CHO cells, in a saturable and concentration-dependent manner (Fig. 2A). Also, as shown in Fig 2B, adhesion to CXCL4 or CXCL4/ CTF was significantly inhibited by monoclonal antibodies LM609, AV1 and B3A against avb3, av and b3, respectively. These results demonstrate that avb3-CHO adhesion on immobilized CXCL4 or CXCL4/CTF is mediated by avb3 integrin. Consistent with these results, addition of 10 mM of EDTA, a strong inhibitor of divalent cation-dependent cell integrin receptors, blocks the adhesion of avb3- CHO cells to CXCL4 or CXCL4/CTF (Fig. 2B). Furthermore, the incubation of cells with two well established cation activators integrins, Mn2+ and Mg2+ [26], prior to adhesion of cells to immobilized CXCL4 or CXCL4/CTF enhances the cell attachment about 25% (Fig. 2C). These findings indicate that immobilized CXCL4 or CXCL4/CTF mediates avb3-CHO cell adhesion in a divalent cation-dependent manner. As with EDTA, addition of RGD-peptides blocks the adhesion of avb3-CHO to CXCL4 or CXCL4/CTF, whereas control RGE peptide had no affect on adhesion (Fig. 2B). These findings suggest that the interaction of avb3-CHO cells with CXCL4 or -CXCL4/CTF is RGD- dependent. This is consistent with the idea that avb3 integrin is a CXCL4 receptor in avb3-CHO cells, since avb3 is RGD dependent. Taken together, these results demonstrate that avb3-CHO cells attachment on immobilized CXCL4 or CXCL4/CTF is a cation- dependent process and is avb3 integrin mediated. CXCL4 and CXCL4/CTF bind to purified integrins. To reinforce the results described above, we studied the direct interaction of CXCL4, or CXCL4/CTF, to integrin in a solid-phase ligand binding assay [27] using a purified human integrin avb3 (protein). As shown in Fig. 3A, soluble avb3 demonstrates a concentration- dependent and saturable binding to immobilized CXCL4 or its peptide CXCL4/CTF. The specificity of the interaction was confirmed by inhibition of CXCL4 or CXCL4/CTF binding to avb3 integrin with the inhibitory av (AV1) and b3 (B3A) antibodies, as well with RGD peptides (Fig. 3B). Thus, these results demonstrate a specific and direct interaction between the angiogenic inhibitor CXCL4 or its peptide CXCL4/CTF with the avb3 integrin. Immobilized CXCL4 or CXCL4/CTF promotes avb3- CHO cell spreading. Confocal analysis showed that immobilized CXCL4 or CXCL4/CTF promoted avb3-CHO cell spreading, focal adhesion and stress fibers formation (Fig. 4). These events were blocked in presence of EDTA or RGD peptides or with antibodies against avb3. In contrast, mock-transfected CHO cells did not appreciably spread and generate stress fibers of actin on CXCL4 or CXCL4/CTF. In addition, when avb3-CHO cells are plated on a scrambled peptide derived (CXCL4CTF-S), or on polylysine, they remained round, and failed to spread and to induce focal adhesion formation (Fig. 4). These results indicate that immobilized CXCL4 or CXCL4/CTF induces avb3-CHO cell spreading through avb3 integrins. Collectively, these results show that CXCL4 or CXCL4/CTF interacts with avb3 integrins and activates postligand binding event downstream to integrins, such as cell spreading. 3/Human endothelial cells (HUVECS) adhere on immobilized CXCL4-, or CXCL4/CTF- through avb3, and additional integrins, avb5 and a5b1 To investigate the interaction of CXCL4 or CXCL4/CTF with endogenous integrins, we tested the interaction of CXCL4 or CXCL4/CTF with integrins expressed on the surface of cultured HUVECs. We first screened the expression of endogenous integrins using a flow cytometry analysis. Accordingly with previous data [28], we found that avb3, avb5 and a5b1 were constitutively expressed on HUVECs cells using anti-human integrin antibodies specific for human integrin antibodies