Study on mechanism of phospholipae A_2 activation.

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(1) Platelet phospholipae A_2 inhibitory protein. 1-Acyl-2-[^<14>4C]arachidonyl GPC was not hydrolyzed by beef platelet lysate (freeze-thawing platelet)(as an enzyme) in the presence of 10 mM Ca^<2+>. The addition of 10 mM Ca^<2+> to [^3H]arachidonic acid-labelled platelet membrane made [^3H]-arachidonic acid released, but the membrane-bound phospholipase A_2 activity was suppressed by beef platelet lysate even in the presence of 10 mM Ca^<2+>. We elucidated the presence of platelet phospholipase A_2 inhibitory protein in beef platelet, which was distinct from lipocortin.(2) Distinct mechanism of ionophore A23187-induced platelet activation in the presence and absence of extracellular Ca^<2+>. In order to study the mechanism of platelet phospholipase A_2 activation, we investigated A23187-induced platelet activation using washed rabbit platelets double-labelled with [^<32>P]phosphoric acid and [^3H]arachidonic acid in the presence and absence of extracellular Ca^<2+>. In the absence of … More extracellular Ca^<2+>, A23187 increased the amount of intracellular Ca^<2+> which was lower than that in the presence of extracellular Ca^<2+>. TPA induced 40 kDa and 20 kDa protein phosphorylation, regardless of extracellular Ca^<2+>. In TPA-treated platelets, A23187-induced arachidonic acid release increased in both the presence and absence of extracellular Ca^<2+>, but inositol phospholipid metabolism was not affected by A23187 in the absence of extracellular Ca^<2+>. We elucidated thus distinct mechanism of A23187-induced platelet activation in the presence and absence of extracellular Ca^<2+>.(3) Occurrence of platelet-activating factor (PAF) in normal rat stomach and alteration of PA level by water-immersion stress. In order to elucidate the mechanism of PAF biosynthesis connected with phospholipase A_2 activation, we detected platelet-activating substance in gastrointestinal areas, which was confirmed by GC/MS analysis. In the normal rat stomach, the level of PAF was high in the antral mucosa. The percentage composition of each molecular species of PAF was 16:OPAF (34%) and 18:OPAF (66%). Application of water-immersion stress affected not only the amounts of PAF but also their molecular heterogeneity in the glandular stomach. The amount of 18:OPAF increased markedly (to 4-fold) in the corpus along with severe lesions after stress for 7 h. These changes might be associated with the pathogenicity of gastric ulcer.(4) Development of a novel scintillation proximity radioimmunoassay for platelet-activating factor measurement. The SPRIA assay system was suitable for the quantitation of 0.03 to 2 pmol of 16:OPAF. The cross-reactivity was high with 1- alkyl-2-acetyl GPC but was very low with PAF analogs and PAF antagonists. The specificity of SPRIA was higher than that of bioassay, quite different from that of the platelet receptor. Less