Effect of vitronectin and plasminogen activator inhibitor-1 heteromers on activation of neutrophil b2 integrins. (A) Using multi-channel flow cytometry, expression of the b2 integrins LFA-1/CD11a and Mac-1/CD11b was analyzed on the surface of neutrophils isolated from the peripheral blood of wild-type (WT) mice undergoing exposure to vitronectin (VN), plasminogen activator inhibitor-1 (PAI-1), uPA, VN-PAI-1, or VN-uPA, panels show quantitative results (mean±standard error of the mean [SEM] for n=4 per group; #P<0.05 vs. unstimulated). (B) Binding of ICAM-1/CD54-Fc to neutrophils isolated from the peripheral blood of WT mice was analyzed upon exposure to VN, PAI-1, uPA, VN-PAI-1, or VN-uPA, panels show quantitative results (mean±SEM for n=6 per group; #P<0.05 vs. unstimulated). VN-PAI-1-elicited binding of ICAM-1/CD54 to neutrophils isolated from the peripheral blood of WT mice was analyzed after application of receptor associated protein (RAP; blocking receptors of the LDL receptor family), blocking anti-LRP-1 antibodies, or different MAPK inhibitors (mean±SEM for n=4-6 per group; #P<0.05 vs. unstimulated). Binding of conformation- specific antibodies ‚KIM127‘ (intermediate and high affinity conformations of b2 integrins) or ‚mAB 24‘ (high affinity conformation of b2 integrins) to human neutrophils was analyzed after application of human VN-PAI-1 (mean±SEM for n=3 per group). MFI: mean fluorescent intensity.