Kinetic and binding parameters for expressed FVIII wild-type and mutant proteins
| Factor VIII . | PSPCPE present . | PSPCPE absent . | ||
|---|---|---|---|---|
| Kd* ± sd (nM) . | kcat† ± sd (min−1) . | Kd‡± sd (nM) . | kcat§ ± sd (min−1) . | |
| WT | 5.4 ± 0.8 | 64.2 ± 2.9 | 133.5 ± 63.3 | 7.96 ± 1.6 |
| Ser558Phe | 6.2 ± 3.1 | 6.5 ± 0.8 | 101.2 ± 15.8 | 0.25 ± 0.01 |
| Val559Ala | 12.4 ± 6.5 | 3.3 ± 0.6 | 90.7 ± 5.8 | 0.26 ± 0.01 |
| Asp560Ala | 8.3 ± 1.3 | 7.5 ± 0.4 | 263.3 ± 82.3 | 1.07 ± 0.14 |
| Arg562Ala | 8.5 ± 1.8 | 57.4 ± 4.1 | 134.4 ± 67.1 | 5.23 ± 0.82 |
| Gln565Arg | 20.9 ± 4.9 | 4.1 ± 0.3 | 132.0 ± 56.9 | 0.27 ± 0.02 |
| Factor VIII . | PSPCPE present . | PSPCPE absent . | ||
|---|---|---|---|---|
| Kd* ± sd (nM) . | kcat† ± sd (min−1) . | Kd‡± sd (nM) . | kcat§ ± sd (min−1) . | |
| WT | 5.4 ± 0.8 | 64.2 ± 2.9 | 133.5 ± 63.3 | 7.96 ± 1.6 |
| Ser558Phe | 6.2 ± 3.1 | 6.5 ± 0.8 | 101.2 ± 15.8 | 0.25 ± 0.01 |
| Val559Ala | 12.4 ± 6.5 | 3.3 ± 0.6 | 90.7 ± 5.8 | 0.26 ± 0.01 |
| Asp560Ala | 8.3 ± 1.3 | 7.5 ± 0.4 | 263.3 ± 82.3 | 1.07 ± 0.14 |
| Arg562Ala | 8.5 ± 1.8 | 57.4 ± 4.1 | 134.4 ± 67.1 | 5.23 ± 0.82 |
| Gln565Arg | 20.9 ± 4.9 | 4.1 ± 0.3 | 132.0 ± 56.9 | 0.27 ± 0.02 |
Experimental conditions for reactions run in the presence of PSPCPE (10 μM) and absence of PSPCPE are described in the legends to Figures 2 and 3, respectively. sd refers to standard deviation based on values from the fitted curves. No significant differences were observed for Kd values between mutants 558-562 and wild type (WT) (P = .1-.8* and P = .2-.99‡), whereas the value for Gln565Arg in the presence of PSPCPE is significant (P = .01). Differences in kcatvalues were significant when comparing the hemophilia A mutants with WT (P < .0001† and P < .001§). No significant difference was noted in comparingkcat values for Arg562Ala and WT (P = .2† and P = .15§).