DETECTION OF AN UNUSUALLY STABLE FIBRINOLYTIC INHIBITOR PRODUCED BY BOVINE ENDOTHELIAL-CELLS
DETECTION OF AN UNUSUALLY STABLE FIBRINOLYTIC INHIBITOR PRODUCED BY BOVINE ENDOTHELIAL-CELLS
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DOI：
10.1073/pnas.80.10.2956
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发表时间：
1983-01-01
期刊：
影响因子：
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通讯作者：
LAWRENCE, D
中科院分区：
文献类型：
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作者：
LOSKUTOFF, DJ;VANMOURIK, JA;LAWRENCE, D
制备纤维蛋白/琼脂膜，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）。一种纤维蛋白制剂在37度孵育后发生自发溶解。C.组织型纤溶酶原激活剂抗体可阻止这种溶解，但尿激酶抗体不能阻止。在NaDodSO4[十二烷基硫酸钠]存在的情况下，用聚丙烯酰胺凝胶电泳分离来自合流内皮细胞的条件培养基。用自发裂解纤维蛋白制备的指示膜（反向纤维蛋白自显影法）对凝胶进行分析。出乎意料的是，随着不透明纤维蛋白膜的清除，在指示凝胶中出现了一个明显的抗裂解区，对应于mw55000。设计了实验来确定指示膜中的抗裂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除相应区域，用缓冲液提取，并通过125i标记的纤维蛋白板法直接检测抗纤溶活性。凝胶提取物以剂量依赖的方式抑制尿激酶介导的纤溶活性，表明存在这种抑制剂。在Triton-X-100洗涤单层提取物和条件培养基中检测抑制剂活性，并随时间积累。内皮细胞抑制剂不仅暴露于NaDodSO4中存活，而且在pH 12孵育或5% (vol/vol) 2-巯基乙醇、6 M尿素、4 M盐酸胍或1 M乙酸处理后也具有活性。在100度加热后，仍有相当大的活性。C 30分钟。培养的牛主动脉内皮细胞明显地合成和分泌一种以前未检测到的、异常稳定的mw55000纤维蛋白溶解抑制剂。反向纤维蛋白自显影为研究这类分子提供了一种方便的方法。
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.degree. 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 [sodium dodecyl sulfate]. 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 as a region corresponding to MW 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.degree. C for 30 min. Cultured bovine aortic endothelial cells apparently synthesize and secrete a previously undetected, unusually stable fibrinolytic inhibitor of MW 55,000. Reverse fibrin autography offers a convenient approach for studying such molecules.