Binding to vWf and specific activity of normal and mutated rfVIII
| fVII variant . | KD (nM)* . | Binding to vWf in plasma (%)† . | fVIII specific activity‡ . |
|---|---|---|---|
| Normal | 1.0 ± 0.3 | 79 ± 1 | 0.6 ± 0.1 |
| Ser2119Tyr | 83.3 ± 28.4 | 5 ± 2 | 0.9 ± 0.2 |
| Ile2098Ser | 7.8 ± 3.2 | 43 ± 6 | 1.1 ± 0.5 |
| Arg2150His | 3.3 ± 1.2 | 56 ± 3 | 0.8 ± 0.4 |
| fVII variant . | KD (nM)* . | Binding to vWf in plasma (%)† . | fVIII specific activity‡ . |
|---|---|---|---|
| Normal | 1.0 ± 0.3 | 79 ± 1 | 0.6 ± 0.1 |
| Ser2119Tyr | 83.3 ± 28.4 | 5 ± 2 | 0.9 ± 0.2 |
| Ile2098Ser | 7.8 ± 3.2 | 43 ± 6 | 1.1 ± 0.5 |
| Arg2150His | 3.3 ± 1.2 | 56 ± 3 | 0.8 ± 0.4 |
The dissociation constants (KD) of factor VIII (fVIII) binding to von Willebrand factor (vWf) were evaluated by Scatchard analysis of fVIII binding to vWf Sepharose. Data are presented as mean ± SD of at least 3 independent experiments.
Recombinant fVIII (0.2 IU/mL) was incubated for 1 hour at 20°C in the plasma of a patient with severe hemophilia A. The samples were diluted 10-fold, and the fraction of fVIII bound to vWf was determined, using a Sepharose coated with an anti-vWf mouse monoclonal antibody, as in Figure 1. Results are expressed as the mean ± SD of triplicate experiments.
fVIII specific activity was evaluated as the ratio between fVIII:c and fVIII:Ag. A chromogenic assay was used to measure fVIII:c. The levels of fVIII:Ag were measured in 3 enzyme-linked immunosorbent assays (ELISAs), using 3 different capture antibodies. For each fVIII variant, 3 evaluations of specific activity were made by determining the ratio between fVIII:c and fVIII:Ag levels as determined in ELISA. Results are expressed as the mean ± SD of specific activity determined in the 3 assays.