Figure 1.
The G haplotype leads to enhanced FV-short splicing and plasma levels. (A) F5 is on chromosome 1. Represented are part of intron 12 to 13 (right hand horizontal arrow), exon 13 (rectangular bar), and part of intron 13 to 14 (left hand horizontal arrow). Therefore, in this schematic, F5 is transcribed from right to left, consistent with F5 being on the reverse strand of chromosome 1, and is transcribed from higher to lower coordinates (shown above, according to the GRCh38). From top to bottom below exon 13, in red, the 4 originally described G haplotype SNVs (left to right: rs6032, rs4525, rs4524, and rs6021); in purple, the rare variants that lead to increased expression of B-domain–truncated FV, including FV-short (above are FV-Amsterdam [left] and ETBD [right]; below is the deletion in FV-Atlanta); the arrows indicate the cryptic acceptor (left) and donor (right) splice sites that lead to FV-short transcripts (coordinates from Zimowski et al24). (B) A bar graph representing the RNA-seq analysis of 4651 participants of the INTERVAL study, stratified by the G haplotype (homozygous for this, F5-G/G, on the left of the x-axis; F5-het in the middle; and homozygotes for the reference sequence on the right). On the y-axis is the proportion of participants, within each group, in which excision events at the FV-short splice sites could be detected. A statistically significant association between the presence of the G haplotype and the splice event was found using logistic regression (P = .003). (C-E) FV-short levels were analyzed by co-IP with TFPI in plasma pools created from plasma from the F5-G/G and F5-ref groups from the NIHR BioResource recall study. Plasma pools were generated from plasma obtained from all study participants (n = 7 and n = 13 for the F5-G/G and F5-ref groups, respectively). A total of 1 mL of each plasma pool was incubated with magnetic beads coated with polyclonal anti-TFPI Abs (PAHTFPI-S). After washing, proteins bound to the beads were eluted with 40 μL of LDS buffer and analyzed by western blotting for FV (AHFV-5146; prolytix) or TFPI (monoclonal anti-K1, K2, and C-terminus; Sanquin). Eluted samples were loaded at 4, 8, and 16 μL onto the gel and run alongside known amounts of recombinant FV-short (rFV-short; C) or TFPIα (rTFPIα; D). The average of the 3 elution volumes was used for quantification from each repeat of the co-IP. As expected, rTFPIα migrated to a slightly higher molecular weight than plasma TFPIα because of the known differences in glycosylation pattern.47 The results from a representative experiment are shown in panels C-D. (E) FV-short coimmunoprecipitated with TFPIα was quantified using rFV-short run on the same western blot as a standard (Bio-Rad Image Lab 6.1). This allowed derivation of a semiquantitative estimate of the amount of FV-short eluted from 1 mL of plasma. Each data point represents a technical replicate (n = 4), and the height of the bar represents the median and the error bar the IQR. ∗P < .05.

The G haplotype leads to enhanced FV-short splicing and plasma levels. (A) F5 is on chromosome 1. Represented are part of intron 12 to 13 (right hand horizontal arrow), exon 13 (rectangular bar), and part of intron 13 to 14 (left hand horizontal arrow). Therefore, in this schematic, F5 is transcribed from right to left, consistent with F5 being on the reverse strand of chromosome 1, and is transcribed from higher to lower coordinates (shown above, according to the GRCh38). From top to bottom below exon 13, in red, the 4 originally described G haplotype SNVs (left to right: rs6032, rs4525, rs4524, and rs6021); in purple, the rare variants that lead to increased expression of B-domain–truncated FV, including FV-short (above are FV-Amsterdam [left] and ETBD [right]; below is the deletion in FV-Atlanta); the arrows indicate the cryptic acceptor (left) and donor (right) splice sites that lead to FV-short transcripts (coordinates from Zimowski et al24). (B) A bar graph representing the RNA-seq analysis of 4651 participants of the INTERVAL study, stratified by the G haplotype (homozygous for this, F5-G/G, on the left of the x-axis; F5-het in the middle; and homozygotes for the reference sequence on the right). On the y-axis is the proportion of participants, within each group, in which excision events at the FV-short splice sites could be detected. A statistically significant association between the presence of the G haplotype and the splice event was found using logistic regression (P = .003). (C-E) FV-short levels were analyzed by co-IP with TFPI in plasma pools created from plasma from the F5-G/G and F5-ref groups from the NIHR BioResource recall study. Plasma pools were generated from plasma obtained from all study participants (n = 7 and n = 13 for the F5-G/G and F5-ref groups, respectively). A total of 1 mL of each plasma pool was incubated with magnetic beads coated with polyclonal anti-TFPI Abs (PAHTFPI-S). After washing, proteins bound to the beads were eluted with 40 μL of LDS buffer and analyzed by western blotting for FV (AHFV-5146; prolytix) or TFPI (monoclonal anti-K1, K2, and C-terminus; Sanquin). Eluted samples were loaded at 4, 8, and 16 μL onto the gel and run alongside known amounts of recombinant FV-short (rFV-short; C) or TFPIα (rTFPIα; D). The average of the 3 elution volumes was used for quantification from each repeat of the co-IP. As expected, rTFPIα migrated to a slightly higher molecular weight than plasma TFPIα because of the known differences in glycosylation pattern.47 The results from a representative experiment are shown in panels C-D. (E) FV-short coimmunoprecipitated with TFPIα was quantified using rFV-short run on the same western blot as a standard (Bio-Rad Image Lab 6.1). This allowed derivation of a semiquantitative estimate of the amount of FV-short eluted from 1 mL of plasma. Each data point represents a technical replicate (n = 4), and the height of the bar represents the median and the error bar the IQR. ∗P < .05.

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