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:<0.3: <0.3. These relative abilities parallel the relative potencies of these compounds as inhibitors of ADP-induced platelet aggregation in vitro. However PGD2, 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.

Authors

Adelaide M. Siegl, J. Bryan Smith, Melvin J. Silver