Specific binding of [3H]-1-O-octadecyl Paf-acether to washed human platelets

[3H]-Paf-acether binds to washed human platelets in a dose-dependent manner. Scatchard analysis reveals two distinct binding sites; a high affinity site with a KD value of 0.259 +/- 0.33 nM (245 +/- 30 sites per platelet) and a lower affinity site with a KD value of 9.22 +/- 1.17 nM (1616 +/- 165 sites per platelet). Association of 3H-Paf-acether to the high affinity receptor is rapid, being maximal within two minutes and remaining constant for at least twenty minutes. Dissociation from the low affinity receptor is also rapid (t1/2: less than 10s) whereas dissociation from the high affinity site is significantly slower (t1/2 : approximately 70s). [3H]-Paf-acether binding is inhibited by unlabelled (R)-C16-Paf (IC50: 0.08 +/- 0.01 nM) greater than (R)-C18-Paf (0.48 +/- 0.03 nM) greater than (RS)-C18-Paf (1.06 +/- 0.19 nM), but remains unchanged in the presence of lyso-C18-Paf at 3.0-300 nM. [3H]-Paf-acether binding and its inhibition by unlabelled (R)-C18-Paf-acether is independent of buffer Ca2+ within the range 0-5.0 mM.