Pkcα and Pkcβ are Positive Regulators of Thrombus Formation in vivo in a Zebrafish (Danio Rerio) Model of Thrombosis

Background: The zebrafish (Danio rerio) is becoming an attractive model organism for 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 by intravital microscopy, without the need for complex surgical techniques, and the capability to rapidly knockdown gene expression using morpholino antisense approaches. Objectives: We have recently shown, in mouse models, that protein kinase PKCα plays a critical role regulating thrombus formation in vivo. PKCβ plays a non-redundant role also in platelet function in vitro, but the function of this gene had not yet been assessed in vivo. Methods: Here we have analysed 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. Results: We show that D. rerio express orthologues of both the PRKCA and PRKCB genes, with high sequence identity. Translation blocking and splice-blocking morpholinos effectively and specifically knockdown expression of these genes and either knockdown leads to attenuated thrombus formation, as assessed by several quantitative parameters including time to initial adhesion and peak thrombus surface area. Conclusions: 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.