Dejana E, Vergara-Dauden M, Balconi G, Pietra A, Cherel G, Donati M B, Larrieu M J, Marguerie G
Eur J Biochem. 1984 Mar 15;139(3):657-62. doi: 10.1111/j.1432-1033.1984.tb08054.x.
Highly purified human fibrinogen was labelled with radioactive iodine and its interaction with cultured human embryo lung fibroblasts (MRC5) was examined. The cell monolayer was incubated at 37 degrees C with 125I-fibrinogen in phosphate/saline buffer containing 1 mM Ca2+ and 1 mM Mg2+. A direct interaction between 125I-fibrinogen and MRC5 was observed. The binding was time-dependent, reached saturation at 10 min and was regulated by the density of the cell monolayer. Non-labelled fibrinogen inhibited the interaction but unrelated proteins, including fibronectin, ovalbumin or myoglobin, did not. Monospecific Fab fragments, directed to fibrinogen, inhibited binding by 55.3% at a 50/1 molar ratio while non-immune Fab produced a 1.5% inhibition at similar concentration. Autoradiography of the display of fibroblast-bound 125I on a 7.5% polyacrylamide gel showed that the extract exhibited electrophoretic bands characteristic of the constitutive B beta and gamma chains of the fibrinogen molecule. An apparent affinity constant, Ka = 6.7 +/- 0.2 X 10(6) M-1, was estimated from binding isotherms. After a 30-min incubation time the interaction between 125I-fibrinogen and the cells was completely reversible and displaceable by a large molar excess of non-labelled fibrinogen. When compared to fibroblasts (MRC5 or W138), cultures of human embryo epithelial cells (EUE) failed to interact with 125I-fibrinogen, providing evidence for the specificity of binding for fibroblast monolayers. Plasmin-degradation fragments D and E, of 100000 and 50000 relative molecular mass respectively, were tested for their capacity to inhibit fibrinogen binding. At a 1/400 125I-fibrinogen/fragment molar ratio, fragment E inhibited binding by 30% while fragment D produced a 3% inhibition only. Altogether, the results demonstrate that human fibroblasts possess specific binding sites for fibrinogen, which exhibit the characteristics of a receptor system regulated by the culture state of the cells. In addition the structural features, which are necessary for the interaction of fibrinogen with the cells, are probably located in the E domain of the molecule.
用放射性碘标记高纯度人纤维蛋白原，并检测其与培养的人胚肺成纤维细胞（MRC5）的相互作用。细胞单层在含有1 mM Ca2+和1 mM Mg2+的磷酸盐/盐缓冲液中，于37℃与125I - 纤维蛋白原一起孵育。观察到125I - 纤维蛋白原与MRC5之间存在直接相互作用。结合呈时间依赖性，10分钟时达到饱和，并受细胞单层密度调节。未标记的纤维蛋白原可抑制这种相互作用，但包括纤连蛋白、卵清蛋白或肌红蛋白在内的无关蛋白质则无此作用。针对纤维蛋白原的单特异性Fab片段，在50/1摩尔比时可抑制55.3%的结合，而在相似浓度下非免疫Fab产生1.5%的抑制作用。在7.5%聚丙烯酰胺凝胶上对与成纤维细胞结合的125I进行放射自显影显示，提取物呈现出纤维蛋白原分子组成性Bβ链和γ链的特征电泳带。根据结合等温线估算出表观亲和常数Ka = 6.7 +/- 0.2 X 10(6) M-1。孵育30分钟后，125I - 纤维蛋白原与细胞之间的相互作用完全可逆，且可被大量摩尔过量的未标记纤维蛋白原取代。与成纤维细胞（MRC5或W138）相比，人胚上皮细胞（EUE）培养物未能与125I - 纤维蛋白原相互作用，这为成纤维细胞单层结合的特异性提供了证据。分别测试了相对分子质量为100000和50000的纤溶酶降解片段D和E抑制纤维蛋白原结合的能力。在1/400的125I - 纤维蛋白原/片段摩尔比下，片段E抑制30%的结合，而片段D仅产生3%的抑制作用。总之，结果表明人成纤维细胞具有纤维蛋白原的特异性结合位点，这些位点表现出受细胞培养状态调节的受体系统特征。此外，纤维蛋白原与细胞相互作用所需的结构特征可能位于分子的E结构域。
