Labeling of human platelet plasma membrane thromboxane A(2) prostaglandin H-2 receptors using SQB, a novel biotinylated receptor probe

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 A(2)/prostaglandin H-2 (TXA(2)/PGH(2)) 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 [H-3]-[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 ([H-3]SQ29,548) specific binding in 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS)-solubilized platelet membranes, with a K-i of 220 nM. The shape of the SQB inhibition binding curve was indistinguishable from that produced by the TXA(2)/PGH(2) 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 TXA(2)/PGH(2) receptors. Furthermore, this SQB-FITC fluorescent labeling was reduced significantly by co-incubation of the platelets with the TXA(2)/PGH(2) 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 TXA(2)/PGH(2), 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 TXA(2)/PGH(2) receptors, which should be of significant value for receptor localization studies in platelets and other tissues.