A new functional role of the fibrinogen RGD motif as the molecular switch that selectively triggers integrin alphaIIbbeta3-dependent RhoA activation during cell spreading.

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 alphaIIbbeta3 occurs exclusively through the synergistic gamma(400-411) sequence, thus questioning the functional role of the RGD recognition site. Here we have investigated the respective role of the fibrinogen gamma(400-411) sequence and the RGD motif in the molecular events leading to ligand-induced alphaIIbbeta3-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 gamma(400-411) sequence (fragment D), and CHO cells expressing resting wild type (alphaIIbbeta3wt), constitutively active (alphaIIbbeta3T562N), or non-functional (alphaIIbbeta3D119Y) receptors. Our data provide evidence that the gamma(400-411) site by itself is able to initiate alphaIIbbeta3 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 beta3 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 beta3 subunit leading to maximal RhoA activation and actin cytoskeleton organization in CHO cells as well as in platelets. Finally, alphaIIbbeta3-dependent RhoA stimulation and cell spreading, but not cell attachment, are Src-dependent and phosphoinositide 3-kinase-independent and are inhibited by the Src antagonist PP2.

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 ␥ 400 -411 sequence, 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 ␥ 400 -411 sequence (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␣ 572-575 (RGDS) (4), and a non-RGD dodecapeptide sequence in the ␥ chain (C-terminal ␥ 400 -411 ) (5). Although fibrinogen binding to ␣v␤3 relies essentially on the A␣ 572-575 RGDS sequence (3), binding to ␣IIb␤3 involves, in addition to the RGDS site, the non-RGD dodecapeptide ␥ 400 -411 sequence (5). Evidence for the functional importance of these fibrinogen motifs in ␤3 integrin ligand binding emerged from biochemical cross-linking and site-directed mutagenesis studies (6), as well as from genetic analysis of naturally occurring ␣IIb␤3 variants in patients with Glanzmann's thrombasthenia (7), characterized by complete loss of fibrinogen binding due to single point mutations within the ␤ propeller of the ␣IIb subunit or the A domain of the ␤3 subunit, that function as contact sites for the ␥ 400 -411 sequence and the RGD motif, respectively. With the recently published crystal structure of the extracellular domains of ␣v␤3 (8) and ␣IIb␤3 (9), the precise contact sites of the RGD motif with the ␤3 subunit have been defined with residue Asp ligated to a Mn 2ϩ held in the metal ion-dependent adhesion site of the ␤A domain and stabilized by additional contacts with residues Tyr-122, Arg-214, and Asn-215 of the ␤A domain.
Despite the higher affinity of the RGDS peptide for ␣IIb␤3 as compared with the ␥ 400 -411 dodecapeptide, experimental evidence has suggested that neither of the two RGD sequences in fibrinogen significantly contributes to the binding of ␣IIb␤3 to either surface-bound or soluble fibrinogen (10 -12), whereas in contrast, binding of ␣v␤3 to fibrinogen appears to rely essentially on the A␣ 572-575 RGDS sequence (3). Soluble or surface-bound fibrinogen interaction with ␣IIb␤3 is believed to be mediated primarily by the ␥ 400 -411 sequence (3,5,10,11,(13)(14)(15)(16), consistent with electron microscopy images showing ␣IIb␤3 associated with the distal ends of fibrinogen comprising the ␥ 400 -411 sequence (17). In addition, fibrinogen deleted of the ␥ 408 -411 AGDV terminal sequence on each ␥ chain produced in vitro (18) or in transgenic mice in vivo (19) is unable to support platelet aggregation, in contrast to fibrinogen mutated in the A␣ chain RGD sites that promotes normal platelet aggregation (10). These data highlight the predominant role of the ␥ chain residues 408 -411 in both ␣IIb␤3-mediated cell adhesion to surface-bound fibrinogen and ␣IIb␤3-mediated platelet aggregation and question the precise functional role of the A␣ 572-575 RGDS sequence in integrin ␣IIb␤3-fibrinogen recognition.
Here we have investigated the respective role of the ␥ 400 -411 sequence and the A␣ 572-575 RGDS site in the molecular events leading to ␣IIb␤3-dependent Chinese hamster ovary (CHO) 4 cell or platelet spreading. Using intact fibrinogen and well characterized proteolytic fragments containing either the ␥ 400 -411 site (fragment D) or the A␣ 572-575 RGDS site (fragment C) as ligands and platelets as well as CHO cells expressing resting wild type (␣IIb␤3wt), constitutively active (␣IIb␤3T562N), or non-functional (␣IIb␤3D119Y) receptors, we report a new functional role of the RGD motif as a molecular switch that triggers an ␣IIb␤3-dependent signaling cascade leading to Src-dependent RhoA activation and cell spreading.
cDNA Constructs, Transfection, and Cell Culture-The pTG2328-␤3D119Y plasmid was obtained from Dr. F. Lanza. The cDNA construct ␤3T562N was generated by PCR mutagenesis using standard procedures and pcDNA3-␤3wt as a template. Expression of wild type, ␣IIb-GFP ␤3, ␣IIb␤3 GFP , and other mutant human ␣IIb␤3 integrins in CHO cells was performed as described previously (21,22). CHO transfectants were cultured under standard tissue culture conditions in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum.
