TABLE 1.
Km, kcat, and kcat/Km values for thrombin hydrolysis of chromogenic substrates in the presence of exosite ligands
The rates of thrombin cleavage of various chromogenic substrates were used to determine the kinetic parameters by Michaelis-Menten analysis. Experiments were performed in the absence or presence of 10 μm HD1, HD22, or hirudin-(54–65). The experiments were repeated three times in duplicate, and the values represent means ± S.E.
| Substrate | Addition | Km | kcat | kcat/Km |
|---|---|---|---|---|
| μm | s−1 | μm−1s−1 | ||
| tGPR-pNA | ||||
| None | 16.4 ± 1.2 | 234.5 ± 4.1 | 14.3 ± 1.0 | |
| HD1 | 14.2 ± 1.4 | 240.3 ± 9.6 | 16.9 ± 1.8 | |
| HD22 | 8.5 ± 0.7 | 192.8 ± 16.3 | 22.7 ± 2.8 | |
| Hirudin-(54–65) | 11.7 ± 2.0 | 251.6 ± 20.3 | 21.5 ± 4.1 | |
| S2238 | ||||
| None | 2.6 ± 0.4 | 101.9 ± 6.8 | 39.8 ± 6.5 | |
| HD1 | 14.3 ± 0.6 | 209.4 ± 6.7 | 14.7 ± 0.8 | |
| HD22 | 12.1 ± 1.7 | 106.8 ± 4.1 | 8.8 ± 1.3 | |
| Hirudin-(54–65) | 3.1 ± 0.1 | 156.5 ± 1.8 | 51.0 ± 2.1 | |
| S2366 | ||||
| None | 282.9 ± 30.6 | 249.5 ± 11.3 | 0.9 ± 0.1 | |
| HD1 | 195.0 ± 18.3 | 217.1 ± 9.4 | 1.1 ± 0.1 | |
| HD22 | 155.4 ± 14.1 | 231.1 ± 13.4 | 1.5 ± 0.2 | |
| Hirudin-(54–65) | 147.1 ± 6.0 | 278.6 ± 3.5 | 1.9 ± 0.1 |