specific for avb3 (LM609), CXCL4 Integrins Angiogenesis avb5 (P1F6), and a5b1 (HA5) (Fig. 5A). We next studied whether avb3, avb5 and a5b1 integrins expressed on HUVECs could bind to CXCL4 or CXCL4/CTF. As shown in Fig. 5B, immobilized CXCL4 or CXCL4/CTF supported HUVECs cell adhesion in a saturable and concentration-dependent manner. Adhesion to CXCL4 or CXCL4/CTF was blocked in presence of RGD peptides or with antibodies against avb3, thus confirming as above, that human endothelial cells adhere to immobilized CXCL4 or CXCL4/CTF through avb3 integrins in an RGD dependent manner (Fig. 5C). Interestingly, blocking antibodies against avb5 (P1F6) and a5b1 (JBS5), as with avb3, significantly reduced the adhesion of HUVECs cells on immobilized CXCL4 or CXCL4/ CTF (Fig. 5C). In contrast, blocking antibody against a2b1-integrin did not affect cell adhesion on immobilized CXCL4 or CXCL4/ CTF (data not shown). Thus, these results demonstrate that endothelial cells adhere to immobilized CXCL4 or CXCL4/CTF through avb3, and other integrins, avb5 and a5b1, which play a crucial role in angiogenesis. 4/Integrin agonists, Mn2+ and PMA, enhance cell spreading, with focal adhesion and stress fibers formation, on immobilized CXCL4 or CXCL4/CTF To further examine the postligand events downstream to integrins induced by immobilized CXCL4 or CXCL4/CTF, we examined in more detail cell spreading. As shown above, confocal analysis shows that immobilized CXCL4 or CXCL4/CTF promoted cell spreading, focal adhesion and stress fibers formation (Fig. 1,4). When the number of cells spread on different immobilized substrates was quantified, we found that immobilized CXCL4 or CXCL4/CTF promoted cell spreading by about 50 to 60% in the absence of integrin agonists. When we analyzed the spreaded cells by confocal microscopic we found that around 40% of these cells spread on CXCL4 or CXCL4/CTF (versus 60% with fibronectin, data not shown) presented focal adhesions with stress fiber formation (Fig. 6). These events were significantly increased (from 40% to 60%) when we added two-well known integrin activators, extracellular (Mn2+) and intracellular (Phorbol 12-Mryristate 13 Acetate (PMA) agonists (Fig. 6). These agonists activate avb3 integrin (convert avb3 to a high-affinity conformation) and stimulate avb3 functions [29], such spreading cells, as well as that of other integrins [30,31]. Incubation of cells with 250 mM of Mn2+ or 100 nM of PMA produced an increase in both the number of attached cells and in cell spreading of about 25% and 30% respectively (Fig. 6). This enhancement was completely blocked by adding EDTA or RGD (data not shown). This finding suggests that the interaction of CXCL4 or CXCL4/CTF to integrins, similar to natural integrin ligands, is subject to integrin function regulation such as affinity modulation (see Discussion). 5/The ability of CXCL4 to bind to integrin is related to its anti-angiogenic action The scrambled control peptide CXCL4/CTF-S fails to inhibit endothelial cell proliferation [32]. We thus tested whether this inactive peptide could bind to integrins. We found that, in contrast with CXCL4/CTF and similar to polylysine, avb3-CHO or HUVECs plated on immobilized CXCL4/CTF-S peptide did not adhere significantly. Cells remained round and failed to assemble focal adhesion and to induce stress fibers (Fig. 1,4). The addition of the two integrin activators, Mn2+ and PMA, had no effect on the number of attached cells or on cell spreading on PF-4/CTF-S as shown by confocal analysis (Fig. 6C). These results suggest that the ability of CXCL4 and CXCL4/CTF to bind integrins is correlated with their anti-angiogenic activity. PLoS ONE | www.plosone.org 3 July 2008 | Volume 3 | Issue 7 | e2657