Cell Adhesion Assays-Adhesion assays were carried out as described previously (23) with minor modifications. Briefly, for each experiment, 3 ϫ 10 4 washed cells were preincubated for 30 min at room temperature with or without inhibitors, then added to the ligand-coated wells (20 g/ml) and cell adhesion was allowed to occur at 37°C. At given time points, the cells were microphotographed in the wells without prior washing of the plates or discharge of non-adherent cells. For all ␣IIb␤3dependent cell adhesion assays, ␣v␤3 receptor function was blocked by the addition of 10 M of the ␣v␤3 inhibitor RO65-5233/001 (Roche Applied Science). For cell adhesion inhibition assays, the cells were either preincubated with 10 M wortmannin (PI3K inhibitor), 10 M PP2 (Src inhibitor), or 10 M PP3 (as a negative control for PP2), all dissolved in Me 2 SO, or with Me 2 SO alone, as a vehicle control (final concentration 0.1%).
Immunofluorescence Microscopy and Flow Cytometry-Cells adherent on glass coverslips precoated with 20 g/ml fibrinogen, fragment D, or fragment C were fixed for 10 min at 4°C with 3% paraformaldehyde, 2% sucrose in PBS, pH 7.4, rinsed twice with PBS, and permeabilized with 0.5% Triton X-100 in PBS, pH 7.4, containing 0.5% heat-denatured bovine serum albumin. Immunofluorescent staining was performed using specific primary antibodies and a fluorescein isothiocyanate-conjugated secondary antibody (Caltag Laboratories, Burlingame, CA). Polymerized actin was stained with tetramethylrhodamine isothiocyanate (TRITC)-phalloidin (Molecular Probes, Leiden, The Netherlands). Microphotographs were taken using a Leica DC 300F digital camera and the Leica IM1000 1.20 software. Flow cytometry analysis of antibody binding to CHO transfectants or platelets was performed as previously described (23).
FAK Tyrosine Phosphorylation and GTPase Activity Assays-Petri dishes were coated with 20 g/ml of fibrinogen, fragment D, or fragment C, blocked with bovine serum albumin and finally washed twice with serum-free Iscove's modified Dulbecco's medium. CHO cells were either kept in suspension or added to the coated Petri dishes. Following incubation at 37°C, adherent cells were chilled on ice and lysed in situ.
For focal adhesion kinase (FAK) tyrosine phosphorylation assays, cells were lysed in buffer containing 2% SDS, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, pH 8.0, 10 mM NaF, 2 mM Na 3 VO 4 , 20 g/ml pepstatin A, 10 g/ml aprotinin and leupeptin, 50 M 4-(2-aminoethyl)benzenesulfonyl fluoride. Phosphorylated FAK was detected by immunoblotting of cell lysates with a polyclonal antibody specific for the phosphorylated FAK residue Tyr-397. The blot was then stripped and reprobed with a FAK-specific polyclonal antibody to monitor total FAK loading. Quantification of FAK tyrosine phosphorylation was performed by densitometric scanning of the autoradiograms (HP ScanJet 5p Scanner and QuantiScan software, Biosoft).
Rho family GTPase activity determination was performed as described by Ren et al. (24). Briefly, adherent cells were lysed in 500 l of lysis buffer containing 25 mM Hepes, pH 7.3, 150 mM NaCl, 5 mM MgCl 2 , 0.5 mM EGTA, 0.5% Triton X-100, 4% glycerol, 20 mM ␤-glycerophosphate, 10 mM NaF, 5 mM dithiothreitol, and a mix of serine and cysteine protease inhibitors (Roche Applied Science), and the cell lysates were incubated for 45 min at 4°C with glutathione-Sepharose beads coated with 20 -30 g of bacterially expressed GST-Rhotekin RBD (25) or GST-PAK1B PBD (26) for the pull-down of GTP-bound RhoA or GTP-bound Rac1, respectively. The GST-Rhotekin RBD and GST-PAK1B PBD constructs were obtained from M. A. Schwartz and J. G. Collard, respectively. Bound Rac1-GTP and RhoA-GTP as well as total Rac1 and RhoA (70 g of total cell lysate) were analyzed by Western blot using mAb against Rac1 or RhoA. Quantification of GTPase activity was performed by densitometric scanning of the autoradiograms, and the relative RhoA or Rac1 activity was expressed as the percentage of the total amount of RhoA or Rac1 present in whole cell lysates.
