Interactions of endothelial cells with fibrin(ogen) are implicated in inflammation, angiogenesis, and wound healing. Cross-linking of the fibrinogen alphaC domains with factor XIIIa generates ordered alphaC oligomers mimicking polymeric arrangement of the alphaC domains in fibrin. These oligomers and those prepared with tissue transglutaminase were used to establish a mechanism of the alphaC domain-mediated interaction of fibrin with endothelial cells. Cell adhesion and chemical cross-linking experiments revealed that oligomerization of the alphaC domains by both transglutaminases significantly increases their RGD (arginyl-glycyl-aspartate)-dependent interaction with endothelial alphaVbeta3 and to a lesser extent with alphaVbeta5 and alpha5beta1 integrins. The oligomerization promotes integrin clustering, thereby increasing cell adhesion, spreading, formation of prominent peripheral focal contacts, and integrin-mediated activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) signaling pathways. The enhanced integrin clustering is likely caused by ordered juxtaposition of RGD-containing integrin-binding sites upon oligomerization of the alphaC domains and increased affinity of these domains for integrins. Our findings provide new insights into the mechanism of the alphaC domain-mediated interaction of endothelial cells with fibrin and imply its potential involvement in cell migration. They also suggest a new role for transglutaminases in regulation of integrin-mediated adhesion and signaling via covalent modification of integrin ligands.