CHARACTERIZATION OF [H-3] APAFANT BINDING TO PAF RECEPTOR ON RABBIT PLATELET MEMBRANES - A COMPARISON OF A MICROPLATE FILTRATION SYSTEM ANDA STANDARD METHOD

This article describes the application of a Microplate Filtration Syst em (MFS) to a binding assay, with the results being compared to those obtained with a conventional 24-Well Filtration Manifold (24WFM). The data reported here characterize the PAF receptor on rabbit platelet me mbranes using [H-3]apafant. The results showed that [H-3]apafant label led a homogenous population of high-affinity binding sites in a concen tration-dependent manner. Binding was very specific, saturable, revers ible, and proportional to receptor concentration. [H-3]Apafant interac ted with membranes in an apparently competitive manner, with pseudo-Hi ll coefficients not significantly different from unity, thus indicatin g that apafant did not interact cooperatively at these binding sites. A number of PAF antagonists (apafant, lexipafant, BN-52021, SCH-37370, SR-27417, UR-12670) inhibited [3H]apafant binding with slopes near un ity and with a rank order of potency in good agreement with their abil ity to inhibit PAF-induced rabbit platelet aggregation, suggesting tha t the sites labelled are functional PAF receptors. C-18-PAF also compe ted with [H-3]apafant for the receptor, but yielded biphasic inhibitio n curves which could be resolved into high- and low-affinity component s. No significant differences were found either in the equilibrium bin ding parameters or in the PAF antagonists affinities obtained with the 24WFM and the MFS. The use of the latter system improved sample handl ing efficiency and shortened overall labor time, thus representing a m ore suitable way to perform receptor binding assays.