Abstract

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.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL026818-08
Application #
3338730
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1980-12-01
Project End
1988-11-30
Budget Start
1987-12-01
Budget End
1988-11-30
Support Year
8
Fiscal Year
1988
Total Cost
Indirect Cost

  Institution

Name
Wake Forest University Health Sciences
Department
Type
Schools of Medicine
DUNS #
041418799
City
Winston-Salem
State
NC
Country
United States
Zip Code
27106

  Publications

Wijkander, J; O'Flaherty, J T; Nixon, A B et al. (1995) 5-Lipoxygenase products modulate the activity of the 85-kDa phospholipase A2 in human neutrophils. J Biol Chem 270:26543-9
O'Flaherty, J T; Tessner, T; Greene, D et al. (1994) Comparison of 1-O-alkyl-, 1-O-alk-1'-enyl-, and 1-O-acyl-2-acetyl-sn-glycero-3-phosphoethanolamines and -3-phosphocholines as agonists of the platelet-activating factor family. Biochim Biophys Acta 1210:209-16
Venable, M E; Olson, S C; Nieto, M L et al. (1993) Enzymatic studies of lyso platelet-activating factor acylation in human neutrophils and changes upon stimulation. J Biol Chem 268:7965-75
Strum, J C; Nixon, A B; Daniel, L W et al. (1993) Evaluation of phospholipase C and D activity in stimulated human neutrophils using a phosphono analog of choline phosphoglyceride. Biochim Biophys Acta 1169:25-9
Daniel, L W; Huang, C; Strum, J C et al. (1993) Phospholipase D hydrolysis of choline phosphoglycerides is selective for the alkyl-linked subclass of Madin-Darby canine kidney cells. J Biol Chem 268:21519-26
Strum, J C; Emilsson, A; Wykle, R L et al. (1992) Conversion of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine to 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. A novel pathway for the metabolism of ether-linked phosphoglycerides. J Biol Chem 267:1576-83
Schmitt, J D; Nixon, A B; Emilsson, A et al. (1992) A facile synthesis of 1-O-alkyl-2-(R)-hydroxypropane-3-phosphonocholine (lyso-phosphono-platelet activating factor). Chem Phys Lipids 62:263-8
Chepenik, K P; Wykle, R L (1992) Synthesis of platelet activating factor and metabolism of related lipids in embryonic cells. Biochim Biophys Acta 1126:192-8
Bauldry, S A; Wykle, R L; Bass, D A (1991) Differential actions of diacyl- and alkylacylglycerols in priming phospholipase A2, 5-lipoxygenase and acetyltransferase activation in human neutrophils. Biochim Biophys Acta 1084:178-84
Nieto, M L; Venable, M E; Bauldry, S A et al. (1991) Evidence that hydrolysis of ethanolamine plasmalogens triggers synthesis of platelet-activating factor via a transacylation reaction. J Biol Chem 266:18699-706

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