ACT-LR whole-blood clotting assay demonstrates phenotypic correction of murine hemophilia A
. | Clotting time, seconds . | . | Estimated FVIII, % normal human . | . | . | ||
|---|---|---|---|---|---|---|---|
| Mouse treatment group . | -hFVIII Ab . | +hFVIII Ab . | -hFVIII Ab . | +hFVIII Ab . | Phenotype, by clotting time . | ||
| Wild-type C57BL/6J | 120-140 | NA | at least 100% | NA | Normal | ||
| FVIII Knockout C57BL/6J-FVIII-/- | more than 400 | more than 400 | less than 0.5% | less than 0.5% | Severe hemophilia | ||
| FVIII Knockout C57BL/6J-FVIII-/- FVIII transposon/SB (▪, Figure 4B) | 187 (± 14) | 248 (± 10) | 10% | 2% | Mild hemophilia | ||
| FVIII Knockout C57BL/6J-FVIII-/- FVIII transposon/SB (▴, Figure 4B) | 186 (± 8) | 350 (± 30) | 10% | less than 0.5% | Mild hemophilia | ||
| FVIII Knockout C57BL/6J-FVIII-/- FVIII transposon/SB (♦, Figure 4B) | 138 (± 10) | 400 (± 12) | 100% | 0.0% | Normal | ||
. | Clotting time, seconds . | . | Estimated FVIII, % normal human . | . | . | ||
|---|---|---|---|---|---|---|---|
| Mouse treatment group . | -hFVIII Ab . | +hFVIII Ab . | -hFVIII Ab . | +hFVIII Ab . | Phenotype, by clotting time . | ||
| Wild-type C57BL/6J | 120-140 | NA | at least 100% | NA | Normal | ||
| FVIII Knockout C57BL/6J-FVIII-/- | more than 400 | more than 400 | less than 0.5% | less than 0.5% | Severe hemophilia | ||
| FVIII Knockout C57BL/6J-FVIII-/- FVIII transposon/SB (▪, Figure 4B) | 187 (± 14) | 248 (± 10) | 10% | 2% | Mild hemophilia | ||
| FVIII Knockout C57BL/6J-FVIII-/- FVIII transposon/SB (▴, Figure 4B) | 186 (± 8) | 350 (± 30) | 10% | less than 0.5% | Mild hemophilia | ||
| FVIII Knockout C57BL/6J-FVIII-/- FVIII transposon/SB (♦, Figure 4B) | 138 (± 10) | 400 (± 12) | 100% | 0.0% | Normal | ||
NA indicates not attempted. Mice (Figure 4B) were sublethally anesthetized with avertin. Blood was collected directly from the heart with a needle and syringe and incubated with or without polyclonal anti-hFVIII antibody for 1 hour at room temperature. Clotting activity in whole blood was then assayed using the ACT-LR method. Anti-hFVIII antibody ablated all (1 of 3) or most (2 of 3) of clotting activity, revealing an hFVIII-dependent effect on clotting time and ruling out significant tissue factor contamination. All mice displayed improved clotting times, indicating partial (2 of 3) or complete (1 of 3) phenotypic correction of murine hemophilia A.