Assessment of active site binding for FXa and prothrombinase
| Probe . | Spec Xa* . | ATIII† . | Pefabloc tPA/Xa† . | |
|---|---|---|---|---|
| Km µM . | kcat s−1 . | k2 ± SD × 103 M–1 s−1 . | KiµM . | |
| Factor Xa | ||||
| wt-FXa | 86 ± 7 | 75 ± 1.5 | 1.67 ± 0.07 | 0.07 ± 0.004 |
| Group 1 | ||||
| FXa-V17M | 600 ± 60 | 62 ± 3.2 | 0.1 ± 0.008 | 1.0 ± 0.09 |
| Group 2 | ||||
| FXa-I16L | 1200 ± 270 | 38 ± 8.0 | 0.03 ± 0.002 | 1.4 ± 0.07 |
| FXa-V17T | >1 mM | >35 | 0.02 ± 0.001 | ND |
| FXa-I16M | >1 mM | >30 | 0.02 ± 0.0008 | 30 ± 1.8 |
| Group 3 | ||||
| FXa-V17S | >1 mM | >15 | 0.01 ± 0.0007 | 100 ± 16 |
| FXa-I16T | >1 mM | >1 | ND | 600 ± 70 |
| Prothrombinase | ||||
| wt-FXa | 160 ± 20 | 115 ± 4.0 | — | 0.3 ± 0.02 |
| Group 1 | ||||
| FXa-V17M | 260 ± 50 | 64 ± 4.9 | — | 0.15 ± 0.006 |
| Group 2 | ||||
| FXa-I16L | 370 ± 30 | 47 ± 1.9 | — | 0.2 ± 0.02 |
| FXa-V17T | 540 ± 60 | 60 ± 3.8 | — | ND |
| FXa-I16M | 500 ± 80 | 55 ± 5.1 | — | 1.15 ± 0.08 |
| Group 3 | ||||
| FXa-V17S | 1100 ± 130 | 41 ± 8.5 | — | 40 ± 4 |
| FXa-I16T | 500 ± 63 | 5.3 ± 0.38 | — | 70 ± 9 |
| Probe . | Spec Xa* . | ATIII† . | Pefabloc tPA/Xa† . | |
|---|---|---|---|---|
| Km µM . | kcat s−1 . | k2 ± SD × 103 M–1 s−1 . | KiµM . | |
| Factor Xa | ||||
| wt-FXa | 86 ± 7 | 75 ± 1.5 | 1.67 ± 0.07 | 0.07 ± 0.004 |
| Group 1 | ||||
| FXa-V17M | 600 ± 60 | 62 ± 3.2 | 0.1 ± 0.008 | 1.0 ± 0.09 |
| Group 2 | ||||
| FXa-I16L | 1200 ± 270 | 38 ± 8.0 | 0.03 ± 0.002 | 1.4 ± 0.07 |
| FXa-V17T | >1 mM | >35 | 0.02 ± 0.001 | ND |
| FXa-I16M | >1 mM | >30 | 0.02 ± 0.0008 | 30 ± 1.8 |
| Group 3 | ||||
| FXa-V17S | >1 mM | >15 | 0.01 ± 0.0007 | 100 ± 16 |
| FXa-I16T | >1 mM | >1 | ND | 600 ± 70 |
| Prothrombinase | ||||
| wt-FXa | 160 ± 20 | 115 ± 4.0 | — | 0.3 ± 0.02 |
| Group 1 | ||||
| FXa-V17M | 260 ± 50 | 64 ± 4.9 | — | 0.15 ± 0.006 |
| Group 2 | ||||
| FXa-I16L | 370 ± 30 | 47 ± 1.9 | — | 0.2 ± 0.02 |
| FXa-V17T | 540 ± 60 | 60 ± 3.8 | — | ND |
| FXa-I16M | 500 ± 80 | 55 ± 5.1 | — | 1.15 ± 0.08 |
| Group 3 | ||||
| FXa-V17S | 1100 ± 130 | 41 ± 8.5 | — | 40 ± 4 |
| FXa-I16T | 500 ± 63 | 5.3 ± 0.38 | — | 70 ± 9 |
The errors in the fitted constants represent ± 2 standard deviations (SDs) for each variant. The data are representative of 2 to 3 independent measurements. Description of the experimental design and reactant concentrations can be found in supplemental Methods.
ND, not determined.
Kinetic parameters for free Xa or prothrombinase were determined from initial velocity studies conducted with Spec Xa or S-2238.
Inhibition kinetics of free Xa were determined from initial velocity studies with Spec Xa or S-2765.