Table 1.
Stoichiometries and Kinetics of Protease Inhibition by Antithrombin Variantsa
| AT variant | SI−H | k a,−H | SIH5 | k a,H5 |
ka,H5/ ka,−H |
SIH50 | k a,H50 |
ka,H50/ ka,H5 |
|---|---|---|---|---|---|---|---|---|
| Thrombin Reactions | ||||||||
| wild type | 1.2 ± 0.1 | (1.1 ± 0.2) × 104 | 1.3 ± 0.1 | (2.3 ± 0.3) × 104 | 2.1 | 1.7 ± 0.1 | (3.4 ± 0.5) × 107 | 1500 |
| V375C/S380C | 1.8 ± 0.2 | (3.8 ± 0.1) × 103 | 2.4 ± 0.3 | (5.0 ± 0.9) × 103 | 1.3 | 2.8 ± 0.1 | (2.2 ± 0.6) × 107 | 4400 |
| L373C/S380C | 1.2 ± 0.1 | (6.4 ± 0.7) × 103 | 1.2 ± 0.1 | (7.7 ± 0.8) × 103 | 1.2 | 1.7 ± 0.1 | (9.2 ± 1.7) × 106 | 1200 |
| K222C/E381C | 1.4 ± 0.1 | (5.3 ± 0.4) × 103 | 2.5 ± 0.1 | (9.0 ± 0.6) × 103 | 1.7 | 14 ± 1 | (4.1 ± 0.4) × 107 | 4600 |
| K222C/K139C | 1.1 ± 0.1 | (1.3 ± 0.2) × 104 | 1.1 ± 0.1 | (8.5 ± 1.2) × 103 | 0.7 | 2.9 ± 0.2 | (2.6 ± 0.3) × 107 | 3100 |
| S380C | 1.0 ± 0.1 | (8.1 ± 0.9) × 103 | – | – | – | 1.5 ± 0.2 | (2.0 ± 0.6) × 107 | 2500b |
| helix D | 1.4 ± 0.1 | (1.1 ± 0.3) × 104 | 1.6 ± 0.1 | (1.3 ± 0.2) × 104 | 1.2 | 1.8 ± 0.1. | (8.5 ± 1.4) × 106 | 650 |
| helix D/L373C/S380C | 1.8 ± 0.2 | (5.9 ± 2.6) × 103 | 2.6 ± 0.1 | (9.1 ± 1.9) × 103 | 1.5 | 3.4 ± 0.2 | (3.0 ± 0.2) × 106 | 330 |
| Y131L | 1.5 ± 0.1 | (1.3 ± 0.2) × 104 | 1.6 ± 0.1 | (2.3 ± 0.2) × 104 | 1.8 | 2.5 ± 0.1 | (2.1 ± 0.3) × 107 | 910 |
| Y131L/V375C/S380C | 60 ± 2 | (7.9 ± 1.0) × 103 | 79 ± 5 | (1.0 ± 0.2) × 104 | 1.3 | 68 ± 7 | (1.3 ± 0.2) × 107 | 1300 |
| S380W | 14 ± 1 | (1.0 ± 0.1) × 104 | 8.8 ± 0.2 | (2.5 ± 0.1) × 104 | 2.5 | 25 ± 1 | ND | – |
| Y131L/S380W | 6.7 ± 0.5 | (9.5 ± 1.1) × 103 | 4.2 ± 0.2 | (1.2 ± 0.1) × 104 | 1.3 | 13 ± 1 | (3.6 ± 0.4) × 106 | 300 |
| Factor Xa Reactions | ||||||||
| wild type | 1.2 ± 0.1 | (4.0 ± 0.8) × 103 | 1.8 ± 0.3 | (1.4 ± 0.3) × 106 | 350 | 2.1 ± 0.1 | (1.8 ± 0.1) × 107 | 13 |
| V375C/S380C | 2.0 ± 0.4 | (5.3 ± 1.3) × 102 | 5.6 ± 0.8 | (1.6 ± 1.0) × 105 | 300 | 5.0 ± 0.3 | (7.0 ± 0.9) × 106 | 44 |
| L373C/S380C | 1.2 ± 0.1 | (6.5 ± 0.7) × 102 | 2.0 ± 0.1 | (2.2 ± 0.3) × 105 | 340 | 2.0 ± 0.2 | (4.0 ± 0.8) × 106 | 18 |
| K222C/E381C | 2.3 ± 0.1 | (4.1 ± 0.4) × 102 | 4.7 ± 0.3 | (7.0 ± 1.8) × 102 | 1.7 | 10 ± 1 | (1.1 ± 0.2) × 105 | 160 |
| K222C/K139C | 1.1 ± 0.1 | (2.6 ± 0.3) × 103 | 2.0 ± 0.1 | (1.5 ± 0.2) × 105 | 58 | 7.7 ± 1.4 | (2.4 ± 0.5) × 106 | 16 |
| S380C | 1.2 ± 0.1 | (1.6 ± 0.2) × 103 | 1.9 ± 0.1 | (6.3 ± 0.7) × 105 | 750 | – | – | – |
| helix D | 1.4 ± 0.1 | (3.1 ± 0.6) × 105 | 2.2 ± 0.3 | (1.0 ± 0.2) × 106 | 3.2 | 5.6 ± 0.8 | (2.4 ± 0.6) × 107 | 24 |
