3H-clonidine binding to membranes of platelets prepared under physiological conditions

A reproducible binding assay has been established to measure alpha 2-adrenoreceptors on membranes of human platelets prepared under physiological conditions. Washed platelet suspensions were obtained from fresh blood by a modified method (Mustard et al., 1972) and membranes were prepared by mechanical homogenization. 3H-clonidine, a selective alpha 2-adrenoreceptor agonist was selected as the ligand in a concentration range of 2-64 nM. Specific binding of 3H-clonidine was defined as the difference between total bound radioactivity and that not displaced by excess cold clonidine (6.4 X 10(-5) M). Platelets from 14 healthy young male volunteers were analysed using this technique, and one high affinity binding site, with KD and Bmax values in the reported range was found (KD: 10.14 +/- 1.95 nM; Bmax: 428 +/- 53 fmol/mg protein (mean +/- S.E.M.)). This technique will be used in future studies that will examine alpha 2-adrenoreceptor changes in specific subgroups of depressed patients.