Table 1. Kinetic parameters for cleavage of fIX by fXIa.
Simulation of full-progress experimental traces was performed with KinTek software (KinTek Explorer Version 2.5) using the Equation shown in the Experimental Procedures section. Km and kcat for activation were calculated from individual rate constants for each step. Values are the mean ± standard deviation for each experiment. Ki was fixed for reactions with fXIa-WT and fXIa/PKA3 based on published SPR results.11
| Cleavage of fIX after Arg145 | ||||||
|---|---|---|---|---|---|---|
| Protease | Substrate | Kcat1 (min-1) | Kd1 (μM) | Km1 (μM) | Catalytic Efficiency 1 (μM-1 · min-1) | Ki1 (μM) |
| fXIaWTa | fIX | 12 ± 1 | 0.10 ± 0.01 | 0.20 ± 0.01 | 60 ± 6 | 0.14b |
| fXIa/PKA3a | fIX | 4.9 ± 0.1 | 4.9 ± 0.2 | 4.9 ± 0.2 | 1.0 ± 0.1 | 3b |
| fXIa-Gly184 | fIX | 13 ± 3 | 0.3 ± 0.1 | 0.7 ± 0.3 | 19 ± 10 | 0.4 ± 0.2 |
| fXIa-Ala183-185 | fIX | 9 ± 1 | 8 ± 1 | 10 ± 2 | 0.9 ± 0.2 | 6 ± 1 |
| fXIa/PKA3-A | fIX | 6.4 ± 0.2 | 1.3 ± 0.1 | 1.4 ± 0.1 | 4.6 ± 0.4 | 1.9 ± 0.1 |
| fXIa/PKA3-B | fIX | 19 ± 2 | 0.06 ± 0.01 | 0.09 ± 0.02 | 200 ± 30 | 0.5 ± 0.1 |
| Cleavage of fIX after Arg180 | ||||||
| Protease | Substrate | Kcat2 (min-1) | Kd2 (μM) | Km2 (μM) | Catalytic Efficiency 2 (μM-1 · min-1) | Ki2 (μM) |
| fXIaWTa | fIX | 35 ± 6 | 0.06 ± 0.01 | 0.08 ± 0.01 | 400 ± 100 | 0.06b |
| fXIa/PKA3a | fIX | 0.6 ± 0.1 | 7 ± 1 | 7 ± 1 | 0.09 ± 0.02 | 3b |
| fXIa-Gly184 | fIX | 40 ± 10 | 0.2 ± 0.1 | 0.2 ± 0.1 | 200 ± 50 | 0.5 ± 0.2 |
| fXIa-Ala183-185 | fIX | 0.4 ± 0.1 | 3 ± 1 | 3 ± 1 | 0.13 ± 0.01 | 0.29 ± 0.01 |
| fXIa/PKA3-A | fIX | 1.7 ± 0.1 | 1.3 ± 0.1 | 1.3 ± 0.1 | 1.3 ± 0.1 | 0.28 ± 0.02 |
| fXIa/PKA3-B | fIX | 39 ± 4 | 0.04 ± 0.01 | 0.07 ± 0.01 | 600 ± 100 | 0.30 ± 0.03 |
a
data for fXIaWT and fXIa/PKA3 are from reference 11.
b
fixed values for Ki are based on data from surface plasmon resonance studies [reference 11].