Fig. 3.
Fig. 3. Binding of 125I-vWF to WT or M239V in the presence of ristocetin. Using the same method as described in the legend to Fig 2, WT and M239V were assayed for binding to 125I-vWF (0.15 μg/mL) in the presence of ristocetin. (A) The inhibitory effect on the binding was evaluated by MoAbs to assess the specificity of the binding. NMC-4 (▨), an anti-vWF MoAb (generous gift of Dr A. Yoshioka); GUR83/35 (), an anti-GPIbα MoAb; and an indifferent antibody (▪) were used. (Left panel) At a low concentration of ristocetin (0.2 mg/mL), WT did not specifically bind to 125I-vWF (P < .05), whereas M239V did (P < .05). (Right panel) At a high concentration of ristocetin (1.0 mg/mL), WT bound to 125I-vWF and M239V binding to vWF was enhanced. Anti-vWF MoAb NMC-4 and anti-GPIbα MoAb GUR83/35 inhibited 125I-vWF binding in all of the conditions shown. The statistical analyses were perfomed using one-way layout ANOVA, which showed that, at 0.2 mg/mL ristocetin, there were no statistically significant difference between the three conditions (control antibody, NMC-4, and GUR83/35) in 125I-vWF binding to NT or WT (P < .05), but there was a difference between the three conditions in the binding to M239V (P < .05). On the other hand, at 1.0 mg/mL ristocetin, there were significant differences between the three conditions in WT and M239V (P < .05), but not in NT (P < .05). (B) 125I-vWF binding was measured in the presence of various concentrations of ristocetin. Nontransfected (NT) medium was used as a control. At low ristocetin concentrations (0.2 to 0.6 mg/mL), the difference between WT and M239V in 125I-vWF binding was marked, whereas the difference was minimized at ristocetin concentrations greater than 1.0 mg/mL. Two-way factorial ANOVA showed significant interaction between the effect of ristocetin concentration and the two recombinant GPIbα fragments (WT or M239V), ie, the two dose-response curves (WT and M239V) were statistically different (P < .05). The results shown are the mean (±SD) of triplicate determinations from one experiment that is representative of four.

Binding of 125I-vWF to WT or M239V in the presence of ristocetin. Using the same method as described in the legend to Fig 2, WT and M239V were assayed for binding to 125I-vWF (0.15 μg/mL) in the presence of ristocetin. (A) The inhibitory effect on the binding was evaluated by MoAbs to assess the specificity of the binding. NMC-4 (▨), an anti-vWF MoAb (generous gift of Dr A. Yoshioka); GUR83/35 (), an anti-GPIbα MoAb; and an indifferent antibody (▪) were used. (Left panel) At a low concentration of ristocetin (0.2 mg/mL), WT did not specifically bind to 125I-vWF (P < .05), whereas M239V did (P < .05). (Right panel) At a high concentration of ristocetin (1.0 mg/mL), WT bound to 125I-vWF and M239V binding to vWF was enhanced. Anti-vWF MoAb NMC-4 and anti-GPIbα MoAb GUR83/35 inhibited 125I-vWF binding in all of the conditions shown. The statistical analyses were perfomed using one-way layout ANOVA, which showed that, at 0.2 mg/mL ristocetin, there were no statistically significant difference between the three conditions (control antibody, NMC-4, and GUR83/35) in 125I-vWF binding to NT or WT (P < .05), but there was a difference between the three conditions in the binding to M239V (P < .05). On the other hand, at 1.0 mg/mL ristocetin, there were significant differences between the three conditions in WT and M239V (P < .05), but not in NT (P < .05). (B) 125I-vWF binding was measured in the presence of various concentrations of ristocetin. Nontransfected (NT) medium was used as a control. At low ristocetin concentrations (0.2 to 0.6 mg/mL), the difference between WT and M239V in 125I-vWF binding was marked, whereas the difference was minimized at ristocetin concentrations greater than 1.0 mg/mL. Two-way factorial ANOVA showed significant interaction between the effect of ristocetin concentration and the two recombinant GPIbα fragments (WT or M239V), ie, the two dose-response curves (WT and M239V) were statistically different (P < .05). The results shown are the mean (±SD) of triplicate determinations from one experiment that is representative of four.

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