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PLATELET ACTIVATING FACTORS

PRESENTATION BY-AISHA SIDDIQUIM.PHARM 1ST YRDEPT.OF PHARMACOLOGYJAMIA HAMDARDNEW DELHI.

Contents

What is platelet activating factor ?Discovery of PAF.Synthesis of PAF.Physiological and Pathophysiological effects of PAF.Platelet aggregation.Thrombus formation.Recent research.Pharmacological actions of PAF.PAF antagonists.

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What is platelet activating factor..??

Platelet-activating factor is a potent biological mediatorthat exerts its effects in a variety of cells and tissues.

Platelet-activating factor, also known as

PAF, PAF- acether or AGEPC (acetyl-glyceryl-ether-phosphorylcholine), is a potent phospholipid activator and mediator of many leukocyte functions, including platelet aggregation and degranulation, inflammation, and anaphylaxis.

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It is also involved in changes to vascular permeability, the oxidative burst, chemotaxis of leukocytes, as well as augmentation of arachidonic acid metabolism in phagocytes. PAF is produced by a variety of cells, but especially those involved in host defense, such as , platelets, endothelial cells, neutrophils,monocytes, and macrophages.

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Discovery of PAF.

In 1970, during a study of immunological mechanisms involving histamine and serotonin release from platelets in immunized rabbits, Henson proposed that 'a soluble factor' was released from leukocytes which stimulated platelets to release vasoactive amines. This observation was confirmed independently bySiraganian and Osler in 1971.

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In 1972, Benveniste, Henson and Cochrane demonstrated that the antibody involved in the immunological reaction described was an IgE class antibody and coined the term 'platelet-activating factor (PAF)' for the soluble factor released from basophils following IgE stimulation.

However, it was not until 1979 that Demopoulos, Pinckard and Hanahan demonstrated that a semisynthetic phosphoacylglycerol, l-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC), had physiochemical as well as biological properties (i.e. aggregation of platelets and secretion of serotonin) indistinguishable from those of naturally-generated PAF.

Synthesis of PAF

The major pathway by which PAF is generated involves the precursor 1-O-alkyl-2-acyl-glycerophosphocholine.PAF is synthesized from this substrate in two steps . Involves the action of phospholipase A2, with the formation of 1-O-alkyl-2-lyso-glycerophosphocholine (lyso-PAF) and a free fatty acid (usually AA) . Eicosanoid and PAF biosynthesis thus is closely coupled, and deletion of cPLA2 in mice leads to an almost complete loss of both prostanoid and PAF generation. The second, rate-limiting step is performed by the acetyl coenzyme-A-lyso-PAF acetyltransferase. PAF synthesis also can occur de novo; a phosphocholine substituent is transferred to alkyl acetyl glycerol by a distinct lysoglycerophosphate acetylcoenzyme-A transferase.

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This pathway may contribute to physiological levels of PAF for normal cellular functions.

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1-The synthesis of PAF may be stimulated during antigen–antibody reactions or by a variety of agents, including chemotactic peptides, thrombin, collagen, and other autacoids.2- PAF also can stimulate its own formation. Both the phospholipase and acetyltransferase are Ca2+-dependent enzymes; thus, PAF synthesis is regulated by the availability of Ca2+.3- The inactivation of PAF also occurs in two steps .1)Initially, the acetyl group of PAF is removed by PAF acetyl hydrolase to form lyso-PAF 2)Lyso-PAF is then converted to a 1-O-alkyl-2-acyl-glycerophosphocholine by an acyltransferase.

1-In addition to these enzymatic routes. PAF-like molecules can be formed from the oxidative fragmentation of membrane phospholipids (oxidized phospholipids).

2-These compounds are increased in settings of oxidant stress such as cigarette smoking. 3-They differ structurally from PAF in that they contain a fatty acid at the sn-1 position of glycerol joined through an ester bond and various short-chain acyl groups at the sn-2 position. 4-Oxidized phospholipids mimic the structure of PAF closely enough to bind to its receptor and elicit the same responses. 5-Unlike the synthesis of PAF, which is highly controlled, oxidized phospholipids production is unregulated; degradation by PAF acetyl hydrolase, therefore, is necessary to suppress the toxicity of oxidized phospholipids.

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6-Levels of PAF acetyl hydrolase (also known as lipoprotein-associated phospholipase a2) are increased in colon cancer, cardiovascular disease & stroke. 7-Polymorphisms have been associated with altered risk of cardiovascular events.

