Zebrafish von Willebrand factor

Abstract

von Willebrand factor (vWF) is a large protein involved in primary hemostasis. A dysfunction in this protein or an insufficient production of the protein leads to improper platelet adhesion/aggregation, resulting in a bleeding phenotype known as von Willebrand disease (vWD). To gain a better understanding of vWF interactions in vivo, the use of zebrafish as a model is ideal because of the transparency of the embryos and larvae. In this article, we examined the presence and function of vWF in hemostasis of zebrafish utilizing a variety of molecular methods. Using RT-PCR and antibody staining, we have shown that vWF mRNA is present in thrombocytes. Through antibody staining, we demonstrated vWF is synthesized in blood vessels. The role of zebrafish vWF in hemostasis was established through knockdown methods using vWF morpholino (vWF MO) antisense oligonucleotides. Embryos injected with vWF MO at the one to four cell stages resulted in a bleeding phenotype. Injection of embryos with vWF MO also caused an increase in time to occlusion within arteries in larvae upon laser induced injury. We then used vWF-specific Vivo-morpholinos (VMO) to induce vWF knockdown in adult zebrafish by targeting the exon homologous to the human exon 28 of the vWF gene. The reduced ristocetin-mediated agglutination of thrombocytes in a plate tilting assay, using blood from adult zebrafish injected with VMO, provided evidence that vWF is involved in the hemostatic process. We also administered desmopressin acetate to larvae and adults which resulted in enhanced aggregation/agglutination of thrombocytes. Zebrafish genome database analysis revealed the presence of GPIbβ gene. It also revealed the exon of zebrafish vWF gene corresponding to exon 28 of human vWF gene is highly similar to the exon 28 of human vWF gene, except that it has an insertion that leads to a translated peptide sequence that separates the two A domains coded by this exon. This exon is also conserved in other fishes. In summary, we established that zebrafish vWF has a role similar to that of vWF found in humans, thus, making zebrafish a useful model for studying the cell biology of vWF in vivo.

Figure 1

Amino acid alignment of vWF protein sequence corresponding to human exon 28. Arrow head points to the region where exon 28 of humans is split in all fishes with the exception of zebrafish. Blue arrow points the regions where exon 28 in humans is split in Stickleback and Medaka. Red arrow points the regions where exon 28 in humans is split in Zebrafish (ENSEMBL database).

Figure 2

Schematic diagram of the RT-PCR amplified cDNA products from exon 26 through exon 29 using RNA collected from zebrafish larvae and PCR products amplified using zebrafish genomic DNA, respectively. Exons are shown as black boxes with the numbering on top corresponding to the human exon numbering. The arrow points to the location of the ‘intron’ according to ENSEMBL database. Forward primers are numbered F1 to F5 whereas the reverse primers are numbered R1 to R4 and are shown beneath the exons. RT-PCR product sizes are shown by lines flanked by solid circles (not drawn to scale). Solid circles represent roughly the primers locations within the exons; dashed lines show the introns removed by splicing. The forward and reverse primer combination is shown in parenthesis followed by the size of the product in base pairs. The agarose gel photographs show the amplified products corresponding to those shown in the schematic diagram. Lanes of the amplified products are marked with the combination of primers; M shows the DNA size markers. Sizes of the amplified products in base pairs are shown by arrows. The cDNA products generated by RT-PCR and the genomic products generated by PCR are marked separately.

Figure 3

Immunostaining of blood vessels. Human von Willebrand factor antibody (vWF-Ab) and rabbit IgG (Control) were used as primary antibodies followed by FITC conjugated secondary antibody in immunostaining. Left and right panels show the brightfield and fluorescence images respectively. Large arrows show caudal artery (upper region) and caudal vein (lower region) whereas the small arrow shows the intersegmental vessels.

Figure 4

Immunostaining of thrombocytes. Human von Willebrand factor antibody (vWF-Ab) and rabbit IgG (Control) were used as primary antibodies followed by FITC conjugated secondary antibody. Left and right panels show the brightfield and fluorescent images respectively. Arrows point to the thrombocytes, all other surrounding cells are red cells.

Figure 5

Larvae generated after injecting vWF MO (bottom panels) and control MO (top panels) into the 1-4 cell-stages of embryos. Arrows indicate the location of bleeding in the head and yolk region.

Figure 6

TTO in 6 dpf zebrafish larvae. TTO using larvae generated after injecting vWF MO and control MO into 1-4 cell-stages of embryos, n=14, p=<0.001 (left panel). TTO using larvae treated with Stimate and untreated controls, n=7, p=<0.001 (right panel).

Figure 7

Analysis of splicing of vWF mRNA after treating the embryos with vWF MO. A and B show representative RT-PCR products from RNA collected from 24-72 hrs post fertilization embryos injected with control MO and vWF MO at 1-4 cell-stages of embryos respectively. Arrows show the size of the RT-PCR products in base pairs (332 for vWF and 220 for EF1-α) on agarose gel photographs. Left most lanes show the DNA size markers. The bar graph shows the 220 bp band/332 bp band densities, n=30, p=<0.001.

Figure 8

Ristocetin mediated thrombocyte agglutination assay. Top panels show thrombocyte aggregation using whole blood from control MO and vWF MO injected adults. Bottom panels show thrombocyte aggregation using whole blood from Stimate treated and untreated control adult zebrafish.

Figure 9

Amino acid alignment of human and zebrafish GP1bβ