Hagen I, Gogstad G O, Brosstad F, Solum N O
Biochim Biophys Acta. 1983 Aug 10;732(3):600-6. doi: 10.1016/0005-2736(83)90237-7.
A possible receptor for thrombin on the platelet membrane has been identified. Whole platelets were treated with 125I-labelled thrombin followed by washing of the platelets, solubilization in Triton X-100, crossed immunoelectrophoresis and autoradiography. A heavily labelled antigen which migrated slightly more slowly than albumin was observed. No corresponding arc was seen on the same immunoplate when stained with Coomassie brilliant blue, indicating that the antigen possessed weak antigenic properties and/or was present in very small amounts. When 125I-labelled thrombin that had been inactivated by phenylmethylsulphonyl fluoride was used, no such labelled arc was seen. The radiolabelled immunoprecipitate does not represent any of the antigens identified hitherto in the immunoelectrophoretic patterns obtained with platelets or platelet material. The electrophoretic mobility of the antigen was influenced neither by neuraminidase treatment of the platelets prior to the 125I-labelled thrombin exposure nor by inclusion of concanavalin A, wheat-germ lectin or lentil lectin in the gel during the first-dimension electrophoresis. This suggests that the antigen does not represent a glycoprotein. Upon subcellular fractionation the radioactively labelled arc was observed in the cytosol fraction following crossed immunoelectrophoresis and autoradiography. Analysis of the secreted proteins after induction of the release reaction with 125I-labelled thrombin revealed labelling of immunoprecipitates representing thrombospondin, albumin and the 'line' form of platelet factor 4. This confirms that stable complexes of 125I-labelled thrombin and platelet proteins can exist in the presence of Triton X-100 and during electrophoresis.
血小板膜上一种可能的凝血酶受体已被鉴定出来。用125I标记的凝血酶处理完整血小板，随后洗涤血小板，在Triton X - 100中溶解，进行交叉免疫电泳和放射自显影。观察到一种标记强烈的抗原，其迁移速度略慢于白蛋白。当用考马斯亮蓝染色时，在同一免疫板上未见到相应的条带，这表明该抗原具有较弱的抗原特性和/或含量极少。当使用已被苯甲基磺酰氟灭活的125I标记凝血酶时，未见到这种标记条带。放射性标记的免疫沉淀物并不代表迄今在用血小板或血小板材料获得的免疫电泳图谱中所鉴定出的任何抗原。该抗原的电泳迁移率既不受125I标记凝血酶处理前血小板经神经氨酸酶处理的影响，也不受一维电泳期间凝胶中加入伴刀豆球蛋白A、麦胚凝集素或扁豆凝集素的影响。这表明该抗原不是糖蛋白。在进行亚细胞分级分离后，交叉免疫电泳和放射自显影显示在胞质溶胶级分中观察到放射性标记条带。用125I标记的凝血酶诱导释放反应后，对分泌蛋白的分析显示代表血小板反应蛋白、白蛋白和血小板第4因子“线状”形式的免疫沉淀物有标记。这证实了在存在Triton X - 100和电泳过程中，125I标记的凝血酶与血小板蛋白能形成稳定的复合物。
