The effects of disruption of 14-3-3ζ binding to GPIbα on cell adhesion to VWF and on PKA inhibitor-induced GPIb-IX activation. (A) 1b9, S166A, S166/609A, or S609A cell lines were perfused through VWF-coated capillaries at 150 s^-1. Transient adhesion (rolling) of these cells was recorded. The number of rolling cells was counted in 30 randomly selected fields of 0.25 mm² and at randomly selected time points. The results shown are the mean ± SD of cell number/mm². (B,C) S166A cells (B) or S166A/S609A cells (C) were preincubated with MPαC; control peptides MαC, MPαCsc, MαCsc; or DMSO, and then perfused into VWF-coated glass capillaries. Numbers of adherent cells were counted at 10 randomly selected time frames and locations (mean ± SD). (D) CHO cells expressing wild-type GPIb-IX (1b9) and mutant GPIb-IX with deletion of the GPIbα C-terminal 14-3-3 binding site (Δ591 and Δ605) were preincubated without or with 100 μM PKI for 15 minutes at 22°C. The cells were then incubated with VWF and ristocetin at 22°C for 30 minutes. VWF binding was detected using the FITC-labeled anti-VWF antibody, SZ29, and flow cytometry. Nonspecific fluorescence was determined by incubating the cells with ristocetin alone. Effects of PKI on enhancing VWF binding in the indicated cell lines were quantified and expressed as the percentage increase in the fluorescence intensity of VWF binding compared with the fluorescence intensity of VWF binding in the absence of PKI. Shown in the figure are the results from 3 separate experiments (mean ± SD).