Remodelling of cell membrane phospholipid denevo 1-0-alkyl-sn-glycero-3-phosphate

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Physiological and pathophysiological effects of PAF

1-Platelet aggregation and secretion, thrombosis.2-Stimulation of neutrophils and macrophages.3-Acute inflammation.4-Asthma and systemic anaphylaxis.5-Endotoxin and immune factor-induced shocks.6-Gastrointestinal ulceration.7-Glycogenolysis and increased portal pressure.8-Pancreatitis.

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9-Cardiac anaphylaxis (negative inotropic effect and increased heart beating rate).10-Pregnancy and ovoimplantation.11-Ovulation.12-Acute lung injury.

Platelet aggregation

1.Platelet aggregate or clump together using fibrinogen of vWF as a connecting agent.

2.The most abundant platelet aggregation receptor is glycoprotein IIb/3A.Activated platelets will adhere, via glycoprotein Ia to the collagen that is exposed by endothelial damage.

3.Aggregation and adhesion act together to form platelet plug.Platelet aggregation is stimulated by ADP,Thromboxane and a2 receptor-activation but inhibited by other inflammatory products like PGI2 and PGD2. Platelet aggregation is enhanced by exogenous administration of anabolic steroids.

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Initially, PAF was found to effect aggregation of platelets at concentrations as low as 10^-11 M, and it induced a hypertensive response at very low levels also. More generally, it is now recognised that its primary role is to mediate intercellular interactions.

For example, by binding to its specific receptor, PAF activates the cytoplasmic phospholipase A2 and phospholipase C. The result of the latter is an increase in intracellular Ca2+ downstream of the cell and activation of protein kinase C.

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It is now known to exert effects on many different types of non-inflammatory biological events and functions, including glycogen degradation, reproduction, brain function and blood circulation.

Thrombus formation.

The function of platelets is the maintainence of haemostasis,this is achievd by the formation of thrombi when damage to endothelium of blood vessels occurs.Conversely ,thrombus formation must be inhibited at times when there is no damage to the endothelium,Activation-the inner surface of blood vessel is lined with a thin layer of endothelial cells,that in normal haemostasis acts to inhibit platelet activation by producing endothelial ADPase, nor-adrenaline and PGI2. Endothelial ADPase clears away ADP,a platelet activator,from platelet surface receptors. Endothelial cells produce a protein called von Willebrand factor,a cell adhesion ligand,which helps endothelial cells adhere to collagen in the basement membrane.Under physiological condition collgen does not pass into the bloodstream;however vWF is secreted constitutively into the plasma by the Endothelial cells that

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Produce it or otherwise is stored within the endothelial cells or in platelets. When endothelial damage occurs platelets comes into contact with exposed collagen and vWF,causing a reduction in secretion of endothelium platelet inhibitors the inner surface of blood vessels is lined with a thin layer of endothelial cells. Under this is a layer of collagen. When the endothelial layer is injured the collagen is exposed. When platelets contact collagen they are activated.

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Recent research

Much recent work has been concerned with the function of PAF as a mediator of inflammation, and in the mechanism of the immune response. 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.

For example, it has a number of proinflammatory 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 multiple sclerosis.

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PAF can activate human inflammatory cells at concentrations as low as 10^-14M.

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, probably via the formation of leukotrienes as secondary mediators. 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.

Pharmacological actions

CVS 1-PAF is a potent dilator in most vascular beds; when administered intravenously, it causes hypotension in all species studied.

2-PAF-induced vasodilation is independent of effects on sympathetic innervation, the renin–angiotensin system, or arachidonate metabolism and likely results from a combination of direct and indirect actions.

3- PAF induces vasoconstriction or vasodilation depending on the concentration, vascular bed, and involvement of platelets or leukocytes.

For example, the intracoronary administration of very low concentrations of PAF increases coronary blood flow by a mechanism that involves the release of a platelet-derived vasodilator.

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4-Coronary blood flow is decreased at higher doses by the formation of intravascular aggregates of platelets and/or the formation of TXA2.

5-The pulmonary vasculature is also constricted by PAF and a similar mechanism is thought to be involved.

6-Intradermal injection of PAF causes an initial vasoconstriction followed by a typical wheal and flare.

7-PAF increases vascular permeability and edema in the same manner as histamine and bradykinin. but PAF is more potent than histamine or bradykinin by three orders of magnitude.

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Platelets

1-PAF potently stimulates platelet aggregation in vitro. 2-While this is accompanied by the release of TxA2 and the granular contents of the platelet, PAF does not require the presence of TxA2 or other aggregating agents to produce this effect.

3-The intravenous injection of PAF causes formation of intravascular platelet aggregates and thrombocytopenia.

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Smooth Muscle1-PAF generally contracts gastrointestinal, uterine, and pulmonary smooth muscle.2- PAF enhances the amplitude of spontaneous uterine contractions; quiescent muscle

contracts rapidly in a phasic fashion.3- These contractions are inhibited by inhibitors of PG synthesis.

