Involvement of serines in the 580-590 domain for 14-3-3ζ binding and integrin activation. (A) GPIb-IX-14-3-3ζ association was studied as described in Figure 1 in CHO GPIb-IX cells where Ser587 and Ser590 were individually substituted to Ala (αA587, αA590) or in cells containing a double mutation (αA587/590). A profound decrease in GPIb-IX-14-3-3ζ association was observed in both cells with a Ser590 to Ala substitution and a milder defect was observed for the single Ala587 mutant. The results are representative of 3 separate experiments. (B-C) GPIb-IX CHO cells expressing wild-type GPIbα (αβIX) or GPIbα with deletions of the 580-590 or 605-610 sequence were allowed to adhere to a VWF matrix in the presence of botrocetin (5 μg/mL) and left to spread for 30 minutes at 37°C. The cells were then fixed in 4% PFA and the actin cytoskeleton was labeled with TRITC-phalloidin. (B) Compared with αβIX cells, αΔ605-610βIX and αΔ580-590βIX cells adhered equally as efficiently but displayed a marked spreading defect. (C) Spreading cells were scored in 5 random fields and expressed as a percentage of total adherent cells. (^***P < .001; Fisher exact test.) Results are the mean (± SEM) of 4 separate experiments. (D) Cells containing Ser to Ala substitutions as described in panel A were allowed to adhere and spread as described in panel B. Spreading cells were scored in 5 random fields and expressed as a percentage of total adherent cells. The level of spreading at 30 minutes in control αβIX cells was lower than in experiments presented in panel B due to a new batch of botrocetin with a lower activity. (^***P < .001; ^*P < .05 Fisher exact test.) Results are the mean (± SEM) of 2 to 3 separate experiments.