Inhibition of GPIb-IX-mediated integrin activation by a dominant-negative mutant of p38. CHO cells expressing GPIb-IX and integrin α_IIbβ₃ (123 cells) were transfected with pCDNA3 vector (123/vector) or Flag epitope-tagged dominant-negative mutant of p38 (123/p38AF). (A,B) 123/vector and 3 different clones of 123/p38AF cells were incubated with Oregon green-labeled fibrinogen in the presence of ristocetin (1 mg/mL) and VWF (20 μg/mL). As a negative control, these cells were also incubated with Oregon green-labeled fibrinogen in the presence of ristocetin alone. Binding of fibrinogen was detected using flow cytometry. Nonspecific binding of fibrinogen was determined by measuring fibrinogen binding in the presence of 1 mM RGDS peptide. Quantitative results from 4 experiments are expressed as mean ± SEM of fibrinogen binding index (total bound fluorescence [geomean]/nonspecifically bound fluorescence [geomean]) as shown in panel A; P < .001 compared with 123 cells. Expression of p38AF as detected by immunoblotting with an anti-Flag M₂ monoclonal antibody is shown in the inset in panel A. Data from a representative experiment are shown in panel B. (C) 123/vector cells and 3 different clones of 123/p38AF cells were analyzed by flow cytometry for expression levels of GPIb-IX using an anti-GPIbα monoclonal antibody, SZ2, and for expression levels of α_IIbβ₃ using a monoclonal antibody against α_IIbβ₃ complex, D57. Note that the expression levels of GPIb-IX and α_IIbβ₃ between these cell lines are comparable.