Labeling of human platelet plasma membrane thromboxane A2/prostaglandin H2 receptors using SQB, a novel biotinylated receptor probe

• Published in: Biochemical pharmacology Vol. 52; no. 5; pp. 763 - 770
• Main Authors: KOMIOTIS, D, WENCEL-DRAKE, J. D, DIETER, J. P, LIM, C. T, LE BRETON, G. C
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Science 13.09.1996
• Subjects: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Biological and medical sciences; Biotin - chemistry; Blood Platelets - chemistry; Bridged Bicyclo Compounds, Heterocyclic; Cell Membrane - chemistry; Cell receptors; Cell structures and functions; Fatty Acids, Unsaturated; Fundamental and applied biological sciences. Psychology; Hormone receptors. Growth factor receptors. Cytokine receptors. Prostaglandin receptors; Humans; Hydrazines - chemistry; Hydrazines - metabolism; Hydrazines - pharmacology; Microscopy, Fluorescence; Molecular and cellular biology; Molecular Probes; Platelet Aggregation Inhibitors - pharmacology; Prostaglandin Endoperoxides, Synthetic - pharmacology; Radioligand Assay; Receptors, Prostaglandin - antagonists & inhibitors; Receptors, Prostaglandin - chemistry; Receptors, Prostaglandin - metabolism; Receptors, Thromboxane - antagonists & inhibitors; Receptors, Thromboxane - chemistry; Receptors, Thromboxane - metabolism; Receptors, Thromboxane A2, Prostaglandin H2; Spectrometry, Fluorescence; Sulfonamides - metabolism; Thromboxane A2 - analogs & derivatives; Thromboxane A2 - pharmacology; Human; Arachidonic acid derivatives; Thromboxane A2; Prostaglandin H2; Biotin; Biomembrane; Chemical labelling; In vitro; Antiplatelet agent; Biological fixation; Platelet; Antagonist; Biological receptor
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/0006-2952(96)00359-0

This study reports the synthesis, biological evaluation, and application of a new biotinylated derivative 1-[[1S-[1 alpha, 2 alpha (Z),3 alpha, 4 alpha]]-7-[3-[[[(1-oxocyclohexylpropyl)amino]acetyl]amino] methyl]-7-oxabicyclo [2.2.1]hept-2-yl]-5-heptenoyl]-2-[hexahydro-2'-oxo-1H-thieno[3',4' d] imidazole-4'-pentanoyl]hydrazine (SQB) of the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor antagonist [1S-[1 alpha,2 alpha(Z),3 alpha,4 alpha]]-7-[3-[[[(1-oxocyclohexylpropyl)amino]acetyl] amino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (SQ31,491). SQB was synthesized by reacting SQ31,491 with biotin hydrazide, and the product was purified by flash chromatography. It was found that SQB specifically inhibited platelet aggregation in response to U46619 with an IC50 of 275 nM. On the other hand, SQB did not inhibit adenosine diphosphate or A23187-induced aggregation. Competition binding studies revealed that SQB produced a concentration-dependent inhibition of [3H]-[1S-[1 alpha, 2 beta (5Z),3 beta, 4 alpha]]-7-[3-[[2[(phenylamino) carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid ([3H]SQ29,548) specific binding in 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS)-solubilized platelet membranes, with a Ki of 220 nM. The shape of the SQB inhibition binding curve was indistinguishable from that produced by the TXA2/PGH2 receptor antagonist BM13.177. Finally, incubation of gel-filtered platelets or platelet-rich plasma with SQB and fluorescein isothiocyanate (FITC)-avidin demonstrated fluorescent labeling of platelet plasma membrane TXA2/PGH2 receptors. Furthermore, this SQB-FITC fluorescent labeling was reduced significantly by co-incubation of the platelets with the TXA2/PGH2 antagonist SQ29,548. Based on the ability of SQB-FITC-avidin to label intact platelets, it can be concluded: (1) that a pool of platelet TXA2/PGH2 receptors resides in the plasma membrane; and (2) that the binding domains for these receptors are oriented at or near the external membrane surface. Collectively, these data demonstrate that SQB is a highly specific probe for TXA2/PGH2 receptors, which should be of significant value for receptor localization studies in platelets and other tissues.