Physicochemical and Functional Identity of Rabbit Platelet-Activating Factor (PAF) Released in Vivo during IgE Anaphylaxis with PAF Released in Vitro from IgE Sensitized Basophils

Abstract

Platelet activating factor (PAF) released in vivo into the plasmas of rabbits during development of IgE-induced systemic anaphylactic shock had indistinguishable physicochemical and functional properties when compared with PAF released in vitro from antigen-stimulated, washed buffy coat cells containing IgE-sensitized basophils. Our findings represent the first documentation for the release of PAF in vivo and strongly support its role as an important chemical mediator involved in acute inflammatory reactions and tissue injury. The intravascular release of PAF during IgE anaphylaxis correlated temporally with the development of intravascular platelet aggregation and the development of thrombocytopenia and neutropenia. Passive transfer experiments indicated that IgE-sensitized basophils alone can initiate the intravascular release of PAF and development of the various platelet and neutrophil alterations similar to those observed during the anaphylactic syndrome in the actively immunized rabbit. The PAF released in vivo or in vitro was completely recoverable by chloroform-methanol-water extraction and was easily and completely phased into a chloroformrich layer after the addition of water. All of the PAF samples studied consistently migrated on thin layer chromatography as a sharp band between sphingomyelin and lysolecithin on silica gel G plates by utilizing a solvent system of chloroform-methanol-water, 65:35:4, v/v. The mobility of the PAF on thin layer chromatography was essentially unaffected when chloroformmethanol-acetic acid-water, 25:15:4:2, v/v, or chloroform-methanol-25% ammonia 65:35:4, v/v, were utilized as the solvent systems. These observations, coupled with the fact that PAF preparations could not be extracted from the chloroform solution with NaHCO₃ argue for a neutral lipid molecule. Functional PAF activity and migratory properties on thin layer chromatography were stable to acid conditions, exposure to air, treatment with periodic acid or sodium nitrite, but were exquisitely labile to methanolic NaOH. The above characteristics would eliminate the occurrence of a structure containing a vinyl ether linkage, a free glycerol, glycol or sugar moiety, but would support a neutral polar lipid containing a fatty acid ester group.