Platelet activating factor (PAF; 1-0-alkyl-2-acetyl-sn-glycero-3 phosphocholine; 1-0-alkyl-2-acetyl-GPC) is a very potent biologically active agent, causing the aggregation and degranulation of rabbit platelets at 10 to the -11 to 10 to the -10 M and the corresponding responses in neutrophils (PMN) at 10 to the -9 M. Evidence indicates PAF is derived from cellular 1-0-alkyl-2-acyl-GPC by deacylation and subsequent acetylation reactions; it is inactivated by an acetylhydrolase and is reacylated to form 1-0-alkyl-2-acyl-GPC. Since PAF is a highly inflammatory agent and appears to be a key mediator of anaphylaxis, it is important to understand how the concentrations of PAF are controlled and by what biochemical mechanism it acts. Our studies indicate PAF activity may be mediated via arachidonate metabolites and that one of the arachidonate metabolites, 5-L-HETE, increase the sensitivity to PAF in the degranulation response of PMN by up to 1000 fold. We propose to investigate these biochemical interrelationships of PAF and arachidonic acid, focusing on the role of 1-0-alkyl-2-arachidonoyl-GPC as a possible metabolic precursor of both PAF and arachidonate metabolites. The enzymes that synthesize and inactivate PAF will be characterized; we will attempt to purify, at least partially, the phospholipase A2, the acetylhydrolase, and possibly others. The mechanism of PAF action will be investigated by using structural analogs of PAF and by exploring the putative receptor for PAF through binding studies. Isolation of the receptor will also be explored. Analogs will be tested as possible blockers of PAF activity, and non-hydrolyzable active analogs of PAF will be used to study the target cells of PAF in vivo; their metabolism will be investigated. The alkyl chains in the PAF synthesized by PMN and platelets will be determined, and the relative activities of chemically synthesized PAF containing defined chains (14:0, 16:0, 18:0, 16:1, 18:1, 18:2) will be compared using various target cells. Finally, the reported activity of 1-0-alkyl-2-acetyl-glycero-3-phosphate will be tested and its biosynthesis explored.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Hematology Subcommittee 2 (HEM)
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Wake Forest University Health Sciences
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