Interaction of Fibrin(ogen) with Fibronectin:  Further Characterization and Localization of the Fibronectin-Binding Site

The interaction of fibronectin with fibrin and its incorporation into fibrin clots are thought to be important for the formation of a provisional matrix that promotes cell adhesion and migration during wound healing. However, it is still unclear whether fibronectin interacts with both fibrin and fibrinogen or fibrin only and whether fibronectin binds exclusively to the fibrin(ogen) αC domains. To address these questions, we studied the interaction of fibronectin with fibrinogen, fibrin, and their proteolytic and recombinant fragments. In both ELISA and surface plasmon resonance (SPR) experiments, immobilized fibrinogen did not bind fibronectin at all, but after conversion to fibrin, it bound fibronectin with high affinity. To test which regions of fibrin are involved in this binding, we studied the interaction of fibronectin with the fibrin-derived D−D:E1 complex and a recombinant αC fragment (residues Aα221−610) corresponding to the αC domain that together encompass the whole fibrin(ogen) molecule. In ELISA, when fibronectin was added to the immobilized D−D:E1 complex or the immobilized αC fragment, only the latter exhibited binding. Likewise, when fibronectin was immobilized and the complex or the αC fragment was added, only the latter was observed to bind. The selective interaction between fibronectin and the αC fragment was confirmed by SPR. The fibronectin-binding site was further localized to the NH2 terminal connector region of the αC domain since in ELISA, the immobilized recombinant Aα221−391 sub-fragment bound fibronectin well while the immobilized recombinant Aα392−610 sub-fragment exhibited no binding. This finding was confirmed by ligand blotting analysis. Thus, the results provide direct evidence for the existence of a cryptic high-affinity fibronectin-binding site in the Aα221−391 region of the fibrinogen αC domain that is not accessible in fibrinogen but becomes exposed in fibrin.