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...

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SummaryHuman normal plasma stimulates prostacyclin (PGI2) production by vascular cells. This plasma activity may be greatly modified in different pathological conditions. The purpose of this study was to characterize some aspects of the mechanism of action of plasma in modulating PGI2 release. Cultured rat aortic smooth muscle cells were used. Citrated plasma from healthy donors stimulated PGI2 production in a concentration-dependent way. Plasma-derived serum containing increasing concentrations of platelets had the same PGI2 stimulating activity as citrated plasma. Plasma stimulation of PGI2 production was accompanied by release of endogenously incorporated arachidonic acid (AA) from the cell membrane. Similarly to AA, plasma induced PGI2 synthesis only once, a second or third challenge producing a reduced response from the cells. Cells stimulated twice with plasma responded to AA like unstimulated cells while cells stimulated twice with AA were poorly responsive to subsequent stimulation with plasma. When the cells were repeatedly stimulated with AA in the presence of plasma no refractoriness was apparent.This study suggests that plasma increases PGI2 synthesis by the release of endogenous substrate from the cell membrane and by protecting the cells from self-inactivation during AA conversion to prostaglandins.

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