TABLE 2.
Stoichiometries and second order association rate constants for reactions of wild-type (WT) and P14 variant antithrombins with proteases in the absence of heparin (SI−H, ka, −H) and presence of pentasaccharide (SI+H5, ka, +H5) or full-length heparins (SI+H50, ka, +H50)
Stoichiometries of inhibition (SI) and association rate constants (ka) ±S.E. for reactions of antithrombin P14 variants with proteases were measured by end point titrations or by time-dependent or heparin-dependent inhibition progress curves under pseudo-first order conditions, respectively, in I 0.15, pH 7.4 sodium phosphate (thrombin and factor Xa) or Hepes/calcium (factor IXa) buffers at 25 °C as described under “Experimental Procedures.” Association rate constants have been corrected for variable inhibition reaction stoichiometries by multiplying apparent rate constants by the SI. ND indicates that a value was not determined.
| P14 variant | SI−H | ka, −H | SI+H5 | ka, +H5 | SI+H50 | ka, +H50 |
|---|---|---|---|---|---|---|
| Thrombin reactions | ||||||
| Val | 1.0 ± 0.1 | (9.4 ± 0.4) × 103 | 1.1 ± 0.1 | (1.1 ± 0.1) × 104 | 1.5 ± 0.1 | (2.0 ± 0.1) × 107 |
| Thr | 1.4 ± 0.1 | (7.5 ± 0.1) × 103 | 1.3 ± 0.1 | (1.3 ± 0.1) × 104 | 1.7 ± 0.1 | (2.7 ± 0.1) × 107 |
| Ala | 1.7 ± 0.1 | (1.2 ± 0.1) × 104 | 1.7 ± 0.2 | (2.3 ± 0.1) × 104 | 1.7 ± 0.2 | (1.5 ± 0.5) × 107 |
| Leu | 1.2 ± 0.1 | (9.4 ± 0.2) × 103 | 2.5 ± 0.1 | (1.3 ± 0.1) × 104 | 3.4 ± 0.2 | (2.1 ± 0.3) × 107 |
| Met | 1.4 ± 0.1 | (8.8 ± 0.1) × 103 | 1.8 ± 0.1 | (1.6 ± 0.1) × 104 | 2.5 ± 0.1 | (2.2 ± 0.1) × 107 |
| Ser (WT) | 1.3 ± 0.1 | (1.1 ± 0.1) × 104 | 1.3 ± 0.1 | (2.2 ± 0.1) × 104 | 1.7 ± 0.1 | (3.4 ± 0.1) × 107 |
| Trp | 13 ± 1 | (1.0 ± 0.1) × 104 | 8.8 ± 0.2 | (2.4 ± 0.1) × 104 | 25 ± 1 | ND |
| Phe | 1.6 ± 0.1 | (9.1 ± 1.2) × 103 | 1.7 ± 0.1 | (2.0 ± 0.1) × 104 | 2.7 ± 0.1 | (1.9 ± 0.2) × 107 |
| Gly | 1.0 ± 0.1 | (6.3 ± 0.2) × 103 | 1.0 ± 0.1 | (1.4 ± 0.1) × 104 | 1.5 ± 0.1 | (2.0 ± 0.1) × 107 |
| Pro | 1.5 ± 0.1 | (1.1 ± 0.1) × 104 | 2.8 ± 0.1 | (2.0 ± 0.1) × 104 | 7.7 ± 0.6 | (2.6 ± 0.8) × 107 |
| His | 10 ± 1 | (2.9 ± 0.1) × 104 | 30 ± 1 | (1.9 ± 0.1) × 104 | 110 ± 6 | (3.7 ± 1.2) × 107 |
| Tyr | 6.8 ± 0.2 | (1.4 ± 0.1) × 104 | 5.6 ± 0.2 | (2.3 ± 0.1) × 104 | 13 ± 1 | (3.2 ± 0.3) × 107 |
| Gln | 2.0 ± 0.1 | (1.2 ± 0.1) × 104 | 11 ± 1 | (1.4 ± 0.1) × 104 | 62 ± 3 | (3.3 ± 0.3) × 107 |
| Asp | 2.2 ± 0.2 | (1.9 ± 0.4) × 104 | 270 ± 20 | (2.0 ± 0.2) × 104 | 150 ± 20 | (1.8 ± 0.2) × 107 |
| Glu | 3.1 ± 0.4 | (1.4 ± 0.5) × 104 | 480 ± 40 | (3.0 ± 0.3) × 104 | 430 ± 40 | (7.6 ± 2.3) × 106 |
| Factor Xa reactions | ||||||
| Val | 1.3 ± 0.1 | (2.6 ± 0.4) × 103 | 5.5 ± 0.4 | (2.5 ± 0.1) × 105 | 3.4 ± 0.2 | (1.3 ± 0.1) × 106 |
