Kinetic parameters of scFv4-20–mediated inhibition of recombinant ADAMTS13 and variants
| Constructs . | scFv4-20 (nM) . | Vmax (FU/s) . | K0.5 (μM) . | αapp (global fit) . |
|---|---|---|---|---|
| FL-AT13 | 0 | 1.05 ± 0.09 | 1.11 ± 0.05 | 1.82 ± 0.06 |
| 0.37 | 1.14 ± 0.08 | 1.45 ± 0.10 | ||
| 1.1 | 0.98 ± 0.09 | 1.22 ± 0.03 | ||
| 3.3 | 0.88 ± 0.08 | 1.71 ± 0.13 | ||
| 10 | 0.36 ± 0.02 | 2.32 ± 0.18 | ||
| MT4 | 0 | 0.65 ± 0.02 | 0.44 ± 0.02 | 1.27 ± 0.07 |
| 0.12 | 0.67 ± 0.07 | 0.56 ± 0.10 | ||
| 0.37 | 0.59 ± 0.04 | 0.50 ± 0.02 | ||
| 1.1 | 0.34 ± 0.05 | 0.50 ± 0.08 | ||
| 3.3 | 0.11 ± 0.02 | 0.92 ± 0.35 | ||
| MDTCS | 0 | 1.42 ± 0.12 | 1.01 ± 0.08 | 1.81 ± 0.39 |
| 1.1 | 1.55 ± 0.14 | 1.39 ± 0.14 | ||
| 3.3 | 1.27 ± 0.14 | 1.26 ± 0.04 | ||
| 10 | 0.89 ±0.09 | 1.77 ± 0.35 | ||
| 30 | 0.26 ± 0.01 | 2.37 ± 0.26 |
| Constructs . | scFv4-20 (nM) . | Vmax (FU/s) . | K0.5 (μM) . | αapp (global fit) . |
|---|---|---|---|---|
| FL-AT13 | 0 | 1.05 ± 0.09 | 1.11 ± 0.05 | 1.82 ± 0.06 |
| 0.37 | 1.14 ± 0.08 | 1.45 ± 0.10 | ||
| 1.1 | 0.98 ± 0.09 | 1.22 ± 0.03 | ||
| 3.3 | 0.88 ± 0.08 | 1.71 ± 0.13 | ||
| 10 | 0.36 ± 0.02 | 2.32 ± 0.18 | ||
| MT4 | 0 | 0.65 ± 0.02 | 0.44 ± 0.02 | 1.27 ± 0.07 |
| 0.12 | 0.67 ± 0.07 | 0.56 ± 0.10 | ||
| 0.37 | 0.59 ± 0.04 | 0.50 ± 0.02 | ||
| 1.1 | 0.34 ± 0.05 | 0.50 ± 0.08 | ||
| 3.3 | 0.11 ± 0.02 | 0.92 ± 0.35 | ||
| MDTCS | 0 | 1.42 ± 0.12 | 1.01 ± 0.08 | 1.81 ± 0.39 |
| 1.1 | 1.55 ± 0.14 | 1.39 ± 0.14 | ||
| 3.3 | 1.27 ± 0.14 | 1.26 ± 0.04 | ||
| 10 | 0.89 ±0.09 | 1.77 ± 0.35 | ||
| 30 | 0.26 ± 0.01 | 2.37 ± 0.26 |
All recombinant proteins expressed in human embryonic kidney (HEK) 293 cells. ScFv4-20 is a single-chain fragment of a variable loop that binds to the spacer domain of ADAMTS13. Reaction conditions: temperature 25°C, pH 6.0. All data are presented as mean ± SEM.
Here, FL-AT13, full-length recombinant ADAMTS13; FU, fluorescence units; MDTCS, a truncated recombinant ADAMTS13 consisting of the first 5 N-terminal domains; MT4, a truncated recombinant ADAMTS13 including MDTCS plus the first 4 TSP1-like repeats.