Figure 7.

In vivo functional evaluation identifies causative variants for human F10 deficiency. (A) Schematic diagram of the human F10 domain structure and position of known and suspected F10 variants associated with bleeding. (B) Multiple sequence alignment of peptides containing human F10 variants shows conservation across vertebrate species. Protein sequences are from human (Homo; NP_000495.1), mouse (Mus; NP_001229297.1), chicken (Gallus; NP_990353.1), and zebrafish (Danio; NP_958870.2). The altered residues are marked by arrows. (C) Human F10 variants were engineered into the orthologous positions of the zebrafish f10 cDNA and placed under the control of the ubi promoter. The expression vectors were injected into the cytoplasm of 1-cell–stage offspring from f10^+/− incrosses. The endothelium of the PCV was injured by laser ablation at 3 dpf, and the time to complete occlusion recorded, after which genotyping was performed. Numbering represents the human amino acid positions. The first 2 variants tested were G262D and C390F, both of which are known to cause clinically significant human F10 deficiency. The subsequent variants, R68C, G173W, ∆T176_Q186, I323M, Q416L, were identified in patients with F10 deficiency and clinically significant bleeding, but are not yet proven to be causative. Although I323M and Q416L showed a trend toward occlusion, none of the variants examined could significantly rescue the hemostatic defect of mutants (P < .001 by Mann-Whitney U test). Horizontal bars represent the median time to occlusion. n ≥ 18 for each variant tested. AP, activation peptide; EGF-1/2, epidermal growth factor-like domains 1/2; Gla, Gla domain; PP, propeptide.