Mac-1 affinity regulation via Coro1A impacts PMN adhesion strengthening and spreading. (A) Quantitative analysis of adherent Coro1A^+/+ and Coro1A^−/− PMNs under flow conditions (1 dyne/cm²) on immobilized fibrinogen in percentage of adherent cells prior to onset of shear stress (100%) at indicated time points (n = 4). #P < .05; ##P < .001; *P < .05 versus initially adherent cells. Mean ± standard deviation. (B) Adhesion strengthening of Coro1A^+/+ and Coro1A^−/− PMNs on immobilized fibrinogen in the presence of a function blocking anti-Mac-1 antibody (clone M1/70, CD11b) or an isotype control antibody (IgG) under flow conditions (1 dyne/cm²). Relative adhesion of Coro1A^+/+ and Coro1A^−/− PMNs in percentage of all interacting PMNs prior to onset of shear stress (pre flow, 100%) (n = 3). *P < .05. Mean ± standard deviation. (C) Flow cytometric analysis of soluble fibrinogen binding to Coro1A^+/+ and Coro1A^−/− PMNs stimulated for 20 minutes with 100 ng/mL of rmCXCL1, 10 μM of fMLP, 5 mM of Mn²⁺, or left unstimulated (minus sign). Percentage of PMN positive for fibrinogen binding was calculated by defining a threshold of fluorescence intensity at which 95% of PMN in the EDTA control were considered negative. Fluorescence histograms (left) and quantitative analysis showing percentage (middle) and median fluorescence intensity (right) of cells with positive fibrinogen binding (n = 6, rmCXCL1, fMLP; n = 3, Mn²⁺). #P < .05; *P < .05 versus unstimulated control. Mean ± standard deviation. (D-E) The fMLP-induced spreading and polarization of Coro1A^+/+ and Coro1A^−/− PMNs upon exposure to immobilized fibrinogen or rmICAM-1 under static conditions (pre flow) or after application of flow (post flow, 1 dyne/cm²) at indicated time points as calculated by measuring the cell area (D) and the aspect ratio (E) (n = 4, fibrinogen, ≥68 Coro1A^+/+ and ≥55 Coro1A^−/− PMNs; n = 3, rmICAM-1, ≥53 Coro1A^+/+ and ≥50 Coro1A^−/− PMNs). Mean ± standard deviation. *P < .05.