Platelet Preparation and Adhesion Assays-Acid-citrate-dextroseanti-coagulated blood (1:7, v/v) was obtained from healthy donors. After centrifugation at 200 ϫ g for 15 min at room temperature, the platelet-rich plasma was carefully removed, adjusted to pH 6.5 with acid-citrate-dextrose-anti-coagulated blood, and centrifuged at 1200 ϫ g for 15 min at room temperature in the presence of 1 M prostaglandin E 1 . Platelets were then gently resuspended and subsequently washed twice in a buffer containing 35 mM citrate acid, pH 6.5, 5 mM glucose, 1 M prostaglandin E 1 , 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 100 mM NaCl.
For adhesion assays, washed resting platelets or platelets stimulated for 20 min at 37°C with either 0.5 mM MnCl 2 or D3GP3 mAb were resuspended in Iscove's modified Dulbecco's medium and added to ligand-precoated glass coverslips saturated with 1% heat-denatured bovine serum albumin. Platelet adhesion was then allowed to occur at 37°C and at given time points platelets were fixed for 15 min at 4°C with 1% paraformaldehyde, 2% sucrose in PBS, pH 7.4. Polymerized actin filaments were visualized by phalloidin staining as described before.

RESULTS
A Two-step Mechanism Underlies ␣IIb␤3-dependent CHO Cell Spreading on Fibrinogen-Integrins ␣v␤3 and ␣IIb␤3 mediate cell adhesion to immobilized fibrinogen through two distinct molecular mechanisms: while ␣v␤3-dependent cell adhesion to fibrinogen relies essentially on the C-terminal A␣ 572-575 RGDS recognition site in the fibrinogen ␣ chain (15), ␣IIb␤3-mediated cell adhesion is mediated primarily through the ␥ chain C-terminal ␥ 400 -411 sequence (11,15). This distinct receptor-ligand interaction could easily be demonstrated in the cell adhesion assay used here by studying the inhibitory effect of the RGDS peptide or various anti-␤3 antibodies on CHO cell clones expressing similar amounts of either ␣v␤3 or ␣IIb␤3, as demonstrated by flow cytometry and Western blot analysis (Fig. 1A). The morphology of CHO cells during cell adhesion is shown in Fig. 1B. Non-adherent cells appeared as small round cells with a refringent peripheral rim, whereas attached cells were enlarged, flattened cells with small protrusions that had lost their peripheral refringency, and fully spread cells exhibited a fibroblastoid morphology.
Because of the presence of a chimeric hamster ␣v/human ␤3 integrin in CHO ␣IIb␤3 cells, control experiments were performed in the presence of 10 M RO65-5233/001, a selective inhibitor of ␣v␤3 and ␣v␤5 integrins, or in the presence of the ␣v␤3-blocking antibody LM609. Whereas these inhibitors completely blocked adhesion of CHO ␣v␤3 cells, they had no effect on CHO ␣IIb␤3 cell adhesion (Fig. 1C). All subsequent ␣IIb␤3-dependent adhesion assays were therefore performed in the presence of RO65-5233/001. The effect of the RGDS peptide or the RGD-containing mAb MA-16N7C2 on the cell morphology is shown in Fig. 1D. Pretreatment of the cells with RGDS or MA-16N7C2 totally inhibited CHO ␣v␤3wt cell spreading as well as cell attachment to intact fibrinogen, whereas these inhibitors prevented essentially CHO ␣IIb␤3wt cell spreading, but still allowed cell attachment. In addition, ␣v␤3-dependent cell adhesion was inhibited with antibodies directed against various epitopes of the ␤3 subunit, whereas these antibodies did not impair ␣IIb␤3-mediated cell adhesion to fibrinogen. Shown is a representative result obtained with the anti-␤3 mAb P37, which prevented ␣v␤3-dependent CHO cell spreading by 80%, but had no major inhibitory effect on ␣IIb␤3-dependent CHO cell spreading (Fig. 1E).
Loss-of-function point mutations have been identified in the ␤3 integrin subunit of Glanzmann's thrombasthenia patients that directly affect the RGD interaction with the ␤3 subunit A domain. Among these, the Asp-119 3 Tyr mutation, which interferes with the ␤3 metal iondependent adhesion site, completely inhibits fibrinogen-dependent platelet aggregation (27). Here we have tested whether CHO cells expressing this mutant ␣IIb␤3D119Y receptor with a non-functional RGD recognition site were still able to interact with immobilized fibrinogen. As expected, CHO ␣IIb␤3D119Y mutants failed to spread on immobilized fibrinogen (28) but underwent attachment, suggesting that the initial step of cell adhesion is unaffected by this mutation and relies on the ␣IIb subunit interaction with the ␥ 400 -411 sequence (Fig. 1F). These results provide evidence that, in contrast to ␣v␤3, the ␣IIb␤3fibrinogen interaction is a two-step mechanism involving two distinct contact sites in fibrinogen that function independently, the first step mediating cell attachment and relying on the ␥ 400 -411 sequence, and the second step leading to cell spreading, and mediated through RGD recognition.
