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

为研究血栓性疾病患者血小板膜上前列腺素E_1/前列环素受体(PGE_1/PGI_2)受体的变化，采用duta-Roy方法纯化了血小板膜PGE_1/PgI_2受体蛋白。简言之，Triton X-100在Tris-HCl缓冲液中增溶PGE_1/PGI_2受体蛋白。含有PMSF的pH 7.4。可溶性膜蛋白经DEAE-Cellofine柱层析后，用超细纤维技术测定各组分的受体活性。在0.7M KCl中洗脱的活性组份被混合在一起。浓缩后，用Sephadex G 200凝胶过滤进一步分级。含有[~3H]PGE_1结合活性的洗脱液呈单峰。对纯化的受体蛋白进行活性组分的浓缩，并用Laemmli‘s法进行SDS-PAGE分析。当凝胶被考马斯亮蓝染上时。观察到一条相对分子质量为190kd的主带和少量较小分子量的附加带。用FPLC系统取代Mono Q系统，未能将受体蛋白纯化到均一。在接下来的实验中，我们计划生产针对190kd受体蛋白的鼠单抗，并利用这种抗体来定量血栓性疾病患者血小板中受体蛋白的数量。

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.