Characterization of [5,6-3H]SQ 29,548 as a high affinity radioligand, binding to thromboxane A2/prostaglandin H2-receptors in human platelets

• Published in: The Journal of pharmacology and experimental therapeutics Vol. 245; no. 3; pp. 786 - 792
• Main Authors: HEDBERG, A, HALL, S. E, OGLETREE, M. L, HARRIS, D. N, LIU, E. C.-K
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Pharmacology and Experimental Therapeutics 01.06.1988
• Subjects: Binding Sites; Binding, Competitive; Biological and medical sciences; Blood Platelets - metabolism; Cell receptors; Cell structures and functions; Fundamental and applied biological sciences. Psychology; Humans; Hydrazines - metabolism; In Vitro Techniques; Kinetics; Molecular and cellular biology; Platelet Aggregation - drug effects; Radioligand Assay; Receptors, Prostaglandin - metabolism; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2; Thromboxane A2 - antagonists & inhibitors; Human; Ligand binding; Radiolabelling; Arachidonic acid derivatives; Saturation; Thromboxane A2; Prostaglandin H2; Competitive inhibition; Characterization; Membrane receptor; Platelet; Substrate specificity; Kinetics
• ISSN: 0022-3565; 1521-0103

The binding of 5,6-3H(1S-[1 alpha, 2 beta(5Z), 3 beta, 4 alpha])-7-[3-([2-[(phenyl amino)carbonyl]hydrazino]methyl)-7-oxabicyclo[2.2.1]hept-2-yl]-5- heptenoic acid to receptors in human washed platelets (WP) and platelet membranes (PM) was characterized with regard to kinetics, saturability and competitive inhibition by putative thromboxane A2/prostaglandin H2 (TP)-receptor ligands. Specific binding of [3H]SQ 29,548 routinely amounted to 90 to 97% of total binding. The rate of association was 1.6 x 10(7) and 2.5 x 10(7) M-1 x min-1 in WP and PM, respectively. The corresponding rate of dissociation was 0.07 and 0.12 min-1, resulting in dissociation constants of 4.1 and 5.8 nM in WP and PM, respectively. Saturable binding to a single class of receptors indicated a receptor density of 2633 fmol/mg of protein in WP (1394 receptors/platelet; kd, 4.5 nM) and 1466 fmol/mg of protein in PM (kd, 11.3 nM). Specific binding of [3H]SQ 29,548 was inhibited by five antagonists (high/low affinity kd values in nanomolar), SQ 29,548 (WP, 5.2; PM, 7.3), SQ 28,668 (WP, 32; PM, 73), SQ 30,741 (WP, 28; PM, 50), BM 13,177 (WP, 140; PM, 4834) and BM 13,505 (WP, 5/379; PM, 11). Two agonists, U 44069 and U 46619, inhibited the binding in a biphasic manner, indicating binding to two receptor sites (approximately 20/80%) with kd values of 4/72 and 4/170 nM, respectively, in WP and 7/136 and 19/502 nM, respectively in PM. The demonstrated high affinity binding of [3H]SQ 29,548 to human platelet TP-receptors should make this radioligand a suitable and potentially useful tool in future studies of function, structure and regulation of TP-receptors.