

Brain: Studies on rat brain have shown that the production of PAF increases in response to convulsant stimuli and ischemia through activation of the remodeling pathway. Phospholipase A2 (PLA2) is activated by increased intracellular calcium concentration during the ischemic event, and this enzyme carries out the first step of the remodeling pathway. Also, protein kinase C (PKC) is activated due to ischemia and can phosphorylate and activate lysoPAF-acetyltransferase – the second enzyme involved in biosynthesis – fully activating the remodeling pathway. One of the effects of PAF produced within the brain is to accelerate synaptic polyphosphoinositide turnover. It also increases the Na+-Ca2+ exchange in the neurolemma and brain synaptosomes. PAF does not cross BBB, and vascular endothelial cells produce smaller amounts of PAF than aortic endothelial cells resulting in lower PMN adherence to brain endothelial cells. Brain endothelial cells have lower activity of PLA2 and have lower production of prostacyclin (PGI2). PAF and PGI2 have antagonizing activities in many aspects, and their balance is crucial in terms of thrombotic and anti-thrombotic effects, respectively. Therefore, brain endothelial cells have low levels of both PAF and PGI2.[23]

Heart: PAF can have direct or indirect effects on the heart. It can directly modify heart rate and contractility. Indirectly, it can stimulate inflammatory or endothelial cells to produce thromboxane A2 (TXA2), LTs, or TNF-alpha, leading to the same modification. These molecules can cause coronary artery constrictions and can even contribute to arrhythmias.[24][25]

Kidneys: The primary source of PAF in the body is the kidneys. Anephric patients and animals undergone bilateral nephrectomy have undetectable levels of PAF in the blood. The PAF receptors are expressed in the highest amount in the renal cortex and least in the inner medulla. In the nephron, the glomerulus contains the highest PAF receptors. The renal effects of PAF are challenging to measure in vivo due to its systemic hemodynamic effects. PAF decreases blood pressure, circulating volume, and cardiac output. As a result, it causes a decrease in renal plasma flow, glomerular filtration rate (GFR), and urine flow, along with increased renal vasculature resistance. There is also an increase in sodium and potassium reabsorption along the nephron.[26][27]

Reproduction: PAF has an essential role in oocyte maturation and successful outcomes of pregnancy. PAF contributes to the maturation of the oocyte. Immature follicles have a low amount of prostaglandin E2 (PGE2) and interleukin-1 (IL-1). PGE2 and IL-1 inhibit PAH acetylhydrolase. Therefore, fewer amounts of inhibitors result in increased degradation of PAF by the activity of PAF acetylhydrolase in immature follicles. As the follicle matures, PGE2 and IL-1 concentration increase resulting in inhibition of PAF acetylhydrolase and accumulation of PAF. PAF accumulation in the follicular fluid may stimulate the production of progesterone, which causes increased production of the proteolytic enzymes - plasmin and collagenase - for follicular wall connective tissue degradation during ovulation. PAF is also associated with successful outcomes of pregnancy after fertilization. Smooth muscles in the ovary may also contract in response to PAF accumulation resulting in extrusion of the oocyte cumulus cell mass.[28] PAF may enhance spermatozoa motility and fertilization success. It has a stimulatory effect on the centrioles of spermatozoa.[29] It also plays an essential role in sperm motility, acrosome reaction, and fertilization.[30]

Mechanism

The mechanism of action of PAF is complicated because of the wide variety of processes it mediates.[31] The receptor for PAF is a G-protein coupled receptor, and it can couple with different types of G-proteins depending on the tissue. Gq-protein activates the phosphatidylinositol (PLA3) pathway. PAFR Signalling through Gi inhibits the conversion of ATP to cAMP. Decreased cAMP impairs cAMP-dependent calcium pumps and leads to increased intracellular calcium.[32]

Products of phosphatidylinositol metabolism, diacylglycerol (DAG), and inositol triphosphate (IP3) enhance the action of protein kinase C (PKC). DAG directly stimulates PKC and IP3 indirectly through calcium by opening intracellular calcium channels in the endoplasmic reticulum. PKC activation leads to many responses, such as the release of arachidonic acid, eicosanoid production, and phosphorylation of protein tyrosine. Metabolites of arachidonic acids can also open membrane calcium channels. In addition to activating PKC, intracellular calcium can also activate intracellular contractile proteins, phospholipase A2 (PLA2), and many other proteins as well. Activation of PLA2 results in the synthesis of prostaglandins, thromboxane A2, leukotrienes, and other inflammatory mediators. These inflammatory mediators are involved in leukocyte activation, chemotaxis, aggregation of cells and platelets, and generation of reactive oxygen species.[33]

