TABLE 2.
Prothrombin titration in prothrombinase assay yielding Km values
The FVa variants were incubated for 4 min with increasing concentrations of prothrombin (2.5-5000 nm) and 5/95 PS:PC phospholipid vesicles (25 μm) at 37 °C. The FVa variants were used at 200 pm final concentrations. Thrombin generation was started by addition of FXa (5 nm final concentration). After 1 min, the reactions were stopped with EDTA, and the generated thrombin determined as described under “Experimental Procedures.” None of the Km values determined for the various FVa variants were significantly different from the WT FVa.
| Factor V variant | Mean ± S.D. | na |
|---|---|---|
| WT | 206 ± 23 | 14 |
| 316A/318A/400A | 229 ± 34 | 3 |
| 320A/321A/323A | 210 ± 29 | 3 |
| 386A/652A/655A | 205 ± 33 | 3 |
| 461A/467A/499A | 200 ± 48 | 3 |
| 501A | 193 ± 18 | 3 |
| 510A | 187 ± 15 | 3 |
| 511D | 190 ± 30 | 3 |
| 501A/510A/511D | 244 ± 5 | 3 |
| 513A | 224 ± 20 | 3 |
| 513A/577A/578A | 199 ± 29 | 3 |
| 577A/578A | 181 ± 40 | 3 |
| 501A/510A/511D/513A | 196 ± 8 | 3 |
| 501A/510A/511D/513A/577A/578A | 181 ± 12 | 4 |
| 1551A/1650A/1683E | 202 ± 15 | 4 |
| 1650A/1665A | 223 ± 36 | 3 |
a
n, number of determinations.