CHO ␣IIb␤3wt Cells Plated on Fragment D Are Blocked at the Cell Attachment Stage-Previous studies have shown that resting ␣IIb␤3 in platelets is able to mediate cell adhesion to intact fibrinogen as well as plasmin-generated fibrinogen fragment D (16,29). To determine the specific involvement of the fibrinogen ␥ 400 -411 recognition site in the two-step adhesion process, we have used fibrinogen fragment D, which contains the ␥ 400 -411 site but is devoid of the RGD motifs, and fragment C, which corresponds to the C-terminal part of the fibrinogen ␣ chain and only contains the A␣ 572-575 RGDS sequence, as ␣IIb␤3 ligands (Fig.  2, A and B). When plated on fragment D, CHO ␣IIb␤3wt cells attached but were unable to spread, in contrast to cells plated on intact fibrinogen. On fragment C, the cells failed to attach and spread, indicating that resting ␣IIb␤3 expressed in CHO cells is unable to interact with the RGDS site exposed in fragment C in the absence of receptor activation through the ␣IIb-␥ 400 -411 interaction (Fig. 2C). A time-course experiment performed over a period of 4 h further showed that the cells plated on fragment D were blocked at this attachment stage (data not shown). These findings support the concept that the fibrinogen ␥ 400 -411 site in fragment D initiates cell attachment but cannot mediate irreversible cell spreading in the absence of the RGD site.
Attachment of CHO ␣IIb␤3wt Cells to Fragment D Induces ␣IIb␤3 Clustering and Recruitment of Intracellular Cytoskeletal and Adaptor Proteins into Focal Complexes-To determine whether CHO ␣IIb␤3wt  cell attachment to fibrinogen fragment D merely corresponded to receptor-ligand interaction or whether this interaction by itself was able to initiate outside-in signaling events leading to ␣IIb␤3 clustering, we investigated the subcellular localization of ␣IIb␤3 in cells plated on fragment D or fragment C. Interestingly, in CHO ␣IIb␤3wt cells attached to fragment D, ␣IIb␤3 was already well organized in small adhesion contacts at the cell periphery, giving the cells a stellar shape morphology (Fig. 3A). However, these cells were essentially devoid of actin stress fibers and exhibited submembranous actin staining, in contrast to cells plated on intact fibrinogen that displayed a fibroblastoid morphology with well structured stress fibers connected to mature focal adhesions. When plated on fragment C, the diffuse staining of ␤3 and the absence of actin filaments confirmed the absence of specific ␣IIb␤3dependent adhesion structures in these cells.
We next analyzed recruitment of typical components of focal adhesions to these ␣IIb␤3-dependent adhesion structures generated on fibrinogen fragment D. To demonstrate that all the immunostained adhesion contacts were also positive for ␣IIb␤3, we used CHO cells expressing an autofluorescent ␣IIb␤3 GFP or ␣IIb GFP ␤3 integrin (22). A perfect colocalization of immunostained paxillin and autofluorescent ␣IIb␤3 GFP is shown in Fig. 3B, demonstrating that on fibrinogen fragment D, only integrin ␣IIb␤3 is engaged in adhesion structures. An identical result was also obtained with cells expressing ␣IIb GFP ␤3 (data not shown). For further experiments, CHO cells expressing wild type ␣IIb␤3 were used. Interestingly, the cytoskeletal proteins talin and vinculin, as well as tyrosine-phosphorylated proteins were found in these adhesion structures (Fig. 3C). These results suggest that the ␣IIb-␥ 400 -411 interaction induces first wave signaling events leading to integrin clustering and to the recruitment of cytoskeletal, scaffolding, and signaling proteins into adhesion structures that resemble focal complexes (30). However, in the absence of an RGD site, the cells are blocked at this attachment stage, suggesting that a synergistic ␣IIb␤3-RGD interaction is required to trigger additional signaling events allowing full cell spreading.

