Platelet-activating factor (PAF) stimulates the lysoPAF acetyltransferase in leukocyte-rich plasma: Use in PAF antagonist studies

Abstract

Addition of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to leukocyte-rich plasma from several species resulted in the rapid and pronounced activation of the PAF biosynthetic enzyme acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (EC 2.3.1.67). Activation of acetyltransferase by PAF occurred in leukocyte-rich plasma from human, chimpanzee, rhesus monkey, and dog. The neutrophil was indicated to be the major cellular source of the activabable acetyltransferase in leukocyte-rich plasma. The induction of acetyltransferase was substantial with 10 nM PAF, and maximal at 10–30 seconds. Measurable acetyltransferase activation was significantly greater when the PAF-activated cells were separated from the plasma by centrifugation before the acetyltransferase assay. This may be due in part to the removal of the PAF-specific acetylhydrolase present in plasma which can cleave the acetyl group from PAF. Measuring PAF activation of acetyltransferase in leukocyte-rich plasma can be useful to determine the potency of PAF antagonists with neutrophils in plasma compared to isolated neutrophils in aqueous buffer, and as anex vivo assay to determine the efficacy and plasma concentration equivalents of antagonists administered to whole animals. The PAF antagonist L-659,989 was shown to be 3–5 times more potent in inhibiting PAF induction of acetyltransferase in isolated human neutrophils than in human leukocyte-rich plasma, with IC₅₀ values of 10 nM and 40 nM, respectively. In theex vivo assay, oral administration of the PAF antagonist L-667, 131 to dogs resulted in very substantial inhibition of PAF induction of acetyltransferase in the leukocyte-rich plasma. Utilizing theex vivo assay, oral administration of 1 mg/kg L-659,989 to rats was found to result in plasma concentration equivalents of approximately 200–300 nM L-659,989. Our findings offer a new approach for charagerizing thein vitro andin vivo efficacy of PAF receptor antagonists and demonstrate that PAF may be able to activate neutrophils in the bloodin vivo, further enhancing PAF synthesis.