NONADRENERGIC IMIDAZOLINE BINDING-SITES ON HUMAN PLATELETS

Human platelets are shown to possess at least two high-affinity, imidazol(in)e-preferring binding sites that are pharmacologically distinct from alpha-2 adrenoceptors. These nonadrenergic sites were radiolabeled even in the presence of a 10 muM norepinephrine mask of alpha-2 adrenoceptors. Heterogeneity at the nonadrenergic sites was demonstrated by comparing [H-3]idazoxan (IDX) binding vs. [I-125]p-iodoclonidine (PIC) binding. Nonadrenergic (I-125]PIC-labeled sites were enriched in platelet plasma membranes, whereas the nonadrenergic sites labeled by [H-3] IDX were codistributed between plasma and internal membranes (nonadrenergic [I-125]PIC-labeled sites had B(max) = 62 fmol/mg in plasma membranes and 20 fmol/mg in internal membranes vs. the [H-3]IDX-labeled sites had B(max) = 141 fmol/mg in plasma membranes and 192 fmol/mg in internal membranes). Furthermore, competition binding studies in the presence of a 10 muM norepinephrine mask revealed major (almost-equal-to 75%) and minor (almost-equal-to 25%) binding components on plasma membranes for [I-125]PIC. Affinities for the major nonadrenergic [I-125]PIC binding site were highly comparable to human subtype-I1 imidazol(in)e receptor sites in the brain stem (rank order: moxonidine > clonidine > cirazoline > IDX > amiloride). However, the minor component of [I-125]PIC binding was similar to a site reported in kidney, having low affinities for all compounds tested, except guanabenz. Finally, a third nonadrenergic internal membrane site, labeled by [H-3]IDX, was consistent with a subtype-I2 imidazol(in)e receptor site (rank order: cirazoline > IDX much greater than amiloride > moxonidine > clonidine). Thus, based on differential subcellular distributions and affinity constants, human platelets appear to possess imidazoline receptors (subtype-I1 imidazol(in)e receptor and subtype-I2 imidazol(in)e receptor), plus a novel guanabenz-sensitive site, as well as an alpha-2A adrenoceptor. These nonadrenoceptor binding sites may explain certain novel platelet aggregatory properties previously ascribed to clonidine and endogenous clonidine-displacing substance(s), and may serve as markers of imidazoline receptors in humans.