The Spontaneously Active ␣IIb␤3T562N Mutant Mediates CHO Cell
Spreading on Fibrinogen Fragment D-Ligand-induced outside-in signaling relies on the long range propagation of conformational changes in the ␤3 integrin subunit that are transmitted from the ligand binding pocket to the cytoplasmic tail, necessary for the formation of mature focal adhesions. These ligand-induced conformational changes can be monitored with well characterized monoclonal antibodies, which identify ligand-induced binding site (LIBS) neoepitopes (31), exposed on the ␤3 integrin subunit following RGD or fibrinogen binding and not present on the unoccupied resting receptor. To determine whether ␣IIb␤3 outside-in signaling necessary for cell spreading requires a physical interaction of the RGD recognition motif with the ␤3 subunit or whether it can be brought about by a conformational change within the ␤3 subunit independent of the RGD interaction, we investigated the adhesive properties of CHO cells expressing the constitutively active ␣IIb␤3T562N receptor (32). As shown in Fig. 4, CHO ␣IIb␤3T562N cells spontaneously bound the fibrinogen-mimetic mAb PAC-1 and bound the LIBS-specific anti-␤3 antibody AP5 in the absence of ligand stimulation, confirming the constitutive high affinity state of the mutant receptor and the conformational change of the ␤3T562N subunit described by Kashiwagi and coworkers (32). An identical result was also obtained with two other anti-LIBS antibodies (data not shown). The most exciting observation, however, was the capacity of CHO ␣IIb␤3T562N cells to undergo full cell spreading on fibrinogen fragment D with the presence of ␣IIb␤3-containing focal adhesions and well organized actin stress fibers (Fig. 4C). A similar result was also obtained with CHO ␣IIb␤3wt cells following activation of ␣IIb␤3 with the activating mAb D3GP3 or AP5 (data not shown). Because, in the absence of the RGD site, a reinforced adhesion due to the RGD-␤3 interaction and necessary for cell spreading could be excluded, we conclude that the structural change in the ␤3T562N subunit mimics the conformational change normally induced following RGD binding to the ␤3 subunit and that this conformational change is sufficient to initiate intracellular signaling events leading to actin stress fiber organization and focal adhesion formation.
␣IIb␤3 Clustering upon Cell Attachment to Fragment D Triggers FAK Tyrosine Phosphorylation-To determine the signaling events that occur during ␣IIb␤3-dependent cell attachment versus cell spreading, we investigated FAK tyrosine phosphorylation on residue Tyr-397 during cell adhesion using an anti-phosphoTyr-397 pAb. As shown in Fig.  5A, FAK phosphorylation increased over a time course of 2 h when CHO ␣IIb␤3wt cells were plated on immobilized fibrinogen, whereas only background FAK tyrosine phosphorylation was observed in CHO ␣IIb␤3wt cells kept in suspension. We next compared FAK tyrosine phosphorylation in CHO cells expressing ␣IIb␤3wt, ␣IIb␤3D119Y, ␣IIb␤3T562N, or in mock transfected cells. As shown in Fig. 5B, CHO ␣IIb␤3wt cells plated for 2 h on fragment D triggered FAK tyrosine phosphorylation, corresponding to ϳ65% of that observed when cells were plated on native fibrinogen. In addition, weak but specific FAK tyrosine phosphorylation could also be observed in CHO ␣IIb␤3D119Y cells attached to fibrinogen, when compared with mock transfected CHO cells plated on fibrinogen or CHO ␣IIb␤3wt cells kept in suspension. More importantly, when CHO ␣IIb␤3T562N cells were tested, the amount of phosphorylated FAK was essentially identical when cells were plated on fragment D or on intact fibrinogen, in line with the immunofluorescence data showing similar receptor clustering in the cells spread on either substrate (Fig. 5C). Altogether, these data provide evidence that clustering of ␣IIb␤3 upon cell attachment to fragment D is sufficient to trigger significant FAK tyrosine phosphorylation and that, under all experimental conditions investigated, FAK phosphorylation correlated closely with the extent of ␣IIb␤3 clustering.
RhoA, but Not Rac1 Activation, Is Dependent on the RGD Interaction with the ␤3 Subunit-Previous studies have shown that cytoskeletal dynamics correlate closely with RhoA activity, with low levels of RhoA activity observed in cells with small focal complexes at the cell periphery and devoid of actin stress fibers, in contrast to high levels of RhoA activity in cells exhibiting stress fibers and focal adhesions (24). Likewise, in human platelets RhoA activation following integrin ␣IIb␤3 engagement leads to actin reorganization (33). Here we have investigated the activity of the small Rho family GTPases Rac1 and RhoA during ␣IIb␤3-mediated cell adhesion to intact fibrinogen and fragment D. In accordance with data shown by others (24), RhoA activity increased over a time course of 2 h on intact fibrinogen, whereas high levels of Rac1 activity could already be detected at early time points (Fig.  6A). To determine whether maximal RhoA activation relied on a conformational change induced following the RGD interaction with the ␤3 subunit, we next compared RhoA and Rac1 activity in CHO ␣IIb␤3wt or ␣IIb␤3T562N cells following a 2-h incubation on intact fibrinogen or fragment D. As shown in Fig. 6B, in CHO ␣IIb␤3wt cells plated on fragment D, the amount of precipitated active RhoA was low as compared with cells plated on intact fibrinogen. More importantly, in CHO ␣IIb␤3T562N cells, identical amounts of active RhoA were present on either intact fibrinogen or fragment D. On the other hand, Rac1 activity was very similar in CHO ␣IIb␤3wt cells attached to fragment D or spread on fibrinogen. These results demonstrate that complete RhoA activation requires an interaction of integrin ␣IIb␤3wt with the fibrinogen RGD motif, whereas Rac1 activity is independent of such an interaction and appears to rely solely on the ␣IIb-␥ 400 -411 recognition. And finally, as expected, the constitutively active mutant ␣IIb␤3T562N was able to circumvent the requirement for an ␣IIb␤3-RGD interaction and to trigger maximal RhoA activity on fragment D.
