A number of RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding and signaling, although it is still unclear whether these two recognition sites function independently, synergistically, or competitively. Experimental evidence has suggested that fibrinogen binding to the RGD-type integrin ␣IIb␤3 occurs exclusively through the synergistic ␥ -411sequence, thus questioning the functional role of the RGD recognition site. Here we have investigated the respective role of the fibrinogen ␥ 400 -411 sequence and the RGD motif in the molecular events leading to ligand-induced ␣IIb␤3-dependent Chinese hamster ovary (CHO) cell or platelet spreading, by using intact fibrinogen and well characterized plasmin-generated fibrinogen fragments containing either the RGD motif (fragment C) or the ␥ -411sequence (fragment D), and CHO cells expressing resting wild type (␣IIb␤3wt), constitutively active (␣IIb␤3T562N), or non-functional (␣IIb␤3D119Y) receptors. Our data provide evidence that the ␥ 400 -411 site by itself is able to initiate ␣IIb␤3 clustering and recruitment of intracellular proteins to early focal complexes, mediating cell attachment, FAK phosphorylation, and Rac1 activation, while the RGD motif subsequently acts as a molecular switch on the ␤3 subunit to trigger cell spreading. More importantly, we show that the premier functional role of the RGD site is not to reinforce cell attachment but, rather, to imprint a conformational change on the ␤3 subunit leading to maximal RhoA activation and actin cytoskeleton organization in CHO cells as well as in platelets. Finally, ␣IIb␤3-dependent RhoA stimulation and cell spreading, but not cell attachment, are Src-dependent and phosphoinositide 3-kinaseindependent and are inhibited by the Src antagonist PP2.Plasma fibrinogen is one of the most abundant soluble adhesion molecules present in blood vessels and serves as a ligand to a variety of vascular cells, including platelets, endothelial cells, and monocytes. Fibrinogen is primarily involved in the maintenance of hemostasis by mediating platelet aggregation, clot formation, and wound healing. In addition, together with thrombin-converted insoluble fibrin, fibrinogen also functions as a component of the extracellular matrix in non-hemostatic normal or pathological processes promoting placenta development, angiogenesis, atherosclerosis, metastasis, as well as a variety of vascular and renal diseases (1). Both fibrinogen and fibrin expose multiple interacting sites that serve as adhesion motifs for vascular cell receptors. Undoubtedly, the first and best characterized of these binding sites are those interacting with the ␤3 integrins, the platelet-specific ␣IIb␤3 fibrinogen receptor (2), and the ␣v␤3 vitronectin receptor (3).Human fibrinogen contains three putative ␤3 integrin binding sites, two RGD motifs within the A␣ chain, A␣ 95-98 (RGDF) and A␣ [572][573][574][575] (RGDS) (4), and a non-RGD dodecapeptide sequence in the ␥ chain (C-terminal ␥ 400 -411 ) (5). Although fibrinogen ...