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 applicati on of a new biotinylated derivative 1-[[1S-[1 alpha,2 alpha(Z),3 alpha ,4 -1H-thieno[3',4'd]imidazole-4'-pentanoyl]hydrazine (SQB) of the thr omboxane A(2)/prostaglandin H-2 (TXA(2)/PGH(2)) receptor antagonist: [ 1S-[1 alpha,2 alpha(Z),3 alpha,4 ]methyl]-7-oxabicyclo[2.2.1]hept-2-yl ]-5-heptenoic acid (SQ31,491). SQB was synthesized by reacting SQ31,49 1 with biotin hydrazide, and the product was purified by flash chromat ography. It was found that SQB specifically inhibited platelet aggrega tion in response to U46619 with an IC50 of 275 nM. On the other hand, SQB did not inhibit adenosine diphosphate or A23187-induced aggregatio n. Competition binding studies revealed that SQB produced a concentrat ion-dependent inhibition of [H-3]-[1S-[1 alpha,2 beta(5Z),3 beta,4 l]h ydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]- 5-heptenoic acid ([H-3 ]SQ29,548) specific binding in olamidopropyl)dimethylammonio]-1-propan e-sulfonate (CHAPS)-solubilized platelet membranes, with a K-i of 220 nM. The shape of the SQB inhibition binding curve was indistinguishabl e from that produced by the TXA(2)/PGH(2) receptor antagonist BM13.177 . Finally, incubation of gel-filtered platelets or platelet-rich plasm a with SQB and fluorescein isothiocyanate (FITC)-avidin demonstrated f luorescent labeling of platelet plasma membrane TXA(2)/PGH(2) receptor s. Furthermore, this SQB-FITC fluorescent labeling was reduced signifi cantly by co-incubation of the platelets with the TXA(2)/PGH(2) antago nist 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)/PG H(2), receptors resides in the plasma membrane; and (2) that the bindi ng domains for these receptors are oriented at or near the external me mbrane surface. Collectively, these data demonstrate that SQB is a hig hly specific probe for TXA(2)/PGH(2) receptors, which should be of sig nificant value for receptor localization studies in platelets and othe r tissues.