Src, but Not PI3 Kinase, Is Involved in ␣IIb␤3-dependent Cell Spreading and RhoA Activation on Immobilized Fibrinogen-An important event in ␣IIb␤3-dependent outside-in signaling is the selective and dynamic recruitment of signaling molecules that ultimately lead to Rho activation and cell spreading. Among these, phosphoinositide 3-kinase (PI3K) (34) as well as tyrosine kinases have been shown to be directly involved (35). Specific tyrosine kinases such as members of the Src family are of particular interest, because they function in proximity of ␣IIb␤3 by directly interacting with the cytoplasmic tail of the ␤3 subunit (36). Here we have used the pharmacological inhibitors wortmannin and PP2 to investigate the potential involvement of PI3K or Src in the ␣IIb␤3 signaling cascade leading to RhoA activation. As shown in Fig.  7A, ␣IIb␤3-dependent cell spreading, but not cell attachment, was completely blocked by the Src inhibitor PP2, and not by PP3 used as a negative control for PP2. Interestingly, the PI3K inhibitor wortmannin had no inhibitory effect. This result correlates with the strongly decreased generation of active RhoA in the cells treated with PP2, whereas RhoA activity was similar in the presence or absence of PP3 or wortmannin (Fig. 7, B and C). Surprisingly however, when CHO ␣IIb␤3T562N cells were tested, complete cell spreading and RhoA activation occurred despite the presence of PP2. These results demonstrate the important role of Src in the signaling cascade downstream of the RGD-␤3 interaction and necessary for RhoA activation. However, they also show that the constitutively active ␣IIb␤3T562N receptor can initiate cell spreading independent of active Src.
Platelets Are Able to Spread on Fibrinogen Fragment D following Activation by Mn 2ϩ -Finally, to further assess our concept (a) that the ␣IIb␤3-fibrinogen interaction is a two-step mechanism and (b) that a physical interaction of the RGD motif with the ␤3 subunit, required for RhoA activation and actin stress fiber organization, can be substituted by Mn 2ϩ activation of platelet ␣IIb␤3, we analyzed attachment and spreading as well as actin polymerization in resting and Mn 2ϩ -activated platelets when plated on either intact fibrinogen or fragment D. As expected, when plated on fragment D, resting platelets attached but failed to spread at early time points, whereas on native fibrinogen plate- let spreading was rapid and almost complete after 5 min with platelets exhibiting a complete reorganized actin cytoskeleton (Fig. 8, A and B). At a later time point, however, platelet spreading on fragment D did also occur due to the release of ␣-granular fibrinogen and dense granule ADP, which has been shown to stimulate phosphatidylinositol 3-kinase independent of ␣IIb␤3 engagement (37,38). When Mn 2ϩ stimulated platelets were tested, spreading on both native fibrinogen and fragment D occurred at early time points, and this result was also obtained when platelets were treated with the ␣IIb␤3-activating mAb D3GP3 (data not shown). Fig. 8C shows that platelets stimulated with either Mn 2ϩ or D3GP3 bound the fibrinogen-mimetic mAb PAC-1, in contrast to nonstimulated platelets, thus confirming the high affinity state of platelet integrin ␣IIb␤3 in stimulated platelets. All together, these results, which are in perfect agreement with our data obtained with ␣IIb␤3-expressing CHO cells, are also in accordance with the previously published data for the role of RhoA in ␣IIb␤3-dependent signaling following platelet spreading on native fibrinogen (33).