The function of PKC is dependent on the cell type. It can activate regulatory protein through phosphorylation and play a role in regulating surface PAF receptors.[33] In some cells, PKC contributes to physiological processes such as secretion and proliferation. In platelets, protein kinase C activated by PAF can cause a change in shape change, aggregation, and degranulation. PAFR Signalling through Gi inhibits the conversion of ATP to cAMP. Decreased intracellular cAMP impairs cAMP-dependent calcium pumps and leads to increased intracellular calcium in platelets, further enhancing their activation.[32]

Another downstream effect of PAF is the activation of the nuclear factor kappa-beta (NF-kB). NF-kB is a transcriptional factor that controls DNA transcription, cytokine production, and apoptosis. In leukocytes, it is a transcriptional factor for immediate-early genes, including monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-alpha), and vascular endothelial growth factor (VEGF).[18] PAF also induces the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM-1), and other adhesion molecules.[31]

PAF also activates the mitogen-activated protein kinase (MAPK) cascade in some tissues such as corneal epithelium and brain.[34][35] MAP-kinases are serine/threonine-specific protein kinases that regulate many functions of the cells, including gene expression, proliferation, division, differentiation, survival, and cell death.[36]

Testing

PAF is not known as the sole mediator of disease in humans. However, it plays a pivotal role in some syndromes and diseases. The activity of PAF and PAF-like lipids is measurable with the activity of lipoprotein-associated phospholipase A2 (Lp-PLA2) or plasma PAF acetylhydrolase.

It is an independent risk marker for cardiovascular disease (CVD). Pro-inflammatory and pro-atherogenic substrates of Lp-PLA2 enhance the expression of this enzyme in macrophages. Therefore, elevated levels might reflect the accumulation of these substrates.[37] The test measures the activity or amount of Lp-PLA2 and can help determine a patient's risk of developing CVD.[38]

Pathophysiology

PAF is not a sole mediator of diseases, but it has a crucial role in specific syndromes and diseases. Most of these conditions result from increased PAF or PAF-like activity, which can be due to increased production of PAF or decrease degradation of PAF or PAF-like lipids. PAF acetylhydrolase is the key enzyme responsible for PAF degradation. PAF acetylhydrolase (PAF-AH) has two intracellular isoforms (PAF-AH 1 and PAF-AH II) and one extracellular form called lipoprotein-associated phospholipase (Lp-PLA2).

PAH-AH I plays a vital role in brain development, sperm production, amyloid-beta precursor trafficking to lysosomes, cancer pathogenesis, protein trafficking, and sorting, and aspirin metabolism.[39] PAH-AH II protects cells from oxidative stress and is responsible for maintaining cell integrity by degrading the oxidized fragments of phospholipids.[40][41][42][43] It can transacetylase PAF to other lipid mediators modifying its cellular function.[44]

Lipoprotein-associated phospholipase (Lp-PLA2) or plasma platelet-activating factor acetylhydrolase (PAF-AH), is mainly associated with LDL (>80%) and HDL (<20%). However, only <1% of LDL particles contain Lp-PLA2. A small subset of LDL particles with Lp-PLA2 is protective against oxidative inactivation. Oxidants are potent and irreversible inhibitors of Lp-PLA2 activity.[45]

The Lp-PLA2 has two main biological activities. It hydrolyzes and inactivates PAF and hydrolyzes PAF-like oxidized lipids found in oxidized LDL. The latter role is crucial in the pathogenesis of atherosclerosis because it results in the production of two pro-inflammatory mediators: lysophosphatidylcholine (lysoPC) and oxidized nonesterified fatty acid (OxNEFA). The low activity of Lp-PLA2 is also associated with patients with asthma attributed to the higher levels of PAF in those patients.

Epidemiological studies show that Lp-PLA2 activity has a protective role in atherosclerotic lesions. These studies of the Japanese population reveal that 4% of their population has the Lp-PLA2 variant with the loss-of-function V279F mutation. People with V279F variant Lp-PLA2 have a higher risk of stroke, MI (in men only), cardiomyopathy (both dilated and hypertrophic), coronary artery disease, cerebral hemorrhage, atherosclerosis, polycystic ovary syndrome, and asthma. Also, Lp-PLA2 gene transfer with adenovirus has shown that plasma expression of Lp-PLA2 is associated with reduced oxidized LDL accumulation in the arteries.

However, a meta-analysis of 32 prospective studies has shown that Lp-PLA2 activity has a strong positive association with the risk of coronary heart disease (CHD) and stroke. The magnitude of the relationship of Lp-PLA2 is similar to non-HDL cholesterol or systolic blood pressure.[46] The discrepancy between epidemiological studies and clinical studies is related to the pathophysiology of atherosclerosis involving the production of pro-inflammatory mediators. The protective role of Lp-PLA2 is due to PAF hydrolysis. However, its role in atherosclerosis is due to hydrolyzing PAF-like oxidized lipids in LDL.  

