Fibronectin (FN) is a large glycoprotein component of the extracellular matrix (ECM). It presents two forms: soluble in plasma or insoluble (fibrillar) within the ECM surrounding the cells in tissues. FN is one of the most important proteins in the ECM, it directly mediates cell adhesion, and therefore is essential for several biological processes such as embryonic development or blood clotting. Its involvement in these processes relay in its interaction with the cellular receptors integrins. The major binding site for integrins in FN is the so-called RGD site located in the 10th FN type III repeat (FNIII10), which is recognized by a5b1, aIIbb3 and all the av-containing integrins. Additionally, the a5b1 and aIIbb3 integrins can also bind the synergy site (DRVPPSRN) in the 9th FN type III repeat (FNIII9). In this work, we aimed to understand the function of these two different FN binding sites in interaction with integrins and fibrillar ECMs formation. When FN fibrillogenesis is disabled either due to insufficient FN production or defective assembly can lead to organ or tissue dysfunction. Integrin interaction with the RGD motif in FNIII10 has been considered the backbone of FN fibrillogenesis. This work has explored new RGDindependent mechanisms for FN fibrillogenesis. The results from this work showed that despite lacking the RGD site, FNdRGD can be assembled in a disorganized fibrillar matrix. The FNdRGD fibrillogenesis takes place through a different binding site located in the 12-14th FN type III repeat (heparinII) through binding of receptors distinct of integrins, the syndecan family of heparan sulphate. Although the RGD sequence in the FNIII10 domain is the key binding site for integrins, the synergy site has been demonstrated in adhesion experiments to cooperate in the interaction of the RGD motif with a5b1 and aIIbb3 in vitro. With the aim of analysing the role of the synergy site in vivo, in this project we analysed a mouse strain in with mutations in the sequence DRVPPSRN. The mutation does not affect mouse development, however the mutant synergy mice (Fn1syn/syn) have prolonged haemorrhages upon vessel injury, indicating that platelet-FN interaction is altered. To further study the function of the synergy site in integrin binding, we developed a series of in vivo and in vitro. In this work we show that the synergy site is not essential for cell adhesion or FN fibrillogenesis, but it is important to strengthen the bond to FN under shear forces. The a5b1 adhesion to FN reinforcement allows regulation of integrin-FN downstream signalling, assembly of focal adhesions and reorganization of the cytoskeleton. Integrin-FN reinforcement through the synergy site also modulates cellular adaptation to different rigidities. Moreover, we could demonstrate that the function of the synergy site can be compensated by av integrins in mesenchymal cells and by fibrinogen, aFibronectin (FN) is a large glycoprotein component of the extracellular matrix (ECM). It presents two forms: soluble in plasma or insoluble (fibrillar) within the ECM surrounding the cells in tissues. FN is one of the most important proteins in the ECM, it directly mediates cell adhesion, and therefore is essential for several biological processes such as embryonic development or blood clotting. Its involvement in these processes relay in its interaction with the cellular receptors integrins. The major binding site for integrins in FN is the so-called RGD site located in the 10th FN type III repeat (FNIII10), which is recognized by a5b1, aIIbb3 and all the av-containing integrins. Additionally, the a5b1 and aIIbb3 integrins can also bind the synergy site (DRVPPSRN) in the 9th FN type III repeat (FNIII9). In this work, we aimed to understand the function of these two different FN binding sites in interaction with integrins and fibrillar ECMs formation. When FN fibrillogenesis is disabled either due to insufficient FN production or defective assembly can lead to organ or tissue dysfunction. Integrin interaction with the RGD motif in FNIII10 has been considered the backbone of FN fibrillogenesis. This work has explored new RGDindependent mechanisms for FN fibrillogenesis. The results from this work showed that despite lacking the RGD site, FNdRGD can be assembled in a disorganized fibrillar matrix. The FNdRGD fibrillogenesis takes place through a different binding site located in the 12-14th FN type III repeat (heparinII) through binding of receptors distinct of integrins, the syndecan family of heparan sulphate. Although the RGD sequence in the FNIII10 domain is the key binding site for integrins, the synergy site has been demonstrated in adhesion experiments to cooperate in the interaction of the RGD motif with a5b1 and aIIbb3 in vitro. With the aim of analysing the role of the synergy site in vivo, in this project we analysed a mouse strain in with mutations in the sequence DRVPPSRN. The mutation does not affect mouse development, however the mutant synergy mice (Fn1syn/syn) have prolonged haemorrhages upon vessel injury, indicating that platelet-FN interaction is altered. To further study the function of the synergy site in integrin binding, we developed a series of in vivo and in vitro. In this work we show that the synergy site is not essential for cell adhesion or FN fibrillogenesis, but it is important to strengthen the bond to FN under shear forces. The a5b1 adhesion to FN reinforcement allows regulation of integrin-FN downstream signalling, assembly of focal adhesions and reorganization of the cytoskeleton. Integrin-FN reinforcement through the synergy site also modulates cellular adaptation to different rigidities. Moreover, we could demonstrate that the function of the synergy site can be compensated by av integrins in mesenchymal cells and by fibrinogen, a plasmatic ECM protein which binds aIIbb3.