DISCUSSION
We have used intact fibrinogen and well characterized proteolytic fragments of fibrinogen containing either the ␥ 400 -411 site (fragment D) or the A␣ 572-575 RGDS site (fragment C) as ligands and platelets as well as CHO cells expressing resting wild type (␣IIb␤3wt), constitutively active (␣IIb␤3T562N), or non-functional (␣IIb␤3D119Y) receptors, to dissect the molecular events leading to ␣IIb␤3-dependent cell spreading and to further determine the functional role of each of the two fibrinogen binding sites during the adhesive process. Our major findings are as follows: the initial stage of cell attachment appears to rely essentially on the fibrinogen ␥ 400 -411 sequence interacting with the ␣IIb integrin subunit, because fibrinogen fragment D, devoid of the RGD recognition sites, is able to support ␣IIb␤3-dependent cell attachment. This ␥ 400 -411 -␣IIb interaction generates preliminary signaling events, which FIGURE 6. RhoA, but not Rac1, activation is dependent on RGD interaction with the ␤3 subunit. A, time course of RhoA or Rac1 activation in CHO ␣IIb␤3wt cells plated on intact fibrinogen at 37°C. Each value obtained for RhoA or Rac1 activity was expressed relative to the highest value observed and arbitrarily fixed to 100%. A representative experiment out of three is shown. B, analysis of RhoA or Rac1 activity in CHO ␣IIb␤3wt or CHO ␣IIb␤3T562N cells plated on fibrinogen or fragment D after a 2-h incubation at 37°C. Mock transfected CHO cells incubated on fibrinogen for 2 h were used as a negative control. Each value obtained for RhoA or Rac1 activity was expressed relative to a scale fixed arbitrarily to 1 for mock transfected cells on fibrinogen and to 100 for ␣IIb␤3 transfected cells on fibrinogen. Each bar represents the mean Ϯ S.D. of three independent experiments. lead to clustering of ␣IIb␤3 in early focal complexes, FAK tyrosine phosphorylation, as well as Rac1 activation. In the absence of the RGD binding site, ␣IIb␤3-mediated cell adhesion is blocked at this cell attachment stage and cannot proceed further, suggesting the requirement for a second signaling event promoting cell spreading. This second signaling event is generated through the subsequent RGDS-␤3 interaction, which relies on receptor activation necessary for fibrinogen RGD recognition. Finally, the observation that the constitutively active ␣IIb␤3T562N receptor, which spontaneously exposes a ␤3 conformation normally induced following RGD binding, is able to mediate complete cell spreading on fibrinogen fragment D, suggests that the premier functional role of the RGD-␣IIb␤3 interaction is to imprint a conformational change on the ␤3 integrin subunit, necessary to initiate second wave intracellular signaling events, such as maximal RhoA activation, required for cell spreading. Finally, we show that these second wave intracellular signaling events are Src-dependent but PI3 kinase-independent. The results reported here thus support a two-step adhesion mechanism responsible for ␣IIb␤3-dependent cell spreading on surface-bound fibrinogen and provide evidence for a sequential interaction of the ␥ 400 -411 site and the A␣ 572-575 RGDS site with ␣IIb␤3 that act synergistically to promote cell spreading. Our results are in good agreement with the recently published crystal structure of ␣IIb␤3 (9), which clearly highlights two distinct contact sites for fibrinogen binding, the specificity determining loop of the ␤3 I domain that interacts with the RGD sequence, and the ␣IIb cap subdomain comprising four insertions in the ␤-propeller, which is necessary for macromolecular recognition of fibrinogen.
The role of the ␣IIb-fibrinogen ␥ 400 -411 interaction in mediating ␣IIb␤3-dependent platelet adhesion to intact fibrinogen and to fragment D has been reported by several authors (16,39). Here we show for the first time that CHO ␣IIb␤3wt cells attached to fibrinogen fragment D exhibit numerous short protrusions with discrete ␣IIb␤3 clusters at the tips of the protrusions and colocalization of major cytoskeletal, adaptor, and signaling proteins such as talin, vinculin, or paxillin. These ␤3 clusters, which also contain tyrosine-phosphorylated proteins, are similar to focal complexes previously described (30). Our data thus provide evidence that the initial ␣IIb-fibrinogen ␥ chain interaction induces intracellular signaling events, independent of RGD-mediated signaling. It has to be emphasized that these results could only be observed in a cell adhesion assay devoid of washing steps. Indeed, the rather loose and reversible cell attachment mediated through the ␣IIb-␥ 400 -411 interaction was disrupted in standard cell adhesion assays including washing steps (16).
Integrin ␣IIb␤3 is an allosteric receptor that can switch from a resting to an active ligand binding receptor (40). In platelets, resting ␣IIb␤3 interacts with immobilized fibrinogen (29,41), whereas activation of ␣IIb␤3 is required for interaction with soluble fibrinogen (42,43). We and others have previously shown that resting ␣IIb␤3 in platelets or CHO cells promotes cell adhesion to native fibrinogen and to fibrinogen fragment D, provided that the ␥ 400 -411 sequence is available for interaction (16,44). These data underscore the synergistic role of the ␥ 400 -411 site in the activation process of integrin ␣IIb␤3, necessary for fibrinogen RGD recognition. Indeed, based on data showing that binding of a high affinity cyclic ␥ 400 -411 site analog to integrin ␣IIb␤3 results in increased LIBS1 antibody binding to the ␤3 subunit (45), we conclude that the ␣IIb-␥ 400 -411 interaction most likely induces in trans conformational changes within the ␤3 subunit, allowing its high affinity interaction with the fibrinogen RGD site. Furthermore, biochemical studies have suggested that the RGD binding site becomes more accessible upon receptor activation (46).
