Antagonism of platelet activating factor receptor binding and stimulated phosphoinositide-specific phospholipase C in...

The objective of this study was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of phosphoinositide-specific phospholipase C (PLC) in rabbit platelets. Saturation binding curves for [3H]PAF indicated that the PAF receptor has a dissociation constant (KD) of 28.72 nM. In comparison, PAF-stimulated PLC activity, as monitored by [3H]inositol triphosphate production, increased at lower concentrations and had an half-maximal effective concentration (EC50) value of 1.5 nM. Unlabeled PAF inhibited [3H]PAF binding competitively and demonstrated two binding sites, a high affinity site with an inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 microM. The inhibitory effects of four PAF antagonists, CV-3988, CV-6209, SRI 63-441 and SRI 63-675 on the binding of [3H]PAF were compared to the effects of the antagonists on PAF-stimulated PLC activity. The four antagonists inhibited [3H]PAF binding almost completely whereas their ability to inhibit PAF-stimulated PLC activity varied. CV-3988, SRI 63-441 and SRI 63-675 had half-maximal inhibitory concentration (IC50) values of 0.28, 0.78 and 0.42 microM, respectively, whereas CV-6209 was more potent at inhibiting [3H]PAF binding (IC50 = 7.73 nM). The SRI 63-441 and SRI 63-675 inhibited PLC totally with an IC50 value of 0.78 and 1.27 microM, respectively. The CV-3988 and CV-6209 showed a maximal PLC inhibition of about 45% with "apparent IC50" values of 1.05 and 0.17 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)