P-Rex 1 is required for E-selectin-mediated slow leukocyte rolling and Gα_i-independent adhesion. (A) Carotid arteries of P-Rex1^−/− mice (n = 3), P-Rex1^−/− mice after injection of a blocking anti-LFA-1 antibody (n = 3) and WT mice (n = 3) were cannulated and connected to autoperfused flow chambers. Average rolling velocity of neutrophils on E-selectin (left) and E-selectin and ICAM-1 (right) is presented as means ± SEM. The wall shear stress in all flow chamber experiments was 5 to 6 dyn/cm². (B) Cumulative histogram of rolling velocities of WT (n = 4) leukocytes before (▼) and after injection of a blocking anti-LFA-1 antibody (Δ) and corresponding values for P-Rex1^−/− (n = 4) leukocytes (● and ○) in inflamed cremaster muscle venules treated with Pertussis Toxin (PTx) and a monoclonal blocking anti-P-selectin antibody. Inset data are means ± SEM. (C) Numbers of adherent cells per square millimeter in TNF-α inflamed cremaster venules after PTx pretreatment in WT (n = 4) and P-Rex1^−/− (n = 4) mice and without PTx (n = 3 each). (D) Number of transmigrated cells per 1.5 × 10⁴ μm² in inflamed cremaster venules of WT (n = 4) and P-Rex1^−/− (n = 4) mice with and without PTx (n = 3 each) as determined by near infrared reflected light oblique transillumination microscopy. (E) Representative images of inflamed cremasteric venules of WT (upper panel) and P-Rex1^−/− (lower panel) mice as visualized using reflected light oblique transillumination microscopy. (F) Cumulative histogram depicting rolling velocities of leukocytes treated with either a cell-penetrating TAT peptide fused to a WT form (n = 3) of Rap1a (○) or to an dominant negative Rap1a (n = 3) with a S17N mutation (●) in inflamed cremaster venules of LysM GFP⁺ mice after pretreatment with PTx and injection of a blocking P-selectin antibody. Inset data are means ± SEM. (G) P-Rex1-knockdown HL-60 cells were generated using short hairpin RNA and knockdown efficiency of P-Rex1 was confirmed by western blot analysis. (H) HL-60 cells were analyzed using a flow chamber adhesion assay with E-selectin and either an antibody specific for the intermediate confirmation of LFA-1 (KIM127) or an isotype control. Adherent cells per field of view were counted and means ± SEM are displayed. (I) Clustering of LFA-1 during slow rolling of leukocytes in inflamed venules of the cremaster muscle in WT (n = 3) and P-Rex1^−/− mice (n = 3). Bars are percentage of clustering cells ± SEM. (J) Representative images of WT (upper panel) and P-Rex1^−/− (lower panel) rolling leukocytes stained with anti-LFA-1 antibody. ^#P < .05.