Selective Binding Site for [3H]Prostacyclin on Platelets

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Abstract
Prostacyclin (PGI2) is the most potent, naturally occurring inhibitor of platelet aggregation known. To determine whether PGI2 is bound by platelets, high specific activity [9-3H]PGI2 was synthesized by iodination and subsequent base treatment of the labeled precursor [9-3H]prostaglandin (PG)F2α methyl ester. Binding experiments were performed at room temperature with normal citrated human platelet-rich plasma that contained [14C]sucrose or [14C]PGF1α as an internal marker for the extracellular space. Binding of [3H]PGI2 plateaued within 2 min and this bond radioactivity could be displaced rapidly by excess nonradioactive PGI2. Scatchard analysis of concentration-dependent binding yielded a hyperbolic plot which appeared to be caused by the existence of two classes of binding sites. The higher affinity class has a dissociation constant of 12.1±2.7 nM and a capacity of 93 (±21)sites per platelet. The lower affinity class had a dissociation constant of 0.909±.236 μM and a capacity of 2,700±700 sites per platelet. The relative ability of PGI2, PGE1, PGE2, and 6-keto-PGF1α to displace [3H]PGI2 initially bound to the higher affinity class of sites were 100:5:2, which is more potent than PGE1 as an inhibitor of aggregation, did not displace bound [3H]PGI2. The higher affinity binding site for PGI2 appears to be the specific receptor through which PGI2 exerts its effect on platelets.