Results 1/Immobilized CXCL4 or CXCL4/CTF induces human endothelial cell spreading, and focal adhesion kinase (FAK) phosphorylation Integrin-mediated cell attachment on cognate integrin ligands, such as ECM proteins, results in cell spreading, focal adhesion formation, and tyrosine phosphorylation of intracellular proteins [21]. When integrin inhibitors such as antibodies are immobilized on a substrate, they act as agonist and similarly activate intracellular events [22,23]. To examine whether immobilized CXCL4 would function as an integrin agonist, HUVECs were plated on coverslips that had been coated with CXCL4. As shown in Fig. 1, immobilized CXCL4 similar to natural integrin ligands fibrinogen or fibronectin, promoted endothelial cells spreading, focal adhesion and stress fibers formation. Furthermore, to determine whether the C- terminus of CXCL4 exhibited similar effects, we used a synthetic peptide encompassing amino-acid sequence 47–70 (CXCL4/CTF). Previous data showed that the peptide retains full anti-angiogenic activity of CXCL4 [5–7]. As shown in Fig. 1, CXCL4/CTF demonstrated similar effects on endothelial cell spreading, focal adhesion and stress fibers formation as full length CXCL4. Furthermore, when HUVECs are plated on a –scrambled peptide containing amino acides derived from CXCL4/CTF (CXCL4/ CTF-S) (that does not exhibit anti-angiogenic activity), or on polylysine (to which cells adhere in an integrin-independent manner), they remained round, and failed to spread and to induce focal adhesion formation (Fig. 1). Focal adhesion kinase (FAK) is a 125 kDa cytoplasmic tyrosine kinase colocalised with integrins at focal adhesion contacts and rapidly becomes phosphorylated and activated upon integrin mediated cell adhesion. As shown above, immobilized CXCL4 or CXCL4/CTF promotes cell spreading with focal adhesion formation (Fig. 1A). We tested whether these events are sufficient to activate (tyrosine phosphorylation) FAK. As shown in (Fig. 1B), attachment of HUVECs to immobilized CXCL4 or CXCL4/ CTF, similar to fibronectin, induced tyrosine phosphorylation of FAK. As expected, attached cell to polylysine, similar to cells in suspension, failed to tyrosine phosphorylate FAK. Taken together, these results suggest that immobilized CXCL4 or CXCL4/CTF serves as an adhesive ligand to endothelial cells and activates postligand binding events downstream of integrins. 2/CXCL4 or CXCL4/CTF mediates avb3-CHO cell adhesion through avb3 integrins avb3 is expressed on activated endothelial cells. In addition, inhibition of avb3 by selective antagonists blocks angiogenesis in response to growth factors in several tumor models [24], indicating that this integrin plays an essential role in tumor growth dependent on angiogenesis. Thus, for all these reasons, we first sought to test the role of avb3 integrin as a putative receptor to the antiangiogenic factor, CXCL4 and its derived peptide. For this purpose, we used a CHO cells transfected with avb3 (avb3-CHO, clone A06) and mock-transfected CHO cells. First, we verified avb3 expression by flow cytometry using a monoclonal antibody specific for the human avb3 (LM609). In accordance with our previous data [25], avb3 integrin is highly expressed in avb3- CHO cells whereas control mock-transfected CHO cells are negative for avb3 expression (data not shown). Immobilized CXCL4 or CXCL4/CTF supported avb3-CHO but not CHO adhesion, in a saturable and concentration- dependent manner. Quantitative cell attachment assays demonstrated that immobilized CXCL4 or CXCL4/CTF supported avb3-CHO cell adhesion, but not mock-transfected CXCL4 Integrins Angiogenesis Figure 1. Immobilized CXCL4 or CXCL4/CTF promotes human umbilical vein endothelial cells (HUVECs) spreading, focal adhesion and stress fiber formation. (A) Analysis of spreading, focal adhesion and stress fibers. HUVECs were plated for 4h on coverslips that had been coated with (25 mg/ml) CXCL4, (25 mg/ml) CXCL4/CTF, (100 mg/ml) fibrinogen, (25 mg/ml), CXCL4/CTF-S and (100 mg/ml) poly-D-lysine. After washing, the cells are fixed. The degree of cell spreading is seen from the phase contrast micrographs (left side) Then the cells were permeabilized and stained to visualize focal adhesion (vinculin staining, green), and actin stress fibers (Rhodamin- Phalloidin staining, red) by confocal microscopy (right side). (B) Analysis of tyrosine phosphorylation of FAK. Effect of HUVEC cell adhesion to CXCL4 or CXCL4/CTF on tyrosine phosphorylation of FAK. HUVECs were maintained in suspension for 60 min (BSA, lane 1) or allowed to attach to integrins ligand (fibrinogen, lane 4) or CXCL4 (lane 2) or CXCL4/CTF (lane 3). Cells lysates containing equal amounts of protein were immunopre- cipitated with anti-FAK antibody. One half of immunoprecipitates was subjected to immunoblotting with anti-phosphotyrosine mAbs, 4G10, and PY20, and the other half was probed with mAb anti-FAK. doi:10.1371/journal.pone.0002657.g001 PLoS ONE | www.plosone.org 2 July 2008 | Volume 3 | Issue 7 | e2657