Protein kinase C alpha and beta are positive regulators of thrombus formation in vivo in a zebrafish (Danio rerio) model of thrombosis

Abstract

The zebrafish (Danio rerio) is becoming an attractive model organism for the assessment of gene function in thrombosis in vivo. Zebrafish, as a thrombosis model, have several advantages, with the capacity to follow thrombus formation at high resolution in real time using intravital microscopy, without the need for complex surgical techniques, and the capability to rapidly knockdown gene expression using morpholino antisense approaches. We have recently shown, in mouse models, that protein kinase C alpha (PKCα) plays a critical role in regulating thrombus formation in vivo. PKC beta (β) plays a non-redundant role also in platelet function in vitro, but the function of this gene had not yet been assessed in vivo. In the present study, we analyzed the function of both PKCα and PKCβ in the zebrafish model in vivo, by live imaging using a laser-induced injury of the main caudal artery in 3-day-old larvae. We showed that D. rerio express orthologs of both the PKCα and PKCβ genes, with high sequence identity. Translation blocking and splice-blocking morpholinos effectively and specifically knockdown expression of these genes and knockdown with either morpholino leads to attenuated thrombus formation, as assessed by several quantitative parameters including time to initial adhesion and peak thrombus surface area. Our data indicate that these two highly related genes play non-redundant roles in regulating thrombosis, an observation that supports our previous in vitro murine data, and suggests unique roles, and possibly unique regulation, for PKCα and PKCβ in controlling platelet function in vivo.