Functional validation of platelet-activating factor receptor sites characterized biochemically by a specific and reproducible [3H]platelet-activating factor binding in human platelets.

In human platelet membranes, [3H]platelet-activating factor(PAF)-C18 binding sites exhibited high affinity (Kd 0.074 +/- 0.005 nM, n = 28 healthy volunteers), saturability, elevated stereoselectivity, marked pharmacological specificity and small intersubject variability. The maximal binding capacity was 215 +/- 12 fmol/mg protein. Saturation of [3H]PAF binding was obtained with 0.3 nM ligand, and its isotherm was compatible with a single class of binding sites. The stereoselectivity for [3H]PAF was clearly indicated by the low displacing potency of enantio-PAF-C16 (the synthetic enantiomer of PAF) that was 5000-fold less potent than PAF. Specific [3H]PAF binding attained 65% with 0.1 nM ligand and was displaced fully not only by cold PAF but also by RP 59227 (Ki = 6.2 +/- 1.3 nM, n = 7), a novel, potent and specific PAF receptor antagonist in a pure enantiomeric form and several other antagonists such as CV-6209, WEB 2086, L-652,731 and BN 52021. Various classical pharmacological agents did not interfere with the [3H]PAF binding. In intact platelets, [3H]PAF binding shared the same properties as those just described for membrane preparations. A functional role for these binding sites was suggested by the high correlation (r = 0.94, P less than .001) between the Ki values for several known PAF antagonists determined in [3H]PAF binding and the IC50 values obtained against PAF-induced aggregation in whole platelets. Thus, the present [3H]PAF binding in human platelet membranes may be a useful pharmacological tool to study possible changes in [3H]PAF binding parameters induced by pathological states for which PAF may be directly or indirectly responsible.