Binding of VWF O-glycosylation variants to recombinant GPIbα and platelets in the presence of ristocetin. (A) Binding of wt and OLG VWF variants to immobilized GPIbα in presence of increasing ristocetin concentrations. A GPIbα-coated plate was incubated with 4nM wtVWF or VWF variants in the presence of increasing ristocetin concentrations (0-1 mg/mL), and bound VWF was detected with polyclonal anti–VWF-HRP antibodies. Results were analyzed by ANOVA followed by Dunnett posttest. At 1 mg/mL, no significant difference in binding to GPIbα between wtVWF and OLG variants was observed. At all concentrations of ristocetin between 0.5 and 0.9 mg/mL, bindings were significantly different for wtWF and the T1255A, Clus1, and DC variants, with P values less than .03. (B) Ristocetin cofactor activity (VWF:RCo) of wt and OLG VWF variants measured by platelet agglutination with 1 mg/mL ristocetin (P = .02). Results are mean ± SEM of minimum 3 independent experiments performed in duplicate. (C) Schematic illustration of VWF monomer with positions of O-linked glycosylation sites indicated, and mutants generated.