Inhibition of the rFVIIa activity in plasma by 283Ser-FVII.

(A) FXa generation curves produced by 2 pM rFVIIa (NovoNordisk, Bagsvaed, Denmark) in diluted (1:20) patient FVII38 plasma (FVIIa/283Ser-FVII molar ratio of 1:100) (▪), Ala294ValdelC homozygote plasma (●), or FVII-depleted plasma (○). (B) FXa generation curves produced by 2 pM rFVIIa in Ala294ValdelC homozygote diluted plasma (1:20) with the addition of conditioned medium containing 0.2 nM (■), 1 nM (▪) r283Ser-FVII, or mock medium (●). Reactions were initiated by adding limiting amount of thromboplastin, 20 nM hFX, and 100 μM FXa fluorogenic substrate, and fluorescence was measured over time. The limiting amount was determined before each assay by evaluating the FXa generation obtained with 2 pM rFVIIa concentration and decreasing the amounts of thromboplastin. The decrease in activity was estimated by comparison of FXa generation rates (relative fluorescence units per minute) between 4 and 8 minutes. Rfu indicates relative fluorescence units.