FV-W1920R affecting the association with PL. (A) FV-PL binding. α-Phosphatidyl-L-serine (5 μg/mL) was added to microtiter wells and air-dried. After blocking, serial dilutions of FV were added to the immobilized PL. Bound FV-WT (open circles) and FV-W1920R (closed circles) was quantified by the addition of AHV-5146, as described in “Materials and methods.” The average values and standard deviations are shown. (B) The effects of PL on prothrombinase activity; FV (WT, open circles; W1920R, closed circles; 2 nM) was activated by thrombin (20 nM) for 1 minute, followed by the addition of hirudin. The reactants were incubated with prothrombin (1.4 μM) and various amounts of PL, followed by initiation by the addition of FXa (10 pM). Aliquots were removed, and the reactions were quenched by the addition of EDTA. Rates of thrombin generation were determined at Abs₄₀₅ after the addition of S-2238. Thrombin generation was quantified by extrapolation from a standard curve prepared using known amounts of thrombin. The plotted data were fitted using the Michaelis-Menten equation. All experiments were performed at least 3 separate times, and the average values are shown.