Table 1. Kinetic parameters for the... | American Society of Hematology

Table 1.

Kinetic parameters for the association of D519V/E665V/K1813A, D519V/E665V, and K1813A FVIII mutants with FIXa or FX derived from FXa generation assays

FVIII mutants	FIXa association		FXa association
	K_d for FIXa (apparent)	Generated FXa	K_m	k_cat	k_cat/K_m
	nM	nM FXa/min per nM FVIII	nM	min⁻¹	(fold)
WT	1.2 ± 0.2	38 ± 5.8	80 ± 11	170 ± 6	2.2 ± 0.3 (1.0)
D519V/E665V/K1813A	0.84 ± 0.04∗	89 ± 6.0∗∗	112 ± 41	332 ± 23∗∗	3.2 ± 1.5 (1.5)
D519V/E665V	0.78 ± 0.1∗	37 ± 8.5	79 ± 16	161 ± 8	2.4 ± 0.8 (1.1)
K1813A	1.0 ± 0.1	59 ± 5.1∗∗	102 ± 23	224 ± 15	2.1 ± 0.3 (0.95)

FVIII mutants	FIXa association		FXa association
	K_d for FIXa (apparent)	Generated FXa	K_m	k_cat	k_cat/K_m
	nM	nM FXa/min per nM FVIII	nM	min⁻¹	(fold)
WT	1.2 ± 0.2	38 ± 5.8	80 ± 11	170 ± 6	2.2 ± 0.3 (1.0)
D519V/E665V/K1813A	0.84 ± 0.04∗	89 ± 6.0∗∗	112 ± 41	332 ± 23∗∗	3.2 ± 1.5 (1.5)
D519V/E665V	0.78 ± 0.1∗	37 ± 8.5	79 ± 16	161 ± 8	2.4 ± 0.8 (1.1)
K1813A	1.0 ± 0.1	59 ± 5.1∗∗	102 ± 23	224 ± 15	2.1 ± 0.3 (0.95)

FVIII (0.5 or 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 (0-5 nM) or by the addition of FIXa (40 nM) and various concentrations of FX (0-400 nM), as described in “Methods.” The kinetic calculations were based on the data shown in Figure 1. Significant differences between WT and FVIII mutants were assessed using the Student t test. Experiments were performed 3 times, and the mean values are shown. The FIXa association was expressed as the apparent dissociation constant for FIXa.

∗

P < .05

∗∗

P < .01

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