Dissociation constants (KI) for EP compound inhibition of blood coagulation proteases in I 0.15, pH 7.4 buffer plus 5mM Ca2+ at 25°C
| Protease . | KI, M . | |||
|---|---|---|---|---|
| EP42675 . | EP217609 . | EP217609–1 . | EP307138–1 . | |
| Thrombin | 6.1 ± 2.0 × 10−11 | 4.3 ± 1.6 × 10−11 | 3.8 ± 0.8 × 10−11 | 5.0 ± 0.9 × 10−11 |
| FXa | 8.2 ± 0.4 × 10−5 | 3.0 ± 0.1 × 10−5 | 1.7 ± 0.2 × 10−5 | 3.5 ± 0.2 × 10−5 |
| FIXa | > 10−5 | > 10−5 | > 10−5 | > 10−5 |
| TF-FVIIa | > 10−5 | > 10−5 | > 10−5 | > 10−5 |
| FXIa | 2.9 ± 0.2 × 10−7 | 1.7 ± 0.1 × 10−7 | 3.2 ± 0.2 × 10−7 | 8.6 ± 0.5 × 10−8 |
| FXIIa | > 10−5 | > 10−5 | > 10−5 | > 10−5 |
| Kallikrein | 2.2 ± 0.1 × 10−5 | 1.3 ± 0.1 × 10−5 | 1.5 ± 0.1 × 10−5 | 1.1 ± 0.1 × 10−5 |
| aPC | 4.0 ± 0.3 × 10−8 | 5.0 ± 0.6 × 10−8 | 5.2 ± 0.4 × 10−8 | 6.3 ± 0.4 × 10−8 |
| tPA | 2 ± 1 × 10−4 | > 10−5 | > 10−5 | > 10−5 |
| Plasmin | 5.2 ± 0.3 × 10−6 | 2.7 ± 0.2 × 10−6 | 1.8 ± 0.2 × 10−6 | 2.6 ± 0.2 × 10−6 |
| Protease . | KI, M . | |||
|---|---|---|---|---|
| EP42675 . | EP217609 . | EP217609–1 . | EP307138–1 . | |
| Thrombin | 6.1 ± 2.0 × 10−11 | 4.3 ± 1.6 × 10−11 | 3.8 ± 0.8 × 10−11 | 5.0 ± 0.9 × 10−11 |
| FXa | 8.2 ± 0.4 × 10−5 | 3.0 ± 0.1 × 10−5 | 1.7 ± 0.2 × 10−5 | 3.5 ± 0.2 × 10−5 |
| FIXa | > 10−5 | > 10−5 | > 10−5 | > 10−5 |
| TF-FVIIa | > 10−5 | > 10−5 | > 10−5 | > 10−5 |
| FXIa | 2.9 ± 0.2 × 10−7 | 1.7 ± 0.1 × 10−7 | 3.2 ± 0.2 × 10−7 | 8.6 ± 0.5 × 10−8 |
| FXIIa | > 10−5 | > 10−5 | > 10−5 | > 10−5 |
| Kallikrein | 2.2 ± 0.1 × 10−5 | 1.3 ± 0.1 × 10−5 | 1.5 ± 0.1 × 10−5 | 1.1 ± 0.1 × 10−5 |
| aPC | 4.0 ± 0.3 × 10−8 | 5.0 ± 0.6 × 10−8 | 5.2 ± 0.4 × 10−8 | 6.3 ± 0.4 × 10−8 |
| tPA | 2 ± 1 × 10−4 | > 10−5 | > 10−5 | > 10−5 |
| Plasmin | 5.2 ± 0.3 × 10−6 | 2.7 ± 0.2 × 10−6 | 1.8 ± 0.2 × 10−6 | 2.6 ± 0.2 × 10−6 |
Dissociation constants were measured by fitting the hyperbolic decrease in the initial velocity of substrate hydrolysis as a function of EP compound concentration to the equation for binding of a competitive inhibitor as described in “Methods” and shown in Figure 3. Fits provided apparent dissociation constants, KI*, which were corrected by dividing by the factor, 1 + [S]o/KM, to obtain KI. Correction factors ranged between 1.1 and 4.3. No correction was required in the case of EP compound binding to thrombin because of the tight binding of the inhibitor and consequent negligible dissociation during measurements of initial velocity.