Hemostasis is a defense mechanism which protects the organism in the event of injury to stop bleeding. Recently, we established that all the known major mammalian hemostatic factors are conserved in early vertebrates. However, since their highly vascularized gills experience high blood pressure and are exposed to the environment, even very small injuries could be fatal to fish. Since trypsins are forerunners for coagulation proteases and are expressed by many extrapancreatic cells such as endothelial cells and epithelial cells, we hypothesized that trypsin or trypsin-like proteases from gill epithelial cells may protect these animals from gill bleeding following injuries. In this paper we identified the release of three different trypsins from fish gills into water under stress or injury, which have tenfold greater serine protease activity compared to bovine trypsin. We found that these trypsins activate the thrombocytes and protect the fish from gill bleeding. We found 27 protease-activated receptors (PARs) by analyzing zebrafish genome and classified them into five groups, based on tethering peptides, and two families, PAR1 and PAR2, based on homologies. We also found a canonical member of PAR2 family, PAR2-21A which is activated more readily by trypsin, and PAR2-21A tethering peptide stops gill bleeding just as trypsin. This finding provides evidence that trypsin cleaves a PAR2 member on thrombocyte surface. In conclusion, we believe that the gills are evolutionarily selected to produce trypsin to activate PAR2 on thrombocyte surface and protect the gills from bleeding. We also speculate that trypsin may also protect the fish from bleeding from other body injuries due to quick contact with the thrombocytes. Thus, this finding provides evidence for the role of trypsins in primary hemostasis in early vertebrates.

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.

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