Effects of vitamin K deficiency and its mechanisms in vertebrate's early development: zebrafish as a model

Disertação de mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Vitamin K (VK) acts as a cofactor of the enzyme γ-glutamyl carboxylase (Ggcx) promoting the γ-carboxylation of VK dependent proteins (VKDP), where a posttranslational conversion of Glu into Gla residues is achieved, providing calcium binding properties to VKDPs. Some VKDPs are involved in blood clotting but also in other processes. During γ-carboxylation, the reduced VK is converted to the epoxide form, which is then recycled back into VK by the enzyme VK epoxide reductase (Vkor). Warfarin inhibits Vkor activity, being used to control blood clotting through the reduction of the γ-carboxylation of coagulation factors in patients who are at risk of venous thromboembolism. However, it might affect the activity of other VKDP. When administered during pregnancy, it induces fetal morbidity and mortality in pregnant women, being this associated with bleeding disorders and skeletal deformities. In this work the effects of VK deficiency in vertebrate’s early development were evaluated using zebrafish (Danio rerio) as a model. Zebrafish (embryos and larvae) were exposed to increasing levels of warfarin (0, 5, 25 and 125 mg L-1) during two critical periods: 0-2.5 (embryonic development) and 2.5-5 (endotrophic larvae) days post fertilization. Larvae exposed to high concentrations of warfarin showed growth retardation, bleeding, underdeveloped swimbladder, pericardial inflammation, disruption of skeletogenesis, and increased mortality. The effects were much more severe when they were exposed to the anticoagulant during the embryonic stage. Regarding skeletogenesis, mineralization of several cranial structures as well as the axial skeleton was reduced. The length of the endochodral structures in the cranial region was also shorter in warfarin exposed larvae. Expression of genes involved in skeletogenesis (sox9, runx2, osx, col2a1, grp1, alp, mgp and bgp) was affected depending on the developmental stage analyzed. Present work brings new insights on the particular mechanisms by which warfarin (and thus, VK deficiency) affects chondrogenesis and osteoblastogenesis.