Kinetic parameters for the interaction of K1813A, K1818A, and K1813A/K1818A mutants with FIXa or FX by FXa generation
| FVIII mutant . | FIXa association . | FX association . | ||||
|---|---|---|---|---|---|---|
| Km . | kcat . | kcat/Km . | Km . | kcat . | kcat/Km . | |
| nM . | min−1 . | (fold) . | nM . | min−1 . | (fold) . | |
| WT | 1.7 ± 0.1 | 95 ± 14 | 55 ± 9.1 (1.0) | 82 ± 21 | 118 ± 20 | 1.5 ± 0.14 (1.0) |
| K1813A | 1.9 ± 0.1 | 137 ± 17∗ | 71.8 ± 11 (1.3) | 89 ± 19 | 154 ± 11 | 1.8 ± 0.3 (1.2) |
| K1818A | 1.2 ± 0.3∗ | 56 ± 7.2∗ | 50 ± 15 (0.91) | 55 ± 3.8 | 57 ± 10∗∗ | 1.1 ± 0.2 (0.73) |
| K1813A/K1818A | 1.3 ± 0.1∗ | 112 ± 16 | 85 ± 18 (1.5) | 86 ± 10 | 128 ± 28 | 1.5 ± 0.5 (1.0) |
| FVIII mutant . | FIXa association . | FX association . | ||||
|---|---|---|---|---|---|---|
| Km . | kcat . | kcat/Km . | Km . | kcat . | kcat/Km . | |
| nM . | min−1 . | (fold) . | nM . | min−1 . | (fold) . | |
| WT | 1.7 ± 0.1 | 95 ± 14 | 55 ± 9.1 (1.0) | 82 ± 21 | 118 ± 20 | 1.5 ± 0.14 (1.0) |
| K1813A | 1.9 ± 0.1 | 137 ± 17∗ | 71.8 ± 11 (1.3) | 89 ± 19 | 154 ± 11 | 1.8 ± 0.3 (1.2) |
| K1818A | 1.2 ± 0.3∗ | 56 ± 7.2∗ | 50 ± 15 (0.91) | 55 ± 3.8 | 57 ± 10∗∗ | 1.1 ± 0.2 (0.73) |
| K1813A/K1818A | 1.3 ± 0.1∗ | 112 ± 16 | 85 ± 18 (1.5) | 86 ± 10 | 128 ± 28 | 1.5 ± 0.5 (1.0) |
FVIII (1 nM) was activated with thrombin (30 nM) for 30 seconds in the presence of 20 μM PL vesicles. FXa generation was initiated by the addition of FX (300 nM) and various concentrations of FIXa (0-40 nM) or by the addition of FIXa (40 nM) and various concentrations of FX (0-400 nM), as described in Methods. Significant differences between the WT and FVIII mutants were investigated using the Student t test. Experiments were performed 3 separate times, and the mean values are shown.
P <.05.
P <.01.