Fig. 3. EphB1/Fc stimulates _vß₃ and ₅ß₁ integrin-mediated endothelial cell attachment. (A) EphB1/Fc stimulates endothelial cell attachment to fibrinogen. 48-well plates were coated with fibrinogen (1 µg/cm²), and endothelial cell attachment was assayed as described in the Materials and Methods. EphB1/Fc stimulated endothelial cell attachment at an optimal concentration of 2 µg/ml and 4 µg/ml for HRMEC and HMEC-1, respectively. An Fc fusion protein control, IgG, is inactive at these concentrations. (B) Upper panel, cell surface expression of integrin proteins in HRMEC and HMEC-1. Eph-B1/Fc does not increase the surface expression of endothelial _vß₃ in HRMEC cells (left panel). _vß₃ is expressed at a low level in HMEC-1 cells, whereas _vß₅ and ₅ß₁ are expressed at much higher levels, as assayed by FACS analysis (right panel). Lower panel, EphB1/Fc-induced endothelial cell attachment is mediated through _vß₃ and ₅ß₁ integrins. Assays of endothelial cell attachment to fibrinogencoated 48-well plates were performed as described in the Materials and Methods. Where indicated, cells were pre-incubated for 15 minutes at 22°C with blocking peptides (100 µg/ml) or anti-integrin antibodies (5 µg/ml) before plating. The data represent means±s.e.m. of three independent experiments. Group comparisons were performed using the Student's t-test. ^*P<0.05 versus IgG control; ^**P<0.05 versus RGE control; ^***P<0.05 versus no inhibitor control, RGE control or avß5 treated; ^****P<0.05 versus no inhibitor control, RGE control or _vß₅ treated.