Studies on the Interaction Between (¹⁴C)-Epinephrine and Isolated Membranes from Human Platelets

Previous studies have reported on the main characteristics of epinephrine’s capacity to stimulate resting platelets. This study was designed to further investigate this problem using (¹⁴C)-epinephrine (^*E) and isolated membranes from human platelets. Membranes were isolated following the method of Barber & Jamieson (1970): a 6 and 2 fold enrichment in p (nitrophenyl) PDEase and ATPase and negligible levels of cytoplasmic marker enzyme activities have been measured. Incubations contained membranes and ^*E (33 mCi/μM) in Tyrode’s solution at pH 7.4; membrane bound ^*E was quantitated by Millipore filtration. Binding was a rapid, reversible and saturable process:It reached equilibrium by 30 min. at 37°C; half-maximum displacement ofE occurred at 5x10⁵M^-l “cold” E; about 400 pmoles of E were bound at saturation /mg of membrane protein/hour with a K_aff = 4.5–5.2x10⁷M^-1. Acid dissociated E from the labelled membranes showed that E structural alteration or degradation does not occur during binding. Impaired binding follows membrane exposure to impermeant -SH groups reagents or to trypsin, chymotrypsin, pronase and beta galactosidase. A specific binding appeared from its inhibition by alpha adrenoceptor agents, whereas beta active compounds had a much lower effect at higher concentrations; E metabolites did not inhibit binding. Mersalyl, a specific inhibitor of Mg²⁺ATPase of the myosin-like platelet protein, Thrombosthenin M, strongly inhibited binding. It thus appears that E interaction with isolated platelet membranes has properties to be expected of binding to alpha adrenoceptors and involves thrombosthenin; binding occurred at E concentrations consistent with those reported to stimulate highly responsive platelets to E, suggesting that changes in stereochemical configuration of isolated membranes may cause exposure of new reactive sites for E.