Studies on PGE_1/PGI_2 receptors in platelets from patients with thrombotic disorders

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The purpose of this report is to study PGE_1/PGI_2 receptors in platelets from patients with thrombotic disorders.PGE_1/PGI_2 receptor proteins in the platelet membranes were purified according to the method of Dutta-Roy. Briefly, PGE_1/PGI_2 receptor proteins were solubilized by Triton X-100 in Tris-HCl buffer. pH 7.4 containing PMSF. The soluble membrane proteins were chromatographed on a DEAE-cellulofine column and the receptor activity of each fraction was assayed by microfiber technique. The active fractions eluted at 0.7m KCl were pooled. concentrated and further fractionated by gel filtration on Sephadex G 200. The eluates containing [^3H] PGE_1 binding activity emerged in a single peak. The active fractions were concentrated and the homogeneity of the purified receptor proteins were tested by SDS polyacrylamide gel electrophoresis by Laemmli's method. When the gels were stained with Coomassie Brilliant Blue. one main band with the molecular weight of 190 kd and a few additional bands with smaller molecular weights were observed. Substitution of Mono Q with FPLC System for DEAE cellulofine failed to purify the receptor protein to homogeneity. In the next experiments we are planning to produce mouse monoclonal antibody against the 190 kd receptor protein, and to quantify the amounts of the receptor protein in platelets from patients with thrombotic disorders by the use of this antibody.