When Lp-PLA2 hydrolyzes PAF-like oxidized lipids in LDL, it generates two pro-inflammatory mediators. These are called lysophosphatidylcholine (lysoPC) and oxidized nonesterified fatty acids (OxNEFA), and they promote plaque development. These pro-inflammatory mediators can recruit and activate leukocytes, impair clearance of dead cells, and induce apoptosis. LysoPC and oxFFA bind to G2A, G protein-coupled receptor that is highly expressed by macrophages in atherosclerotic lesions.[47][13] LysoPC is a direct chemoattractant for monocytes, and it also induces the expression of VCAM-1 and ICAM-1 for monocyte adhesion. It causes macrophages and smooth muscles to proliferate and causes endothelial dysfunction in various arteries. OxNEFA can cause platelet aggregation and vasoconstriction.[48] Lp-PLA2 promotes instability of atherosclerotic plaques because vulnerable and ruptured plaques with necrotic core and apoptotic macrophages contain the highest expression of Lp-PLA2. Stable and less advanced plaques do not express Lp-PLA2.

Lp-PLA2 is a reliable plasma biomarker for cardiovascular disease, and its activity is an independent predictor of coronary heart disease in the general population.[48] Animal studies have shown that darapladib, drug inhibiting Lp-PLA2, reduces lypoPC content in lesions in the coronary artery. It also reduced the expression of pro-inflammatory needed for macrophage and T-cell recruitment and functioning.[49][50] Several large-scale phase-III clinical trials showed that darapladib reduced the rate of major coronary events and total coronary events in patients with atherosclerosis. However, it did not reduce the risk of cardiovascular death, stroke, or myocardial infarction.[51]

Increased PAF activity in the lungs is associated with the pathogenesis of asthma. It mediates bronchoconstriction, bronchial hyperreactivity, mucous secretion, and inflammatory cell infiltration. Aerosolized inhaled PAF causes hyperresponsiveness with methacholine challenge in healthy and asthmatic patients. It increases blood flow through poorly ventilated areas in the lungs. Clinical trials in Japan with PAF receptor antagonist, apafant, showed dose-dependent clinical improvement in clinical symptoms in 8-week treatment in patients with mild to moderate asthma.[52]

The deficiency of plasma PAF-AH is associated with the severity of asthma. A V279F polymorphism is a loss-of-function mutation of plasma PAF-AH in the Japanese population, and it is associated with an increased risk of developing asthma and atopy compared to wild type. There are also other genetic mutations associated with polymorphisms of the plasma PAF-AH gene found in German and British individuals.[20]

Clinical Significance

Sepsis: PAF mediates the pathogenesis of severe sepsis. Bacterial endotoxins can induce PAF production resulting in severe hypotension and end-organ damage.[53] Pafase, recombinant PAF-AH (rPAF-AH), was shown to reduce mortality compared to control in patients with severe sepsis in phase IIB trial.[54] However, the phase III trial was terminated after the interim analysis and showed that it did not reduce mortality.[55]

Stroke: Epidemiological studies have shown that PAF-AH deficiency or mutations in PAF-AH may be a genetic risk factor for stroke.[56]

Cancer: PAF is associated with tumors and cancer cells. The receptor density for PAF is more in cancer cells, and PAF receptor antagonists are known to decrease tumor growth.[57]

Intrauterine Insemination: PAF enhances intrauterine insemination pregnancy rates when inoculated with sperm with normal semen parameters.[58] It does not improve pregnancy rates with sperm with mild male factor infertility parameters.[59]

Diabetes: Plasma PAF-AH (Lp-PLA2) levels will elevate in patients with type 2 Diabetes Mellitus compared to patients with dyslipidemia and healthy patients. Statin therapy in diabetic patients decreases plasma PAF-AH activity.[60] The decrease could be due to the anti-atherogenic effects of statins.[61]

Kidney Disease: Excessive PAF production by mesangial cells results in glomerular damage and can cause glomerulosclerosis and proteinuria. When PAF receptors on podocytes are activated, it results in loss of nephrin, decreased production of proteoglycan, and cytoskeletal rearrangements. As a result, anion charge of the glomerular basement membrane decreases and results in proteinuria.[62]

Allergies: PAF also mediates allergic rhinitis symptoms through its vascular effects. Rupatadine is a second-generation antihistamine with anti-PAF activity approved for the treatment of allergic rhinitis and chronic urticaria. It inhibits mast cell activation by inhibiting both histamine and PAF. When compared to antihistamines without anti-PAF activity, rupatadine is more effective in blocking nasal symptoms.[63]