| helix D/L373C/S380C | 2.3 ± 0.1 | (6.9 ± 1.0) × 103 | 4.2 ± 0.3 | (4.0 ± 0.4) × 105 | 58 | 5.1 ± 0.6 | (1.3 ± 0.3) × 106 | 3.3 |
| Y131L | 1.7 ± 0.2 | (4.7 ± 0.9) × 104 | 1.8 ± 0.2 | (8.4 ± 1.6) × 105 | 18 | 1.8 ± 0.2 | (2.6 ± 0.4) × 106 | 3.1 |
| Y131L/V375C/S380C | 65 ± 5 | (1.5 ± 0.3) × 103 | 180 ± 3 | (5.4 ± 0.3) × 105 | 360 | 69 ± 2 | (2.0 ± 0.1) × 106 | 3.7 |
| S380W | 28 ± 2 | (1.3 ± 0.2) × 104 | 82 ± 4 | (6.0 ± 1.0) × 105 | 46 | 55 ± 2 | (2.9 ± 0.4) × 106 | 4.8 |
| Y131L/S380W | 19 ± 1 | (8.9 ± 1.2) × 104 | 32 ± 2 | (5.4 ± 1.0) × 105 | 6.1 | 28 ± 1 | (2.8 ± 0.3) × 106 | 5.2 |
| Factor IXa Reactions | ||||||||
| wild type | 1.0 ± 0.1 | (1.8 ± 0.3) × 102 | 1.0 ± 0.1 | (2.5 ± 0.4) × 104 | 140 | 1.1 ± 0.1 | (6.0 ± 0.8) × 106 | 240 |
| V375C/S380C | ND | (3.9 ± 0.1) × 100c | 5.6 ± 0.4 | (7.8 ± 1.1) × 103 | >360c | 7.3 ± 0.7 | (1.2 ± 0.3) × 107 | 1500 |
| L373C/S380C | ND | (2.1 ± 0.8) × 100c | 1.4 ± 0.1 | (7.6 ± 1.5) × 103 | >2600c | 1.4 ± 0.1 | (4.8 ± 0.9) × 105 | 63 |
| K222C/E381C | ND | (0.2 ± 0.1) × 100c | ND | (0.2 ± 0.1) × 100c | 1 | 2.8 ± 0.2 | (4.5 ± 0.9) × 102 | >800c |
| K222C/K139C | ND | (5.2 ± 0.1) × 10c | 2.0 ± 0.2 | (7.6 ± 1.0) × 103 | 150 | – | – | – |
| S380C | 1.0 ± 0.1 | (2.7 ± 0.2) × 10 | 1.0 ± 0.1 | (1.3 ± 0.1) × 104 | 480 | – | – | – |
| helix D | 1.4 ± 0.1 | (1.8 ± 0.2) × 104 | 1.3 ± 0.4 | (2.9 ± 1.4) × 104 | 1.6 | – | – | – |
| helix D/L373C/S380C | ND | (1.2 ± 0.1) × 10c | 2.3 ± 0.1 | (9.2 ± 0.6) × 103 | >330c | – | – | – |
Stoichiometries of inhibition (SI) and second-order association rate constants (ka) for antithrombin–protease reactions were determined in I = 0.15 sodium phosphate buffer for thrombin reactions or in I = 0.15 Hepes/calcium buffer for FXa and FIXa reactions, all at pH 7.4 and 25 °C in the absence of heparin (−H), in the presence of heparin pentasaccharide (+H5), or in the presence of full-length heparin (+H50), as described in Materials and Methods. All measurements were made at least in duplicate with errors representing the range or standard error. Heparin allosteric activation rate enhancements are indicated by the kH5/k−H ratio, and heparin bridging rate enhancements are indicated by the kH50/kH5 ratio.
The ratio represents ka,H50/ka,−H.
Apparent values of ka because slow reaction rates precluded measurement of SIs. Lower limits of rate enhancements are based on corrections of apparent ka values assuming SIs no greater than those measured in the presence of heparin.