Loskutoff D J, van Mourik J A, Erickson L A, Lawrence D
Proc Natl Acad Sci U S A. 1983 May;80(10):2956-60. doi: 10.1073/pnas.80.10.2956.
Fibrin/agar films were prepared and used to detect plasminogen activators produced by cultured bovine aortic endothelial cells (fibrin autography). One preparation of fibrin underwent spontaneous lysis upon incubation at 37 degrees C. This lysis was prevented by antibodies to tissue-type plasminogen activator but not by antibodies to urokinase. Conditioned medium from the confluent endothelial cells was fractionated by polyacrylamide gel electrophoresis in the presence of NaDodSO4. The gels were analyzed on indicator films prepared with the spontaneously lysing fibrin (reverse fibrin autography). Unexpectedly, as the opaque fibrin film cleared, a distinct lysis-resistant zone appeared in the indicator gel at a region corresponding to Mr 55,000. Experiments were devised to determine whether the lysis-resistant zone in the indicator film reflected the presence of a cellular inhibitor in the polyacrylamide gel. The corresponding region was excised from a polyacrylamide gel, extracted with buffer, and tested directly for antifibrinolytic activity by the 125I-labeled fibrin plate method. Urokinase-mediated fibrinolytic activity was inhibited by the gel extract in a dose-dependent manner indicating the presence of such an inhibitor. Inhibitor activity was detected in Triton X-100 extracts of washed monolayers and in conditioned medium, where it accumulated with time. The endothelial cell inhibitor not only survived exposure to NaDodSO4 but also was active after incubation at pH 12 or treatment with 5% (vol/vol) 2-mercaptoethanol, 6 M urea, 4 M guanidine hydrochloride, or 1 M acetic acid. Considerable activity also remained after heating at 100 degrees C for 30 min. These results indicate that cultured bovine aortic endothelial cells synthesize and secrete a previously undetected, unusually stable fibrinolytic inhibitor of Mr 55,000. Reverse fibrin autography offers a convenient approach for studying such molecules.
制备了纤维蛋白/琼脂膜，并用于检测培养的牛主动脉内皮细胞产生的纤溶酶原激活剂（纤维蛋白自显影法）。一种纤维蛋白制剂在37℃孵育时发生自发溶解。这种溶解可被抗组织型纤溶酶原激活剂的抗体阻止，但不能被抗尿激酶的抗体阻止。汇合的内皮细胞的条件培养基在十二烷基硫酸钠存在下通过聚丙烯酰胺凝胶电泳进行分级分离。凝胶在由自发溶解的纤维蛋白制备的指示膜上进行分析（反向纤维蛋白自显影）。出乎意料的是，随着不透明的纤维蛋白膜变清，在指示凝胶中对应于Mr 55,000的区域出现了一个明显的抗溶解区。设计实验以确定指示膜中的抗溶解区是否反映了聚丙烯酰胺凝胶中细胞抑制剂的存在。从聚丙烯酰胺凝胶中切下相应区域，用缓冲液提取，并通过125I标记的纤维蛋白平板法直接检测抗纤溶活性。凝胶提取物以剂量依赖性方式抑制尿激酶介导的纤溶活性，表明存在这种抑制剂。在洗涤过的单层细胞的Triton X-100提取物和条件培养基中检测到抑制剂活性，其活性随时间积累。内皮细胞抑制剂不仅在暴露于十二烷基硫酸钠后仍存活，而且在pH 12孵育或用5%（体积/体积）2-巯基乙醇、6 M尿素、4 M盐酸胍或1 M乙酸处理后仍有活性。在100℃加热30分钟后也仍有相当的活性。这些结果表明，培养的牛主动脉内皮细胞合成并分泌一种先前未检测到的、异常稳定的Mr 55,000的纤溶抑制剂。反向纤维蛋白自显影为研究此类分子提供了一种便捷的方法。
