vWf interaction with the G233V/M239V cell line in the absence of ristocetin.

(A) Wild-type, G233V, M239V, V234G, and G233V/M239V cells were incubated with 0 or 4 μg/mL vWf in the absence of ristocetin. Bound vWf was detected using an FITC-conjugated anti–human vWf antibody, followed by flow cytometric analysis. (B) CHO cells (wild type, G233V, M239V, and G233V/M239V) were stirred for 5 minutes in the presence of 10 μg/mL human vWf without ristocetin. G233V/M239V was also pretreated for 10 minutes with 5 μg/mL AK2 before the addition of vWf (G233V/M239V + AK2). Cells were fixed and mounted onto glass microscope slides, and differential interference contrast images were captured as described in “Materials and methods.” (C) Aggregation traces for wild-type cells incubated with 10 μg/mL vWf in the presence (+/+) and absence (+/−) of 1 mg/mL ristocetin are presented. Traces for the G233V/M239V mutant incubated with vWf in the absence of ristocetin (+/−) and after pretreatment with AK2 (+/− and AK2) are also presented. Aggregation traces are representative of 3 experiments.