Specific binding of [3H] dihydrokadsurenone to rabbit platelet membranes and its inhibition by platelet activating factor (PAF) and PAF receptor antagonists

Kadsurenone inhibits specifically and competitively the specific binding of [3H] PAF to rabbit platelet membranes with a K/sub B/ of 9 x 10-8M. 5-propyl analog of kadsurenone (dihydrokadsurenone) retains roughly the same potency as kadsurenone. To confirm that the inhibitory effect on the specific [3H] PAF binding by kadsurenone, dihydrokadsurenone or other PAF receptor antagonists is due to the blockage of the receptor site, [3H] dihydrokadsurenone was synthesized. A saturable specific [3H] dihydrokadsurenone binding was found with an equilibrium dissociation constant (K/sub D/) of 16.48 (+/- 0.03) nM. The total number of detectable binding sites is 2.32 (+/- 0.09) pmol/mg protein, which is exactly identical to the B/sub max/ of the PAF receptor sites (2.30 +/- (0.40) pmol/mg protein) under identical assay conditions. Both C16 and C18-PAF fully displaced the specific [3H] dihydrokadsurenone (5 nM) binding with an identical ED50 of 10-9M. Dihydrokadsurenone and kadsurenone also displaced the specific binding with roughly the same potency (ED50 = 3.6 x 10-8M). L-652,731 is more potent with an ED50 of 2 x 10-8 M. Ono-6240 and CV-3988 also displaced the binding with ED50 of 10-7 and 4 x 10-7 M respectively. These results strongly suggest that PAF and these PAF receptor antagonists interact with a common binding site in the PAF receptor