High affinity specific binding sites for tritiated platelet-activating factor in canine platelet membranes: counterparts of platelet-activating factor receptors mediating platelet aggregation.

In canine platelet membranes, tritiated platelet activating factor (PAF) labels in a saturable and reversible manner a single population (nH = 0.97) of binding sites. The affinity of this binding was high (Kd = 0.23 +/- 0.02 nM, n = 4, and 0.21 +/- 0.05, n = 8, determined by kinetics or saturation experiments, respectively), and the density of binding sites (Bmax) was 911 +/- 31 fmol/mg of protein (n = 8). [3H]PAF binding was entirely reversed by unlabeled PAF (10 microM). [3H]PAF exhibited stereoselective discrimination inasmuch as it was poorly displaced by enantio-PAF, the PAF enantiomer that does not occur naturally. Furthermore, the displacing potency of the (+)-enantiomer of the PAF antagonist 52770 RP against [3H]PAF was 45 times higher than that of the (-)-enantiomer. [3H]PAF binding displayed a remarkable specificity in that it was not affected by a variety of classical pharmacological agents. However, this binding was displaced by several PAF receptor antagonists such as 59227 RP, CV-6209, Ro 19-3704, 52770 RP, brotizolam, WEB 2086, SRI 63-441, L-652,731, alprazolam, triazolam, and BN 52021. The Ki of the 16 studied antagonists ranged from 7.9 nM (59227 RP, most potent) to 16.8 microM (BN 52021, least potent). The possible biological significance of our binding procedure was assessed by correlating the potencies of 16 PAF antagonists as [3H]PAF displacers in dog platelet membranes and as inhibitors of PAF-induced platelet aggregation in washed canine platelets. This analysis revealed the existence of a highly significant correlation (r = 0.82, p less than 0.001) between biochemical and functional tests. However, two compounds (Ro 19-3704 and BN 52021) were found to be located outside the confidence limits when the probability level of belonging to the regression line was set at 0.01. In conclusion, this study provides evidence that [3H]PAF binding in canine platelet membranes exhibits the required properties for a valid binding procedure. Furthermore, the labeled sites are likely to be the counterparts of platelet receptors that, when activated by PAF, induce aggregation.