Specific binding of human fibrinogen to cultured human fibroblasts

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Abstract
Highly purified human fibrinogen was labeled with radioactive I and its interaction with cultured human embryo lung fibroblasts (MRC5) was examined. The cell monolayer was incubated at 37.degree. 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-labeled 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 nonimmune 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 .times. 106 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-labeled 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 100,000 and 50,000 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. Human fibroblasts apparently 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.