Diagram of the novel splicing event, sequencing results of the F5 gene, and direct interaction between FV Amsterdam and TFPI. (A) cDNA sequencing from the polymerase chain reaction product of the proband revealed a sequence that is not concordant with the reference transcript from National Center for Biotechnology Information (NM_000130.4). The F5 mutation created a novel donor site at the coding position 2588, which together with an aberrant acceptor site at position 4457 of exon 13 (same as reported for the East Texas bleeding disorder) leads to a shorter F5 transcript. The new sequence is missing 1869 nucleotides in exon 13 (c.2588_4456del) that results in an in-frame loss of 623 amino acids. Diagram of the gene is not to scale. Adapted from Vincent et al.2 (B) Sequencing results of the new transcript found in the proband. The novel donor splice site is indicated by a backslash. (C) Plasma of individuals was incubated with anti-TFPI-coated beads. Bound proteins were eluted, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotted with antibodies against FV. Variant forms of FV can be visualized as indicated. NP, normal plasma.
