Characterization of [5,6-3H]SQ 29,548 as a high affinity radioligand, binding to thromboxane A2/prostaglandin H2-receptors in human platelets.
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.
Hedberg
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A
Department of Pharmacology, Squibb Institute for Medical Research, Princeton, New Jersey.
Hall
S E
SE
Ogletree
M L
ML
Harris
D N
DN
Liu
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EC
D001665:Binding Sites; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic; D005231:Fatty Acids, Unsaturated; D006801:Humans; D006834:Hydrazines / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D010974:Platelet Aggregation / Q000187:drug effects; D011869:Radioligand Assay; D011982:Receptors, Prostaglandin / Q000378:metabolism*; D017482:Receptors, Thromboxane; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D013928:Thromboxane A2 / Q000037:antagonists & inhibitors*
D019086:Bridged Bicyclo Compounds, Heterocyclic; D005231:Fatty Acids, Unsaturated; D006834:Hydrazines; D011982:Receptors, Prostaglandin; D017482:Receptors, Thromboxane; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D013928:Thromboxane A2; C045749:SQ 29548