CXCL4 Integrins Angiogenesis Figure 5. CXCL4-, or CXCL4/CTF-integrin mediates HUVECs adhesion through endogen avb3, avb5 and a5b1 integrins. A. Expression profile of integrins in HUVECs cells analyzed by flow cytometry. HUVECs were incubated with primary antibodies specific for avb3 (LM609), or avb5 (P1F6), or a5b1 (HA5) and antibody binding was detected with FITC-labeled secondary antibody as described under Materials and Methods. Cells stained with secondary antibody (2u Ab) only were used as a negative control. B. Dose dependence of HUVECs cell adhesion to CXCL4 or CXCL4/CTF. HUVECs were plated onto microtiter wells coated with the indicated concentrations of CXCL4 or CXCL4/CTF and cell attachment was analyzed as described in Material and Methods C. Cell adhesion to CXCL4 and CXCL4/CTF is avb3, avb5 and a5b1 integrins dependent. Cells were incubated with the indicated antibodies or RGD-, RGE peptides before plating to wells coated with CXCL4 or CXCL4/CTF. Anti-integrin antibodies used were: anti- avb3 (LM609), anti-av (AV1), anti-b3 (B3A), anti-avb5 (P1F6), anti-a5b1 (JBS5), anti-a5 (P1D6) and anti-b1 (CD29). Cell attachment was analyzed as above. Error bars represent the mean6SD, *, P#0.001; **, P#0,0001; #, P#0,00001 compared to CXCL4 or CXCL4/CTF in the presence of IgM control antibody; n = 4 independent experiments. doi:10.1371/journal.pone.0002657.g005 PLoS ONE | www.plosone.org 7 July 2008 | Volume 3 | Issue 7 | e2657