EC50 (± SEM) for DHG inhibition of the mean velocity index for thrombin generation in factor IX–deficient plasma (μM)
| Protein . | Factor IX(a) WT . | Factor IX(a) R170A . | Factor IX(a) R233A . |
|---|---|---|---|
| Zymogen | 0.38 ± 0.01 | 0.86 ± 0.06 | 3.55 ± 0.47 |
| Protease | 0.38 ± 0.01 | 1.02 ± 0.02 | 2.98 ± 0.64 |
| Protein . | Factor IX(a) WT . | Factor IX(a) R170A . | Factor IX(a) R233A . |
|---|---|---|---|
| Zymogen | 0.38 ± 0.01 | 0.86 ± 0.06 | 3.55 ± 0.47 |
| Protease | 0.38 ± 0.01 | 1.02 ± 0.02 | 2.98 ± 0.64 |
Thrombin generation was initiated with 0.2 pM human TF, 8.3 μM PC:PS vesicles, and 40 μg/mL CTI (plasma concentrations) in factor IX–deficient plasma supplemented with 100% (90 nM) recombinant factor IX WT, R170A, and R233A in the presence of increasing DHG (0-10 μM). For factor IXa–initiated assays, TF was omitted, and 100 pM recombinant factor IXa was added. Duplicate wells were averaged to determine individual thrombin generation curves. The relative velocity index was plotted versus DHG concentration, and EC50 values for each replicate (n = 3) were determined as described in “Fluorogenic assay for detection of plasma thrombin generation.” The mean and standard error of the EC50 values were determined from the 3 independent replicates.
Statistical significance analysis: EC50 values for each zymogen were not significantly different from their respective protease (Student t test, P > .2). EC50 values for factor IX(a) R170A and R233A were significantly different from factor IX(a) WT (Student t test, P ≤ .01). EC50 values for factor IX(a) R170A were significantly different from factor IX(a) R233A (Student t test, P < .02).