Rap1 activation does not promote SS RBC adhesion to laminin via the α4β1 integrin. (A) SS RBC adhesion to laminin stimulated via the Epac/Rap1 pathway is not RGD-dependent. SS RBCs in perfusion media were treated with 100 μM 8CPT-2-Me for 20 minutes or with 1 mM of either RGDW or RGEW peptide for 30 minutes before the 20-minute 8CPT-2-Me treatment. While still in the presence of the indicated reagents, the cells were then flowed over chambers coated with 0.75 μg laminin in a flow adhesion assay. Results are expressed as mean ± SE from 4 separate experiments. (B) SS RBC adhesion to laminin is not dependent on the LDV sequence. SS RBCs were untreated or pretreated with 1 mM EILDV peptide or 1 mM EILEVPST peptide for 30 minutes. The SS RBCs and the SS RBC/peptide mixture were then treated with 100 μM 8CPT-2-Me for 20 minutes and flowed over 0.75 μg laminin in a flow adhesion assay. Results are expressed as mean ± SE from 3 separate experiments. (C) The α4β1 integrin does not mediate SS RBC adhesion to laminin. SS RBCs in perfusion media either were not pretreated or were preincubated with either 1 μg/mL α4 and β1 integrin subunit-blocking antibodies or an equivalent concentration of IgG control antibody for 30 minutes and then 100 μM 8CPT-2-Me was added to the RBC/antibody mixture for 20 minutes. The SS RBCs, still in the presence of antibody and 8CPT-2-Me, were flowed across chambers coated with 0.75 μg immobilized laminin. Results are expressed as mean ± SE from 5 separate experiments. (D) Rap1 activation via Epac does not promote adhesion to the α4β1 selective substrate VCAM-1. Cells were treated with either 100 μM 8CPT-2-Me or 100 μM 4N1K peptide for 20 minutes. The SS RBCs, still in the presence of the indicated pharmacologic agents, were then flowed over chambers coated with 3 μg of immobilized VCAM in a flow adhesion assay. Results shown are combined data ± SE from 2 separate experiments.