Paf binding sites: Characterization by [ 3h]52770 rp, a pyrrolo[1,2- c]thiazole derivative, in rabbit platelets

Abstract

52770 RP, the N-(3-chlorophenyl)-3-(3-pyridinyl)-1H, 3H-pyrrolo[1,2- c]thiazole-7-carboxamide, displaces in a potent, specific and competitive manner [ 3H]PAF from its binding sites on rabbit platelets. Since 52770 RP is not structurally related to PAF and has low liposolubility with respect to PAF, it was selected as a potential radioligand for PAF receptor sites. [ 3H]52770 RP displayed high-affinity, specificity, as well as saturable and displaceable binding to a single class of recognition sites in intact platelets and crude platelet membranes. In these preparations, the values of binding parameters were, respectively, 8.5 and 7.6 nM for K d, 0.2 pmol/5 × 10 7 platelets and 3.66 pmol/mg protein for B max and 0.96 and 0.91 for nH. Inasmuch as the (+)-52770 RP was 300-fold more potent than the (−)-isomer at displacing [ 3H]52770 RP in intact platelets, the studied binding site manifested stereospecific discrimination. A variety of pharmacological agents including pro- and anti-aggregant compounds did not exhibit affinity for [ 3H]52770 RP binding sites. In contrast, PAF, some of its active analogues and several recognized PAF antagonists (BN 52021, brotizolam, L-652,731, triazolam), displaced the [ 3H]52770 RP binding. Studies carried out using [ 3H]PAF demonstrated that 52770 RP was approximately 4- and 200-fold more potent than L-652,731 and BN 52021 respectively, as a PAF-receptor antagonist. In washed rabbit platelets, the rank order of potency ( K i) for several analogues of 52770 RP, to displace [ 3H]PAF from its binding sites, was highly correlated ( r = 0.96) to their ability to antagonize [ 3H]52770 RP binding. In functional studies, 52770 RP antagonized not only the PAF-induced aggregation in washed rabbit platelets but also the hypotension evoked by PAF in the anesthetized rat. In this respect, it was 26 and 2 times more potent than L-652,731, respectively. In conclusion, [ 3H]52770 RP might represent a novel interesting tool for furthering our understanding of the role of PAF binding sites in pathophysiological processes.