4-PAF does not affect tracheal smooth muscle but contracts airway smooth muscle. Most evidence suggests that another autacoid (e.g., LTC4 or TxA2) mediates this effect of PAF.

5-When given by aerosol, PAF increases airway resistance as well as the responsiveness to other bronchoconstrictors.

6-PAF also increases mucus secretion and the permeability of pulmonary microvessels; this results in fluid accumulation in the mucosal and submucosal regions of the bronchi and trachea.

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Kidney1-When infused intrarenally in animals, PAF decreases renal blood flow, glomerular

filtration rate, urine volume, and excretion of Na+ without changes in systemic hemodynamics

2-These effects are the result of a direct action on the renal circulation.3-PAF exerts a receptor-mediated biphasic effect on afferent arterioles, dilating them at

low concentrations and constricting them at higher concentrations. 4-The vasoconstrictor effect appears to be mediated, at least in part, by COX products,

whereas vasodilation is a consequence of the stimulation of NO production by endothelium.

Stomach1-In addition to contracting the fundus of the stomach, PAF is the most potent known ulcerogen.2-When given intravenously, it causes hemorrhagic erosions of the gastric mucosa that

extend into the submucosa.

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Pathophysiological function of PAF

PAF generally is viewed as a mediator of pathological events.

Dysregulation of PAF signaling or degradation has been associated with some human diseases, aided by data from genetically modified animals.

Platelets1-Since PAF is synthesized by platelets and promotes aggregation, it was proposed as the mediator of cyclooxygenase inhibitor–resistant, thrombin-induced aggregation.

2-However, PAF antagonists fail to block thrombin-induced aggregation, even though they prolong bleeding time and prevent thrombus formation in some experimental models.

3-Thus, PAF does not function as an independent mediator of platelet aggregation but contributes to thrombus formation in a manner analogous to TxA2 and ADP.

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1-The proinflammatory actions of PAF and its elaboration by endothelial cells, leukocytes, and mast cells under inflammatory conditions are well characterized.

2-PAF and PAF-like molecules are thought to contribute to the pathophysiology of inflammatory disorders, including anaphylaxis, bronchial asthma, endotoxic shock, and skin diseases.

3-The plasma concentration of PAF is increased in experimental anaphylactic shock, and the administration of PAF reproduces many of its signs and symptoms, suggesting a role for the autacoid in anaphylactic shock.

4- In addition, mice overexpressing the PAF receptor exhibit bronchial hyperreactivity and increased lethality when treated with endotoxin.

5-PAF receptor knockout mice display milder anaphylactic responses to exogenous antigen challenge, including less cardiac instability, airway constriction, and alveolar edema; they are, however, still susceptible to endotoxic shock.

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6-Despite the broad implications of these observations, the effects of PAF antagonists in the treatment of inflammatory and allergic disorders have been disappointing.

7- Although PAF antagonists reverse the bronchoconstriction of anaphylactic shock and improve survival in animal models, the impact of these agents on animal models of asthma and inflammation is marginal.

8-Similarly, in patients with asthma, PAF antagonists partially inhibit the bronchoconstriction induced by antigen challenge but not by challenges of exercise, or inhalation of cold air.

9-These results may reflect the complexity of these pathological conditions and the likelyhood that other mediators contribute to the inflammation associated with these disorders.

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PAF Antagonists.

1.Ginkgolide B

Biological ActivityPlatelet-activating factor (PAF) receptor antagonist.Inhibits neutrophil degranulation and superoxide production in vitro and inhibits bronchoconstriction and is neuroprotective following oral administration in vivo.

2.PCA 4248

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Biological ActivityA specific PAF antagonist, more potentthan the ginkolide BN-52021. Inhibits rabbit platelet aggregation and has no significant activity on Ca2+ channels. Active in vivo, antagonizing PAF-induced systemic hypotension and protein-plasma extravasation in rats.

3.WEB 2086

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Alternative Name: Apafant

Potent, selective platelet-activating factor (PAF) receptor antagonist. Displays anti-inflammatory, antiangiogenic and anticancer activity. Inhibits growth and proliferation of breast cancer cells.

References

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Demopoulos, C. A., Pinckard, R. N. and Hanahan, D. J. (1979) J. Biol. Chem. Henson, P. M. (1970) J. Exp.

Med. Siraganian, R. P. and Osler, A. G. (1971) J. Immunol. Platelet activating factor and signal transduction. ,136,1356-1377.

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).

Yost, C.C., Weyrich, A.S. and Zimmerman, G.A. The platelet activating factor (PAF) signaling cascade in systemic inflammatory responses. Biochimie, 92, 692-697 (2010).

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