APC resistance testing of recombinant FV variants.
(A) 14 nM FV was diluted 1:5 in FV-depleted plasma. Equal amounts of plasma and APTT reagent were mixed and incubated for 5 minutes. Clotting was started by addition of CaCl2 with or without APC. A ratio was calculated between the clotting time in the presence and absence of APC. (B) FV was activated with RVV-V (final concentration, 0.5 μg/mL) at 37°C for 45 minutes and diluted in FV-deficient plasma to a concentration of 1.25 nM. APC resistance test was performed as described above. (C) Correction of APC resistance by recombinant FV. Plasma (12 μL) from a person with homozygosity for FV Leiden was mixed with 36 μL FV-deficient plasma and 12 μL recombinant FV (14 nM). As a negative control, 12 μL TBS with 0.2% BSA was used instead of the FV. APC resistance tests were performed as described above.