The most exciting result, however, concerns integrin ␣IIb␤3T562Nmediated cell spreading on fibrinogen fragment D, devoid of the RGD recognition site. Because it is commonly accepted that both the synergistic site and the RGD site are necessary for integrin-dependent anchorage of cells to RGD ligands, our results provide evidence that irreversible ␣IIb␤3-dependent cell spreading can be achieved in the absence of the RGD site, provided that ␣IIb␤3 is in the active state and has undergone an additional conformational change. As previously reported, introduction of the T562N mutation into the ␤3 subunit induces spontaneous PAC-1 binding as well as constitutive exposure of LIBS neoepitopes normally only exposed following RGD binding. By mediating CHO cell spreading on fragment D, the ␣IIb␤3T562N receptor circumvented the requirement for a ␤3-RGDS interaction, suggesting that the conformational change introduced into the ␤3 subunit by the T562N mutation most probably mimicked the conformational change normally imprinted on ␤3 following RGDS binding, and required for the initiation of the second wave of signaling events responsible for stress fiber organization and focal adhesions formation.
By using platelets as a more physiological cell model relevant to hemostasis and thrombosis, we have been able to confirm the data observed with CHO transfectants. Indeed, resting platelets attached but did not spread onto fibrinogen fragment D, at an early time point, whereas antibody or Mn 2ϩ activation of ␣IIb␤3 was sufficient to induce platelet spreading and actin reorganization. However, these results could only be observed at early time points, because the obvious platelet release of dense and ␣-granule constituents such as ADP and fibrinogen circumvented the signaling events initiated through ␣IIb␤3-fibrinogen fragment D interaction. The CHO cell model used here thus provides an excellent tool to precisely dissect the signaling events that specifically occur during ␣IIb␤3-mediated cell attachment and cell spreading. Using FAK tyrosine phosphorylation assays as well as intracellular immunofluorescence labeling of phospho-FAK (data not shown), we provide evidence that FAK phosphorylation on residue Tyr-397 already occurred in the early focal complexes observed in ␣IIb␤3wt-expressing CHO cells attached to fragment D. These data indicate that integrin clustering is sufficient to mediate the recruitment of FAK, allowing phosphorylation of residue Tyr-397 in FAK by a transautophosphorylation mechanism, necessary for the recruitment of Src family tyrosine kinases (47,48) and other signaling and cytoskeletal molecules. Full FAK Tyr-397 autophosphorylation was observed when cells were spread on intact fibrinogen. Thus, FAK tyrosine autophosphorylation correlated with integrin clustering, in agreement with our earlier results showing a strong correlation between the extent of mutant ␤3 clustering and the level of ␤3-triggered FAK tyrosine phosphorylation (21).
Integrin-dependent rearrangement of the cytoskeleton is highly influenced by the activity of the Rho family GTPases RhoA, Rac1, and Cdc42 (49). For example, RhoA promotes the formation of actin stress fibers and focal adhesions, whereas Rac1 promotes the formation of lamellipodia and membrane ruffling, and Cdc42 causes actin microspike formation and filopodia development. In contrast to Cdc42 and Rac1, the regulation of RhoA during cell adhesion is biphasic with a rapid and transient inhibition of RhoA activity upon integrin engagement, followed by a subsequent reactivation leading to the assembly of contractile actin-myosin filaments (stress fibers) associated with mature focal adhesions (24). Although the mechanisms linking integrins to the regulation of RhoA are still not fully understood, it has been shown that the extracellular domain of the integrin ␤ subunits is critical in stimulating RhoA activity (50,51). In human platelets, evidence has been provided that RhoA is not required for the adhesion of resting platelets (33) but plays an important role in regulating the stability of integrin ␣IIb␤3 adhesion contacts under high shear stress (52). Here we show for the first time that the RGD-␤3 interaction functions as a molecular switch and triggers maximal RhoA activation, most likely by imprinting a conformational change on the ␤3 integrin subunit, whereas the ␣IIb-␥ 400 -411 interaction mainly induces Rac1 activation. Our results further demonstrate that the signaling cascade initiated through the RGD-␤3 subunit interaction and leading to RhoA activation and cell spreading is dependent on active Src, but not PI3K, suggesting an important role of Src in the downstream activation of RhoA. These results are in line with the recent data reported by the group of Shattil, showing that Src activation occurs through its direct binding to the C-terminal part of the cytoplasmic tail of the ␤3 subunit of ␣IIb␤3 and thus becomes a major regulator of integrin-dependent outside-in signaling (36,53,54). Because Src is also a known regulator of the downstream guanine nucleotide exchange factors (GEFs) of Rho, such as Vav GEFs that regulate RhoA (55), Src signaling appears to link integrins to RhoA, as shown here with the Src inhibitor PP2, which strongly reduced the generation of active RhoA in CHO ␣IIb␤3 cells seeded on intact fibrinogen.
Finally, based on our data, we suggest that the two-step mechanism described here for ␣IIb␤3-mediated cell adhesion to fibrinogen could serve as a general model applicable to RGD-type integrins that rely on a synergistic site for full receptor function: one apparent function of the synergistic site would be to allow RGD-type integrins to bind a ligand preferentially over other RGD-containing proteins (56 -58) and to mediate ligand-specific attachment resulting in the activation of the receptor necessary for subsequent RGD recognition. The major functional role of the RGD site would be to initiate additional outside-in signaling events necessary for full cell spreading.