FIGURE 4.
Extrinsic FXase assay (a) and stoichiometry of relipidated TF (b). a, varying concentrations of FX were incubated with FVIIa (5 nm) in complex with rTF1–243 (●), rTF1–243“D” (○), rTF1–263G (■), rTF1–263D (□), pTF (♦), and pTFD (◇) (0.1 nm). FXa generation was measured and calculated from the FXa standard curve (inset). b, the stoichiometry of relipidated TF was analyzed in the TF·FVIIa amidolytic assay. Varying concentrations of rTF1–263D (□) and pTFG (♦) were incubated with FVIIa (0.05 nm) in HBS, 0.1% polyethylene glycol, 2 mm CaCl2, pH 7.4, for 10 min. Fluorogenic substrate was added (50 μm), and the rate of substrate hydrolysis was recorded. The graph represents the calculated 1:1 stoichiometry of the TF·FVIIa complex. Each point is a mean of two experiments ± 1 S.D.