Figure 3.
Figure 3. APC cleavage at Arg506. Recombinant FV variants (0.8 nM) were incubated with thrombin (0.5 U/mL) for 10 minutes at 37°C, and hirudin (final concentration, 1 U/mL) was then added to inhibit the thrombin. APC was subsequently added together with 10:20:70 PS/PE/PC phospholipids (final concentration, 25 μM). Subsamples were withdrawn at different time points, and FVa activity was measured in the PTase assay in the presence of 1 nM FXa, 0.5 μM prothrombin, and 50 μM 10:90 PS/PE phospholipids. The values were normalized to the value of FVa activity at time zero for each reaction. (A) FVa-Arg306Gln/Arg679Gln, □; FVa-Arg306Gln/Ile359Thr/Arg679Gln, ▴. Final APC concentration was 0.025 nM. (B) FVa-WT, □; FVa-Ile359Thr, ▴. Final APC concentration was 0.033 nM. Each data point represents the mean of 3 independent experiments performed in duplicate. Error bars represent ± SD.

APC cleavage at Arg506. Recombinant FV variants (0.8 nM) were incubated with thrombin (0.5 U/mL) for 10 minutes at 37°C, and hirudin (final concentration, 1 U/mL) was then added to inhibit the thrombin. APC was subsequently added together with 10:20:70 PS/PE/PC phospholipids (final concentration, 25 μM). Subsamples were withdrawn at different time points, and FVa activity was measured in the PTase assay in the presence of 1 nM FXa, 0.5 μM prothrombin, and 50 μM 10:90 PS/PE phospholipids. The values were normalized to the value of FVa activity at time zero for each reaction. (A) FVa-Arg306Gln/Arg679Gln, □; FVa-Arg306Gln/Ile359Thr/Arg679Gln, ▴. Final APC concentration was 0.025 nM. (B) FVa-WT, □; FVa-Ile359Thr, ▴. Final APC concentration was 0.033 nM. Each data point represents the mean of 3 independent experiments performed in duplicate. Error bars represent ± SD.

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