(A–D) A pool of plasma from affected family members was subjected to either FV or TFPI depletion to analyze whether TFPI was specifically associated with FV-short. (A and B) Plasma (300 μl) was incubated with 900 μl Streptavidin-coated magnetic beads carrying biotinylated polyclonal antibodies against TFPI (AHTFPI-S). After 2 hours at 11°C, the mixture was centrifuged, the supernatant diluted 1:40 in sample preparation buffer, and 10 μl added for Western blot analysis using a monoclonal antibody against FV (A, AHV-5146) or an antibody against TFPI (B, ATFPI-5138 against Kunitz 1 domain). The beads were washed once in HNSBSA and bound TFPI eluted with 300 μl sample preparation buffer; 10 μl of a 1/20 dilution was loaded and analyzed with antibodies against FV (A) and TFPI (B). Aff, affected; sup, supernatant, ip, immunoprecipitate (C and D) Affected plasma (1 ml) was either loaded to a 1-ml HiTrap column with coupled MK30 against the FV B domain or a 1-ml HiTrap column with coupled polyclonal anti-FV IgG (#8806) (αFV). After 30 minutes incubation, the plasma was eluted and analyzed by Western blotting for FV (C) or TFPI (D). The lanes in C and D were run on the same gel but were noncontiguous. (E and F) Unaffected plasma (5 ml) was immunoprecipitated with 1 ml anti-TFPI magnetic beads, essentially as in A and B. The starting plasma and the supernatant (10 μl of 1/10 dilution) were analyzed with antibodies against FV (E, AHV-5146) or TFPI (F, ATFPI-5138). The beads were washed and eluted with 100 μl sample preparation buffer, and 10 μl undiluted sample was analyzed for FV (E) and TFPI (F).