Adhesion of endothelial cells (EC) to surfaces can be enhanced by supplementing the integrin-mediated adhesion with high-affinity streptavidin (SA) that links a biotinylated EC to a biotinylated surface. Biotin pullout from the EC membrane limits the effectiveness of this treatment, leading to a predominance of EC detachment by cohesive failure. In this study we investigated whether a RGD-SA mutant that links SA to EC integrin receptors, and eliminates EC biotinylation, improves EC adhesion. Suspended EC were incubated with the RGD-SA mutant prior to cell seeding, primarily via attachment to the RGD binding site on alpha(v)beta(3) integrin. RGD-SA-incubated EC were subsequently seeded onto a surface preadsorbed with a mixture of fibronectin (Fn) and biotinylated bovine serum albumin (b-BSA). Results showed EC adhesion supplemented with the RGD-SA-biotin system significantly increased cell retention under flow, critical shear stresses for detachment, focal contact area, and force per bond relative to SA used with biotinylated EC. These increases were accompanied by significant reductions in membrane fragments left behind following EC detachment, which suggested cohesive failure via cell membrane rupture was significantly reduced, and enhanced phosphorylation of focal adhesion kinase, which suggested activation and clustering of integrin receptors. Together, these results show that the integrin-independent augmentation of EC adhesion using SA-biotin can be further improved through use of an RGD-SA mutant.