Selective Binding Site for [<sup>3</sup>H]Prostacyclin on Platelets

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

doi:10.1172/JCI109292

Selective Binding Site for [³H]Prostacyclin on Platelets

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

Published February 1, 1979 - More info

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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)F_2α methyl ester. Binding experiments were performed at room temperature with normal citrated human platelet-rich plasma that contained [¹⁴C]sucrose or [¹⁴C]PGF_1α 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-PGF_1α 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.