Figure 3. CXCL4 or CXCL4/CTF binds to purified avb3 integrins in a concentration-dependent and specific manners in solid- phase ligand binding assay. A. avb3 integrin was added to wells coated with CXCL4 or CXCL4/CTF as indicated, and incubated overnight at 4uC. Anti-av, peroxidase-conjugated anti-rabbit IgG antibodies, and TMB substrate were used to detect bound integrin. B. Soluble RGD-, RGE peptides or inhibitory anti-integrin antibodies were added to block the interaction between CXCL4 or CXCL4/CTF and avb3 integrin. Bound integrin was detected as above. Anti-integrin antibodies used were: anti-av (AV1) and anti-b3 (B3A). Error bars represent the mean6SD, *, P#0.005; **, P#0,0005 compared to CXCL4 or CXCL4/CTF in the presence of IgM control antibody; n = 3 independent experiments. doi:10.1371/journal.pone.0002657.g003 6/Soluble CXCL4 or CXCL4/CTF inhibits cell adhesion to immobilized fibronectin and vitronectin To investigate whether CXCL4 or CXCL4/CTF could inhibits cell adhesion to integrin ligands, we tested the effect of soluble CXCL4 or CXCL4/CTF on avb3-CHO and HUVECs cells adhesion to specific avb3 and a5b1 ligands, vitronectin and CXCL4 Integrins Angiogenesis fibronectin respectively. We found that the pre-incubation of cells with soluble CXCL4 or CXCL4/CTF induces cell rounding, and significantly inhibits cell adhesion to immobilized fibronectin and vitronectin, in a concentration-dependent manner (Fig. S1). CXCL4 or CXCL4/CTF did not affect cell adhesion to collagen I (data not shown). These results suggest that soluble CXCL4 and CXCL4/CTF act as antagonists of avb3 and a5b1 integrins to their ligands. 7/immobilized CXCL4 or CXCL4/CTF promotes and soluble CXCL4 or CXCL4/CTF inhibits endothelial cell migration in an integrin-dependent and -specific manners Cell migration is an essential step in the angiogenesis process [13]. Previous studies showed that CXCL4 or CXCL4/CTF block endothelial cell migration in response to FGF-2 or VEGF in stimulation in vitro [7,33], a process thought to mediate the anti- angiogenic effect of CXCL4 in vivo. On the other hand, avb3 [34], and a5b1 [17] integrins have a crucial role in cell migration during angiogenesis. Taking into account our findings, we determined whether CXCL4 and its antiangiogenic derived peptide modulates endothelial cell migration through avb3 and/or a5b1 integrins. Our finding showed that soluble CXCL4 or CXCL4/CTF inhibited HUVECs cells migration through fibronectin a reported avb3 and a5b1 ligand [13], toward VEGF, in a dose depending manner (Fig. 7A). This migration is similarly inhibited by adding RGD peptides or by the blocking integrin antibodies, anti-avb3 LM609 or anti-a5b1 JBS5 (Fig. 7A). These results are consistent with previous data [7,33], and suggest that CXCL4 or CXCL4/ CTF inhibits endothelial cell migration in an integrin dependent manner. To study the functional significance of the specific CXCL4- integrin interaction, we examined the capacity of CXCL4 to modulate cells functions under conditions in which these functions are strictly dependent on integrins, and not any other agents. For this purpose, we use a haptotactic Boyden chamber assay to assess cell migration in the absence of growth factors, or other soluble chemoattractants (such as VEGF). Using this assay, previous data showed that immobilized integrin inhibitors act as functional agonists and similarly promote integrin functions as cell migration [22]. On the other hand, cell migration is efficiently inhibited by the same integrin inhibitors when given in solution to the cells [22]. We demonstrated that CXCL4 or CXCL4/CTF is similarly capable of modulating endothelial cell migration. Thus, HUVEC cells under serum free-medium migrated in a haptotactic Boyden chamber assay through a CXCL4- or CXCL4/CTF-coated membrane and this migration was significantly inhibited by RGD peptides or by the blocking integrin antibodies, anti-avb3 LM609 or anti-a5b1 JBS5 (Fig. 7B). In control experiments, blocking antibody against a2b1-integrin did not affect cell migration through CXCL4- or CXCL4/CTF-coated membrane (Fig. 7B). Soluble CXCL4 or CXCL4/CTF, in turn, interferes with integrin-dependent cell migration. As shown in (Fig. 7C), soluble CXCL4 or CXCL4/CTF, but not the inactive peptide CXCL4/CTF-S, inhibited cell migration on immobilized fibro- nectin, in a concentration-dependent manner. In control exper- iments, RGD peptides or blocking antibodies against av or a5b12integrin inhibited endothelial cell motility on fibronectin (Fig. 7C). Soluble CXCL4 and CXCL4/CTF, or antibodies against av and a5b1 integrins, did not affect cell migration on collagen I, on which the cells migrated in a manner dependent on non-RGD binding b1 integrins (data not shown). Thus, immobi- lized CXCL4 or CXCL4/CTF promotes and soluble CXCL4 or PLoS ONE | www.plosone.org 5 July 2008 | Volume 3 | Issue 7 | e2657