Quantification of plasma- and platelet-derived FV levels in an FV-deficient patient prior to and subsequent to administration of FFP. (A) Plasma-derived FV antigen and activity was measured prior to (time = 0 hours) and subsequent to FFP administration, at the times indicated, using a double antibody competitive radioimmunoassay (circles). The FV concentrations between 0 and 2 hours (circles, dashed line) were extrapolated based on the human FV turnover rate in a nonhuman primate model as described in “Study design.” Platelet-derived FV (triangles) was measured using a quantitative western blot described in “Study design” using washed platelets lysed with triton X-100 in the presence of leupeptin. Platelet lysates were treated with thrombin (2 U/mL, 10 minutes, 37°C) to convert all platelet-derived FV and its partial activation products to FVa. Following sodium dodecyl sulfate–polyacrylamide gel electrophoresis, immunoblotting was performed using a mixture of an anti-FV heavy chain and an anti-FV light chain monoclonal antibody. (B) FV in plasma was immunoblotted prior to (0 hours) and subsequent to FFP administration (6, 24, 96, and 168 hours) as described above. The position of single chain FV (SC) is indicated. C, plasma from an unaffected individual. (C) Platelet-derived FV was immunoblotted in whole platelet lysates following its conversion to FVa prior to (0 hours) and subsequent to FFP administration (6, 24, and 96 hours), as described above. The positions of the FVa heavy chain (HC) and light chain (LC) are indicated.