APC-mediated inactivation of FV 306 variants.

FV (final concentration, 0.8 nM) was incubated with 0.5 U/mL thrombin for 10 minutes at 37°C. APC (final concentration, 0.2 nM) was added to the reaction mixtures, which also contained phospholipids (PS/PE/PC wt/wt/wt 10/20/70) at a final concentration of 25 μM. Experiments were performed in the absence (open symbols) and presence (closed symbols) of 100 nM protein S. At intervals, samples were drawn, and the FVa degradation was stopped by one fifth dilution in ice-cold HNBSACa. FVa activity was measured with the PTase assay. FVa activity was related to the activity observed before the addition of APC. (A) WT; (B) 306G; (C) 306T; (D) 306Q. The following final FVa concentrations in U/mL (mean ± SD) were used: WT, 0.04 ± 0.005; 306G, 0.042 ± 0.004; 306T, 0.033 ± 0.002; and 306Q, 0.039 ± 0.01. Plotted values represent the mean of 3 individual experiments; error bars represent SD.