APC cofactor activity of FV-A2086D on APC-induced FVIIIa degradation. (A) FVIIIa inactivation. FVIII (6 nM) with PL (20 μM) was activated by thrombin (5 nM), followed by the addition of hirudin (2.5 U/mL). Generated FVIIIa was incubated either with mixtures of APC (1 nM), PS (10 nM), and FV-WT or FV-A2086D (0-2 nM) for 20 minutes. FXa generation was initiated by the addition of FIXa (2 nM) and FX (200 nM) for 1 minute. Values of FXa generation in the absence of FV were regarded as 100%. All experiments were performed at least 3 times, and the average values are shown. (B) FVIIIa inactivation and A1 cleavage at Arg³³⁶ in FVIIIa by APC. rFVIII (6 nM) with PL (20 μM) was activated by thrombin (5 nM) for 30 seconds, followed by the addition of hirudin (2.5 U/mL). Generated FVIIIa was incubated with mixtures of APC (2 nM), PS (10 nM), with or without FV-WT or FV-A2086D (2 nM) for the indicated times. (Ba) FXa generation was initiated by the addition of FIXa (2 nM) and FX (200 nM) for 1 minute. The data were fitted using an equation of single exponential decay (dashed lines). All experiments were performed at least 3 times, and the average values are shown. The rate constants (min⁻¹) obtained were FV-WT, 0.19 ± 0.02; FV-A2086D, 0.13 ± 0.05; no FV, 0.11 ± 0.04. (Bb) The same samples as in panel B were analyzed on 8% gels, followed by western blotting using an anti-A1 mAbC5 immunoglobulin G. (Bc) Band densities of intact A1^1-372 observed from panel b were measured by quantitative densitometry. The density before the addition of APC was regarded as 100%.