Clonidine p-isothiocyanate, an affinity label for α₂-adrenergic receptors on human platelets

Exposure of intact human platelets or platelet membranes to the clonidine analog clonidine p-isothiocyanate (clonidine-NCS), followed by extensive washing, results in the loss of [³H]yohimbine binding to platelet α₂-receptors. In addition, exposure of intact platelets to clonidine-NCS, followed by extensive washing, results in the loss of epinephrine-induced inhibition of adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in frozen-thawed platelets and in purified platelet membranes. This effect is dependent on time and concentration (T(1/2) at 30°C is <15 min; half-maximal effect occurs with clonidine-NCS at <10μM). Clonidine-NCS appears to interact by irreversibly blocking the platelet α₂-receptors because it abolishes α₂-receptor effects of adenylate cyclase activity (i.e., epinephrine-induced inhibition of basal and prostaglandin E₁-stimulated activity) while not altering other cyclase activity (basal, prostaglandin E₁-stimulated, and NaF-stimulated) and its effect on both [³H]yohimbine binding and epinephrine-induced inhibition of adenylate cyclase can be specifically prevented by α-agonists [(-)-epinephrine and clonidine] and α-antagonists (yohimbine and phentolamine). These observations indicate that clonidine-NCS is an effective affinity label for platelet α₂-receptors.