6 research outputs found
FVIII splice isoforms.
<p><b>a: Variants identified by ExonMine</b>: Variants 1 and 2 correspond to major and minor RefSeq isoforms; variants 3–5 to expressed sequence tag (EST) sequences deposited in Genbank. Note none of the alternate variants encode the FVIII mAb epitopes. <b>b: Variants identified in EC</b>. Simultaneous expression of variants 1–4 in HPAEC (PA), HPMEC (PM), and HUVEC (H). Gels: φx, HaeIII-digested φx marker, C<sup>A</sup> negative water control for HPAEC/HUVEC, C<sup>M</sup> negative water control for HPMEC. The apparent difference in size of variant 4 is an artefact due to gel running (note differential site of 194 marker band in first and last lanes). Cartoons: Thin and thick arrows indicate sites of PCR and sequencing oligonucleotide primers respectively. Sequence chromatograms were obtained using nested reverse internal primers in exon 23 (variants 1–3) or exon 3 (variant 4; low concentration first round product sequenced). Note V5 sequences (exons U1-1-2-3) were not amplified from EC in any reaction.</p
Lung expression of FVIII.
<p>Serial sections of frozen normal human lung tissue from two donor blocks (a and b) stained with <b>i)</b> control IgG<sub>1</sub>, <b>ii</b>) anti-CD31, or <b>iii</b>) anti-FVIII (C5). The 200x images are representative of data from all five donors.</p
Immunofluorescence images of FVIII expression by EC.
<p><b>a</b>: Sequential confocal fluorescence microscopy images in primary human EC. <b>a</b> and <b>b</b>: Representative HPMEC and HPAEC control images using To-Pro-3 nuclear counterstain (first panel, monochrome), murine control IgG<sub>1</sub> (second panel monochrome) and merged images (third panel: To-Pro-3 nuclear counterstain white, control IgG<sub>1</sub> red) using maximum gain used for imaging. <b>c</b>) Comparison of proportion of <b>i</b>) <b>HPMEC</b> and <b>ii</b>) <b>HPAEC</b> expressing vWF and FVIII protein (expression levels defined in methods). <b>d, e, f, g</b>) Sequential confocal fluorescence microscopy images comparing vWF (monochrome in first panel); FVIII (monochrome in second panel, specific mAb as denoted), and merged images (third panel; vWF green, anti-FVIII reacting protein red) in <b>d</b>): HPMEC, and <b>e, f, g</b>): HPAEC. FVIII mAb images displayed here are representative of all FVIII mAbs examined, and all cell lots. Note yellow merged images suggesting degree of FVIII/vWF colocalisation in <b>e, f, g</b>, with white colouring denoting the nuclei (TO-PRO3 nuclear counterstain). Scale bars indicate 5 µm.</p
Secreted and cell surface FVIII.
<p><b>a</b>) <b>Quantification of FVIII on the surface of EC.</b> Relative fluorescence intensity (RFI) of confluent HUVEC, HPAEC and HPMEC stained with FVIII mAb C2, compared to EC from the same well in which C2 was omitted. <b>b</b>)<b> Quantification of HPAEC surface FVIII: bi</b> Comparison of EC RFI for HPAEC from the same well treated with IgG<sub>1</sub> control, or C2 mAb; p values calculated by Mann Whitney. <b>bii-iii</b>. Representative raw data plots of log expression (FL1 log) versus forward scatter as a marker of cell size for HPAEC from the same well treated with <b>bii</b>) IgG<sub>1</sub> control, <b>biii</b>) C2 mAb. <b>c</b>)<b> Quantification of HPAEC surface vWF. ci</b>) Comparison of EC RFI for HPAEC from the same well treated with IgG<sub>1</sub> control, or vWF pAb, p values calculated by Mann Whitney. <b>cii-iii</b>. Representative raw data plots of log expression (FL1 log) versus forward scatter as a marker of cell size for HPAEC from the same well treated with <b>cii</b>) IgG<sub>1</sub> control, <b>ciii</b>) vWF pAb. <b>d</b>) <b>Quantification of FVIII:Ag in control and EC-conditioned media by ELISA</b>. M<sub>1</sub> denotes control EC media (2% FCS), M<sub>2</sub> denotes control MEC media (5% FCS). M<sub>1</sub> control data are presented twice for clarity. <i>P</i> values are presented for the 48 hour data sets of EC from passages 4 and 5. Differences at 24 hours did not reach statistical significance. <b>e</b>)<b> Manual FVIII:c assay standard curve</b>. Ln FVIII, logarithm of FVIIIc activity (U/ml). Note clotting time in samples exceeded 240 seconds.</p
Evolutionary conservation of alternatively spliced exons.
<p><b>a–d</b>)<b>:</b> Sequence conservation on the UCSC Genome Browser for sequences flanking <b>a</b>) exon 22 encoding part of the FVIII light chain; <b>b</b>) exon 1, <b>c</b>) alternate first exon U1, and <b>d</b>) alternate first exons 22A and 22B. Note that the FVIII gene is on the complementary strand, hence 5′ to 3′ is represented right to left. Dotted lines above the exons represent genomic DNA distances; red boxes denote exact sites of exons. <b>e,f</b>)<b>:</b> Sequence conservation spanning <i>int22h</i> repeats in <b>e</b>) intron 22 (<i>int22h-1</i>, note positions of exons 22A and 22B), and <b>f</b>) telomeric X chromosome repeats <i>int22h-2</i> and <i>int22h-3</i>.</p
FVIII genomic and domain structure.
<p>FVIII undergoes a complex series of steps between primary transcript synthesis and eventual activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009154#pone.0009154-Pipe1" target="_blank">[20]</a>. The 9 kb ‘full length’ 26 exon primary mRNA transcript is translated to a 360 kDa polypeptide chain which is translocated to the ER and Golgi for post-translational processing including B domain proteolysis to generate the mature heavy and light chains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009154#pone.0009154-Becker1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009154#pone.0009154-Pipe2" target="_blank">[22]</a>. Bars indicate the epitope sites for mAbs C2, C5, C6 and C8 which react with the 360 kDa precursor, and the heavy (C2, C6, C8) or light chains (C5) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009154#pone.0009154-Rotblat1" target="_blank">[23]</a>. Dotted lines indicate the sites of previously described transcriptional silencing regions (see text).</p