| Thr | 2.1 ± 0.1 | (1.7 ± 0.2) × 103 | 2.4 ± 0.1 | (4.0 ± 0.2) × 105 | 2.0 ± 0.1 | (1.6 ± 0.2) × 106 |
| Ala | 1.1 ± 0.1 | (1.7 ± 0.4) × 103 | 1.6 ± 0.3 | (7.4 ± 2.1) × 105 | 1.7 ± 0.1 | (6.0 ± 0.4) × 106 |
| Leu | 1.8 ± 0.1 | (2.6 ± 0.2) × 103 | 41 ± 1 | (6.0 ± 0.8) × 105 | 16 ± 1 | (8.8 ± 1.6) × 105 |
| Met | 1.6 ± 0.1 | (1.9 ± 0.4) × 103 | 7.9 ± 0.1 | (4.8 ± 1.7) × 105 | 3.9 ± 0.7 | (4.3 ± 0.2) × 106 |
| Ser (WT) | 1.2 ± 0.1 | (4.4 ± 0.2) × 103 | 1.4 ± 0.1 | (4.6 ± 0.5) × 105 | 1.4 ± 0.1 | (1.5 ± 0.2) × 106 |
| Trp | 28 ± 2 | (1.3 ± 0.5) × 104 | 81 ± 5 | (5.8 ± 1.0) × 105 | 55 ± 2 | (2.9 ± 0.4) × 106 |
| Phe | 2.2 ± 0.3 | (1.6 ± 0.1) × 104 | 5.1 ± 0.4 | (5.6 ± 0.3) × 105 | 4.4 ± 0.2 | (2.5 ± 0.3) × 106 |
| Gly | 1.0 ± 0.1 | (1.6 ± 0.1) × 104 | 1.4 ± 0.1 | (6.2 ± 0.6) × 105 | 1.3 ± 0.1 | (1.6 ± 0.1) × 106 |
| Pro | 1.8 ± 0.1 | (3.6 ± 0.3) × 104 | 8.3 ± 0.1 | (4.6 ± 0.1) × 105 | 10 ± 1 | (1.4 ± 0.1) × 106 |
| His | 18 ± 1 | (4.6 ± 0.6) × 104 | 170 ± 10 | (4.9 ± 0.1) × 105 | 180 ± 10 | (4.8 ± 0.3) × 106 |
| Tyr | 29 ± 2 | (7.4 ± 1.0) × 104 | 38 ± 2 | (8.5 ± 1.6) × 105 | 25 ± 2 | (6.8 ± 0.2) × 106 |
| Gln | 5.4 ± 0.3 | (9.0 ± 1.1) × 104 | 180 ± 10 | (7.4 ± 1.1) × 105 | 120 ± 5 | (2.7 ± 0.2) × 106 |
| Asp | 4.1 ± 0.1 | (8.2 ± 1.0) × 105 | 740 ± 30 | (8.1 ± 0.4) × 105 | 440 ± 30 | (3.9 ± 0.9) × 106 |
| Glu | 7.9 ± 1.9 | (1.3 ± 0.2) × 106 | 2100 ± 400 | (1.5 ± 0.7) × 105 | 1300 ± 300 | (1.7 ± 0.3) × 106 |
| Factor IXa reactions | ||||||
| Val | 1a | (4.5 ± 1.1) × 100 | 1.2 ± 0.1 | (8.5 ± 1.3) × 103 | ||
| Thr | 1a | (1.7 ± 0.3) × 101 | 1.2 ± 0.1 | (1.6 ± 0.6) × 104 | ||
| Ala | 1.0 ± 0.1 | (1.3 ± 0.1) × 101 | 1.0 ± 0.1 | (1.4 ± 0.2) × 104 | ||
| Leu | 1.0 ± 0.1 | (3.9 ± 0.3) × 101 | 1.4 ± 0.1 | (1.3 ± 0.3) × 103 | ||
| Met | ND | ND | ND | ND | ||
| Ser (WT) | 1.1 ± 0.2 | (1.8 ± 0.1) × 102 | 1.0 ± 0.1 | (2.4 ± 0.2) × 104 | ||
| Trp | 1.8 ± 0.2 | (2.7 ± 0.4) × 102 | 3.2 ± 0.1 | (9.2 ± 0.1) × 102 | ||
| Phe | 1.3 ± 0.2 | (4.3 ± 0.6) × 102 | 1.0 ± 0.1 | (5.7 ± 1.0) × 103 | ||
| Gly | 1.0 ± 0.1 | (6.6 ± 0.6) × 102 | 1.0 ± 0.1 | (2.1 ± 0.1) × 104 | ||
| Pro | 1.1 ± 0.1 | (8.5 ± 0.9) × 102 | 1.2 ± 0.1 | (4.5 ± 0.7) × 103 | ||
| His | 1.8 ± 0.1 | (1.0 ± 0.1) × 103 | 9.6 ± 2.6 | (9.6 ± 4.0) × 102 | ||
| Tyr | 1.5 ± 0.1 | (1.7 ± 0.1) × 103 | 2.0 ± 0.2 | (1.4 ± 0.2) × 103 | ||
| Gln | 1.4 ± 0.1 | (2.4 ± 0.2) × 103 | 6.1 ± 0.3 | (2.5 ± 0.4) × 103 | ||
| Asp | ND | ND | ND | ND | ||
| Glu | 1.7 ± 0.1 | (1.9 ± 0.4) × 104 | 710 ± 120 | (4.2 ± 0.7) × 103 | ||
a SI could not be measured because of slow reaction kinetics and was assumed to be 1 based on the SI measured for the pentasaccharide-activated antithrombin reaction.