monoclonal antibody (10 mg/ml) specific for avb3 (LM609), or avb5 (P1F6), or a5b1 (HA5). After washing, the cells were incubated for 20 min on ice with FITC-conjugated goat anti- mouse IgG, washed again, and analyzed on a FACSCalibur flow cytometer (Becton Dickinson Biosciences). As a negative control, samples were incubated with the secondary antibody alone. Cell Attachment Assay Cell attachment was assayed as described [52]. Microtiter wells (Immulon 2, ThermoLabsystems) were coated for 2 h at 37uC with the indicated concentrations of proteins. The wells were blocked for 1 h with 1% BSA in PBS. Cells were briefly trypsinized followed by washes with serum-containing medium and serum-free medium containing 0,5% BSA. Cells were suspended in 3,56105 cells/ml in serum-free medium containing 0,5% BSA and incubated in the presence or absence of 10 mM EDTA, 25 mg/ml RGD, RGE peptides or 10 mg/ml of anti- integrin antibodies for 15 min at room temperature. To inhibit adhesion on various ECMs proteins with soluble CXCL4 or CXCL4/CTF, CXCL4 or CXCL4/CTF were incubated with cells 30 min in suspension prior plating onto wells, as indicated in the figures. 100 ml of cells suspensions were added to the coated wells, and the plates were incubated at 37uC for 60 min. Non attached cells were removed by washing with PBS and then fixed and stained in 30% methanol, 10% acetic acid, containing 0,1% Coomassie blue. After extensive washing, the cells were lysed in 1% SDS, after which the amount of lysed cells were quantified by measuring their absorbance at 620 nm. Background absorbance observed in the wells coated with BSA was deducted from the values obtained. Each time point represented three independent experiments performed in triplicate. Solid-Phase Ligand-Binding Assay Solid-phase ligand binding assay was performed as described previously with minor modifications [27]. Immulon 2 microtiter wells were coated with 25 mg/ml CXCL4 or CXCL4/CTF in PBS overnight at 4uC. The wells were blocked with 1% BSA in walsh buffer at room temperature for 1 h. Octylglucoside avb3 integrin was added on coated wells and incubated for 2 h at 37uC. Unbound integrin molecules were extensively washed with walsh buffer containing 0,05% Tween 20. The bound integrin molecules were incubated with 1/1000 dilution of a polyclonal anti-av cytoplasmic domain antibody (Chemicon) for 1 h at room temperature. After extensive washes with walsh buffer-0,05% Tween 20, the bound antibodies were detected by using Peroxidase-Conjugated goat anti-rabbit IgG (Dako). TMB liquid substrate (sigma) was added to the wells, the reactions were stopped with 0,5 M H2SO4, and absorbance was measured at 450 nm. Background absorbance in the wells coated with BSA was deducted from the values obtained. Cell Spreading and Confocal Microscopy Analysis Cells were washed twice with serum-free medium, suspended to the serum-free medium, and plated on coverslips that had been coated with (25 mg/ml) CXCL4, (25 mg/ml) CXCL4/CTF, (25 mg/ml) CXCL4/CTF-S, (100 mg/ml) fibrinogen and (100 mg/ml) poly-D-lysine and incubated at 37uC for 4 h. After removing nonadherent cells, the adherent cells were fixed in 4% parafolmaldehyde, permeabilized with 0,2% Triton X-100 in PBS, and stained with monoclonal anti-vinculin antibody, FITC-anti- mouse IgG and rhodamine-phalloidin [53]. Cells were analyzed by confocal laser scanning microscopy and photomicrographs were prepared using Adobe Photoshop 8. CXCL4 Integrins Angiogenesis Analysis of Tyrosine Phosphorylation of FAK Tyrosine Phosphorylation of FAK was assayed as described [54]. HUVECs were plated for 60 min on dishes that had been coated with 5 mg/ml of BSA or (100 mg/ml) fibrinogen or (25 mg/ml) CXCL4 or (25 mg/ml) CXCL4/CTF or (100 mg/ml) poly-D-lysine. After 60 min, non-adherent cells from BSA-coated plates were diluted 1:1 with PBS, sedimented at 100 g for 5 min and washed once with PBS before lysis in complete RIPA buffer (1% Triton X- 100, 1% sodium deoxycholate, 0.1% SDS, 158 mM NaCl, 10 mM Tris, pH 7.4, 1 mM Na2EGTA, 1 mM sodium vanadate, 0.5 mM leupeptin, 0.25 mg/ml pefabloc, 5 mg/ml aprotinin). The adherent cells from plates coated with fibrinogen or CXCL4 or CXCL4/CTF were rinsed twice with PBS, lysed on the plates with ice-cold complete RIPA buffer and scrapped into microcentrifuge tubes. Lysates were incubated for 30 min on ice and clarified supernatants for immunoprecipitation. Equal amounts of protein from each lysate (200 mg of protein) were immunoprecipitated with rabbit antiserum specific for FAK (Santa Cruz). One half of immunoprecipitates were subjected to western blotting with anti-phosphotyrosine mAbs, 4G10 (BD Transduction Laboratories) and PY20 (Upstate Biotechnology), and the other half was probed with mAb anti-FAK (BD Transduction Laboratories). Immunoreactive bands were detected by enhanced cheminuluminescence detection. Endothelial cell Migration Haptotactic cell motility was measured by using a modified Boyden chamber (BD, Falcon) as previously described [22]. The undersurface of the membrane filter was coated with 25 mg/ml of CXCL4 or 25 mg/ml CXCL4/CTF or various matrix proteins. After washing the membranes with PBS, nonspecific adhesion sites were saturated with 1% BSA at 22uC for 1 h. HUVECs (36104) were added to the upper chambers in serum-free medium and were allowed to migrate to the underside of the chamber for 4 h at 37uC. Cell migration was measured after 4 h of incubation at 37uC. All non migrated cells were removed from the upper face of the membrane filter with the cotton swab and cells migrated to the lower face were fixed and stained in 30% methanol, 10% acetic acid, containing 0,1% Coomassie blue. Subsequently, the number of stained migrated cells (to the lower face of the membrane filter) was counted. Migration results are expressed in terms of the average number of cells/high-magnification microscopic field. To inhibit migration on CXCL4 or CXCL4/CTF, cells with pretreated with 10 mg/ml of anti-integrin antibodies for 30 min in suspension prior to plated in chambers. To inhibit migration on various matrix proteins with soluble CXCL4 or CXCL4/CTF, CXCL4 or CXCL4/CTF were incubated with cells 30 min in suspension prior adding to the upper chambers, as indicated in the figures. In chemotactic cell motility experiments, VEGF (5 ng/ml) was added in the lower chambers. To inhibit migration on fibronectin to VEGF with soluble CXCL4 or CXCL4/CTF, CXCL4 or CXCL4/CTF were incubated with cells 30 min in suspension prior adding to the upper chambers. In all experi- ments, nonspecific migration was determined using BSA as a ligand and was subtracted from the values of relative cell migration obtained on different matrix proteins. Statistical Analysis Student’s test was used to determine the significance of differences. Supporting Information Figure S1 Soluble CXCL4 or CXCL4/CTF inhibits cell adhesion to fibronectin and vitronectin. HUVECs and avb3- PLoS ONE | www.plosone.org 12 July 2008 | Volume 3 | Issue 7 | e2657