Specific binding of human fibrinogen to cultured human fibroblasts

Abstract

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" C with 1251-fibrinogen in phosphate/saline buffer containing 1 mM Ca2+ and 1 mM Mgzc. A direct interaction between 1251-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 1251 on a 7.5 % polyacrylamide gel showed that the extract exhibited electrophoretic bands characteristic of the constitutive BP and y chains of the fibrinogen molecule. An apparent affinity constant, K, = 6.7 0.2 x lo6 M-', was estimated from binding isotherms. After a 30-min incubation time the interaction between '251-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 '251-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 1/400 251-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 ofithe 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.Fibrinogen is a major plasma protein, involved in haemostatic and thrombotic disorders. It is a substrate for thrombin in the blood coagulation cascade and it is transformed into fibrin after a proteolytic activation by the enzyme. Fibrinogen is also essential in the initial haemostatic plug formation as it regulates platelet aggregation via its interaction with specific inducible receptors on the platelet membrane [l]. In addition several observations have implicated the molecule or its physiological derivatives in different cellular functions. This includes the immune response [2, 31, the acute-phase reaction [4] and cell proliferation [5]. A common mechanism for the participation of fibrinogen in these biological processes may involve a direct interaction of the molecule with surface-exposed or induced receptors.Fibroblasts are important in wound healing and play a role in the inflammatory response. Fibrin depositi...

Increased Plasminogen Activator Inhibitor-1 in Keloid Fibroblasts May Account for their Elevated Collagen Accumulation in Fibrin Gel Cultures

“…47 It has been shown that fibroblasts are able to bind to fibrin using integrins containing the ␣ v subunit. 17,48,49 It is, however, not excluded in the current study that fibronectin may be involved in the binding of fibroblasts to the fibrin gel matrix through ␣ 5 ␤ 1 integrin, 21 because fibrinogen used in the study contains a trace amount of fibronectin (Ͻ 0.1 g/mg of fibrinogen), and 10% fetal calf serum (which contains fibronectin) was used in the collagen synthesis assay. Therefore, the difference in uPA and PAI-1 expression between fibrin and collagen gels may be mediated by a difference in ␣ v -containing integrin or ␣ 5 ␤ 1 binding to fibrin/fibronectin and/or ␣ 2 ␤ 1 binding to collagen.…”

Immunologic relationship between platelet membrane glycoprotein GPIIb/IIIa and cell surface molecules expressed by a variety of cells.

“…Thus, the GPIIb/IIIa-related molecules may constitute a family of structurally and antigenically related adhesion receptors with a recognition specificity for Arg-Gly-Asp sequences. Consistent with this hypothesis is the existence of fibrinogen binding sites on fibroblasts (50) and endothelial cells (51) as well as platelets, although the role of the GPIIb/IIla-related molecules in these interactions remains to be established. Bacterial surface molecules that mediate cell adhesion have been termed "adhesins" (52).…”

Interaction between fibrinogen and cultured endothelial cells. Induction of migration and specific binding.

“…Thus, the fg molecule may express interfaces with ECs and may influence EC migration, but direct evidence supporting this concept is still lacking. An attractive hypothesis is that the participation of the molecule in endothelial function is mediated via specific binding sites; the identification of receptor systems for fg on platelets (5,6), macrophages (7,8), and fibroblasts (9,10) may support this concept.…”