Zebrafish von Willebrand factor

Abstract

Abstractvon 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 gen 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.

Targeted mutagenesis of zebrafish antithrombin III triggers disseminated intravascular coagulation and thrombosis, revealing insight into function

“…[9][10][11][12] Zebrafish (Danio rerio) have been widely used to study hemostasis, demonstrating conservation and function of the structural components of coagulation, including fibrinogen 13,14 and von Willebrand factor, 15,16 and the ability to develop thrombosis in response to a laser-induced injury. 14,17 Embryonic development is external, rapid, and transparent, allowing unfettered access to studies of the circulatory system.…”

Simple and Rapid Quantification of Thrombocytes in Zebrafish Larvae

“…10 Orthologs for many mammalian platelet proteins, such as VWF and CD41, are also present. [11][12][13] Transgenic technology has allowed the development of zebrafish with fluorescently tagged cell populations, including a thrombocyte line. This strain expresses green fluorescent protein (GFP) under regulation of the cd41 promoter, and it has been used for multiple studies that have led to novel insights into platelet function.…”

Animal Models of Thrombosis From Zebrafish to Nonhuman Primates

“…145 Similarly, the vascular endothelium in zebrafish possesses several factors found in human endothelial cells. 146 Therefore, the overall machinery responsible for hemostasis and thrombus formation seems to have evolved in earlier vertebrates and seems to be conserved throughout evolution. This conservation is especially important in identifying novel factors in thrombosis so that factors identified in zebrafish can be studied in murine and nonmurine species and eventually be translated into targets for antithrombotic drugs.…”

“…155 Similarly, the Vivo-Morpholino knockdown method was introduced to knockdown different genes, such as vWF and factor VII in adult zebrafish. 146,156 Recently, the zinc finger nuclease knockout method was used to mutate the antithrombin III gene, which modeled disseminated intravascular coagulation in zebrafish. 157 With all these available tools, it should be possible to discover more novel factors that participate in thrombosis.…”

“…157,162 The recent introduction of the 146 Gregory et al, 147 Hughes et al, 154 Day et al, 155 Khandekar and Jagadeeswaran, 156 Tournoij et al, 158 Williams et al, 159 and O'Connor et al 161 …”