SPR kinetic analysis for the interaction between FXIII-A2B2 activation forms binding to immobilized fibrinogen, fibrin, and the GST captured fibrinogen αC fragments 1 (233-425) and 9 (371-425)
| Analyte . | Immobilized ligand . | Equilibrium dissociation constant (Kd1), nM* . | Association constant ka1 (1/Ms) . | Dissociation constant kd1 (1/s) . | Association constant ka2 (1/Ms) . | Dissociation constant kd2 (1/s) . |
|---|---|---|---|---|---|---|
| Unactivated | GSTαF1 | 7.3 ± 6.3 | 1.97 ± 1.52E+05 | 1.43 ± 0.68E-03 | 4.78 ± 8.00E+02 | 1.81 ± 2.35E-01 |
| FXIII-A2B2 | GSTαF9 | 30.9 ± 23 | 1.32 ± 1.03E+05 | 4.09 ± 3.39E-03 | 2.22 ± 3.54E+03 | 1.85 ± 1.24E-01 |
| (no calcium) | Fibrinogen | 11.0 ± 4.7 | 5.97 ± 1.54E+05 | 6.57 ± 1.02E-03 | 2.68 ± 3.57E+03 | 1.50 ± 1.75E-01 |
| Fibrin | 35.2 ± 4.0 | 3.66 ± 0.91E+05 | 1.29 ± 0.46E-02 | 9.01 ± 8.35E+03 | 3.68 ± 2.76E-01 | |
| Thrombin | GSTαF1 | 3.7 ± 0.3 | 1.64 ± 0.06E+05 | 6.00 ± 0.33E-04 | 7.35 ± 2.78E+01 | 1.81 ± 0.72E-01 |
| Cleaved A2B2 | GSTαF9 | 5.4 ± 1.8 | 1.27 ± 0.66E+05 | 6.91 ± 4.33E-04 | 7.51 ± 3.03E+01 | 1.29 ± 0.67E-01 |
| (no calcium) | Fibrinogen | 2.9 ± 2.1 | 8.67 ± 1.62E+05 | 2.48 ± 1.34E-03 | 6.09 ± 5.17E+03 | 5.95 ± 2.45E-01 |
| Fibrin | 6.1 ± 1.0 | 5.80 ± 3.15E+05 | 3.53 ± 3.22E-03 | 6.07 ± 4.61E+03 | 6.05 ± 0.48E-01 | |
| Thrombin | GSTαF1 | 21.9 ± 2.2 | 1.10 ± 0.19E+06 | 2.41 ± 0.16E-02 | 5.20 ± 1.77E+02 | 1.61 ± 0.48E-01 |
| Cleaved A2B2 | GSTαF9 | 7.3 ± 1.8 | 1.95 ± 0.07E+06 | 1.42 ± 0.32E-02 | 2.59 ± 2.17E+03 | 3.09 ± 2.67E-01 |
| (with calcium) | Fibrinogen | 10.7 ± 0.9 | 1.19 ± 0.37E+06 | 1.28 ± 0.41E-02 | 1.41 ± 1.19E+04 | 2.11 ± 1.64E-01 |
| Fibrin | 14.5 ± 3.1 | 1.26 ± 0.32E+06 | 1.83 ± 0.69E-02 | 1.71 ± 1.07E+04 | 2.24 ± 1.24E-01 |
| Analyte . | Immobilized ligand . | Equilibrium dissociation constant (Kd1), nM* . | Association constant ka1 (1/Ms) . | Dissociation constant kd1 (1/s) . | Association constant ka2 (1/Ms) . | Dissociation constant kd2 (1/s) . |
|---|---|---|---|---|---|---|
| Unactivated | GSTαF1 | 7.3 ± 6.3 | 1.97 ± 1.52E+05 | 1.43 ± 0.68E-03 | 4.78 ± 8.00E+02 | 1.81 ± 2.35E-01 |
| FXIII-A2B2 | GSTαF9 | 30.9 ± 23 | 1.32 ± 1.03E+05 | 4.09 ± 3.39E-03 | 2.22 ± 3.54E+03 | 1.85 ± 1.24E-01 |
| (no calcium) | Fibrinogen | 11.0 ± 4.7 | 5.97 ± 1.54E+05 | 6.57 ± 1.02E-03 | 2.68 ± 3.57E+03 | 1.50 ± 1.75E-01 |
| Fibrin | 35.2 ± 4.0 | 3.66 ± 0.91E+05 | 1.29 ± 0.46E-02 | 9.01 ± 8.35E+03 | 3.68 ± 2.76E-01 | |
| Thrombin | GSTαF1 | 3.7 ± 0.3 | 1.64 ± 0.06E+05 | 6.00 ± 0.33E-04 | 7.35 ± 2.78E+01 | 1.81 ± 0.72E-01 |
| Cleaved A2B2 | GSTαF9 | 5.4 ± 1.8 | 1.27 ± 0.66E+05 | 6.91 ± 4.33E-04 | 7.51 ± 3.03E+01 | 1.29 ± 0.67E-01 |
| (no calcium) | Fibrinogen | 2.9 ± 2.1 | 8.67 ± 1.62E+05 | 2.48 ± 1.34E-03 | 6.09 ± 5.17E+03 | 5.95 ± 2.45E-01 |
| Fibrin | 6.1 ± 1.0 | 5.80 ± 3.15E+05 | 3.53 ± 3.22E-03 | 6.07 ± 4.61E+03 | 6.05 ± 0.48E-01 | |
| Thrombin | GSTαF1 | 21.9 ± 2.2 | 1.10 ± 0.19E+06 | 2.41 ± 0.16E-02 | 5.20 ± 1.77E+02 | 1.61 ± 0.48E-01 |
| Cleaved A2B2 | GSTαF9 | 7.3 ± 1.8 | 1.95 ± 0.07E+06 | 1.42 ± 0.32E-02 | 2.59 ± 2.17E+03 | 3.09 ± 2.67E-01 |
| (with calcium) | Fibrinogen | 10.7 ± 0.9 | 1.19 ± 0.37E+06 | 1.28 ± 0.41E-02 | 1.41 ± 1.19E+04 | 2.11 ± 1.64E-01 |
| Fibrin | 14.5 ± 3.1 | 1.26 ± 0.32E+06 | 1.83 ± 0.69E-02 | 1.71 ± 1.07E+04 | 2.24 ± 1.24E-01 |
Data were evaluated with BIAcore 3000 BIAevaluation 4.1 software fitted to a bivalent analyte model with the use of blank reference and buffer subtracted data. The bivalent analyte model; A + B ↔ AB1: AB1 + B ↔ AB2, where A is the analyte and B is the ligand, results in 2 sets of rate constants; association (ka1/ka2), dissociation (kd1/kd2), and equilibrium dissociation constant (Kd1/Kd2). Kd1 (AB1) is clearly stronger than Kd2 (AB2) and is therefore the primary interaction considered in this study. The low-affinity second interaction (AB2 [Kd2]) is less relevant than AB1, because it can only occur after AB1 formation and is generally thought of as a stabilizing interaction, supporting the formation of AB1. For each rate constant the average and SD of triplicate experiments are shown.
χ2 < 5 for all analysis shown.