American Society of Hematology

Figure 2.

$Figure 2. Healthy and Del2201 BDD-rFVIII interactions with VWF and phospholipids. (A) CHO cells transfected with expression vectors for healthy and Del2201 BDD-rFVIII were incubated for 16 hours in the presence of various concentrations of VWF. FVIII activity in the cell culture supernatant was determined using a chromogenic assay (mean ± SD). (B) CHO cells transfected with expression vectors for healthy and Del2201 BDD-rFVIII were incubated for 16 hours in the presence of various concentrations of VWF as in panel A. FVIII:Ag in the cell culture supernatant was determined by ELISA (mean ± SD). (C) BDD-rFVIII binding to VWF. Various concentrations of VWF bound to Sepharose beads were incubated with healthy, Del2201, and Ser2119Tyr BDD-rFVIII (0.5 IU/mL). Following centrifugation, FVIII was measured in the supernatant of VWF Sepharose (free FVIII) and of control Sepharose (total FVIII) by using a FVIII chromogenic assay. FVIII bound to VWF was calculated by subtracting free FVIII from total FVIII. The fraction of FVIII bound to VWF is expressed as the percentage of total FVIII (mean ± SD). (D) Activity of BDDr-FVIII was evaluated in a chromogenic assay by using various concentrations of synthetic phospholipid vesicle, representative of PLs expressed at the surface of activated platelets. At each PL concentration, FXa generation was compared with that observed in the presence of a known concentration of healthy plasma FVIII. For each FVIII variant, results are expressed as a percentage (mean ± SD) of the FVIII activity measured at the highest PL concentration tested (4 μM).$

Healthy and Del2201 BDD-rFVIII interactions with VWF and phospholipids. (A) CHO cells transfected with expression vectors for healthy and Del2201 BDD-rFVIII were incubated for 16 hours in the presence of various concentrations of VWF. FVIII activity in the cell culture supernatant was determined using a chromogenic assay (mean ± SD). (B) CHO cells transfected with expression vectors for healthy and Del2201 BDD-rFVIII were incubated for 16 hours in the presence of various concentrations of VWF as in panel A. FVIII:Ag in the cell culture supernatant was determined by ELISA (mean ± SD). (C) BDD-rFVIII binding to VWF. Various concentrations of VWF bound to Sepharose beads were incubated with healthy, Del2201, and Ser2119Tyr BDD-rFVIII (0.5 IU/mL). Following centrifugation, FVIII was measured in the supernatant of VWF Sepharose (free FVIII) and of control Sepharose (total FVIII) by using a FVIII chromogenic assay. FVIII bound to VWF was calculated by subtracting free FVIII from total FVIII. The fraction of FVIII bound to VWF is expressed as the percentage of total FVIII (mean ± SD). (D) Activity of BDDr-FVIII was evaluated in a chromogenic assay by using various concentrations of synthetic phospholipid vesicle, representative of PLs expressed at the surface of activated platelets. At each PL concentration, FXa generation was compared with that observed in the presence of a known concentration of healthy plasma FVIII. For each FVIII variant, results are expressed as a percentage (mean ± SD) of the FVIII activity measured at the highest PL concentration tested (4 μM).

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