Abstract 17985: Deletion of Tissue Factor Pathway Inhibitor in Zebrafish Leads to Enhanced Thrombosis, Atherosclerosis, and Myocardial Infarction Injury
Background: The extent to which tissue factor pathway inhibitor (TFPI) may regulate the extrinsic clotting cascade, atherogenesis, and MI is uncertain. Reverse genetic studies in the mouse, including deletion of the TFPI Kunitz-1 (K1) domain, have been hindered by intrauterine lethality. We sought to determine the role of TFPI in an alternative animal model, by knocking out the zebrafish ortholog tfpia.
Methods and results: High-efficiency transcription activator-like effector nucleases (TALENs), targeting the 5’ region of tfpia, were microinjected into 1-cell zebrafish embryos. Sequence analysis of genomic DNA derived from adult caudal fin tissue revealed indels causing a frameshift mutation and premature stop codon prior to K1. Somatic mutants were outcrossed to WT fish, and the embryos were screened for germline mutations. Founders (F1) were raised and incrossed to produce embryos (F2), including approximately 25% with the desired biallelic null mutation (tfpia-/-) that were physically and developmentally indistinguishable, but functionally disparate from heterozygous (tfpia+/-) or WT (tfpia+/+) siblings. At 6 days post-fertilization (dpf), F2 tfpia-/- larvae exhibited a reduction in time to occlusion of the dorsal aorta after exposure to ferric chloride, which was more significant in 6-dpf F3 tfpia-/- larvae (generated by incrossing F2 tfpia-/- adults) lacking a possible maternal effect. Furthermore, F2 tfpia-/- fish had in vivo evidence of increased fatty streaking as late larvae, after receiving a high cholesterol diet, and enhanced cardiac injury as adults, 4 days after cryoinjury-induced MI.
Conclusions: Deletion of tfpia does not significantly effect zebrafish development, but leads to hypercoagulability in early larvae, enhanced atherogenesis in late larvae, and increased injury related to MI in adults, indicating a fundamental role for TFPI in the pathogenesis of cardiovascular disease.
- © 2013 by American Heart Association, Inc.