Effect of sulfhydryl reagents on PAF binding to human neutrophils and platelets

The effects of sulfhydryl reagents on the binding characteristics of platelet-activating factor (PAF) to PAF receptors in human neutrophils and platelets were examined. Simultaneous incubation of neutrophil or platelet membranes with radiolabeled PAF and N-ethylmaleimide (NEM), p-(chloromercuri)benzenesulfonic acid (PCMBS), or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) resulted in a dose-dependent inhibition of PAF specific binding to the membranes. PCMBS, a membrane-impermeable compound, also inhibited PAF specific binding to intact neutrophils and platelets. In the presence of PCMBS (0.1 mM) or NEM (20 mM), the ability of PAF to displace bound [3H]PAF from neutrophil membranes was reduced. Scatchard analysis of data from saturation binding experiments using neutrophil membranes indicated that the inhibitions of PCMBS (0.1 mM) and NEM (20 mM) on PAF specific binding were due to a decrease in the receptor affinity with no significant change in the number of binding sites. The results suggest that free sulfhydryl group(s) may be associated with the agonist binding site of PAF receptors in human neutrophils and platelets.