Targeted mutagenesis of zebrafish antithrombin III triggers disseminated intravascular coagulation and thrombosis, revealing insight into function.
Pathologic blood clotting is a leading cause of morbidity and mortality in the developed world, underlying deep vein thrombosis, myocardial infarction, and stroke. Genetic predisposition to thrombosis is still poorly understood, and we hypothesize that there are many additional risk alleles and modifying factors remaining to be discovered. Mammalian models have contributed to our understanding of thrombosis, but are low throughput and costly. We have turned to the zebrafish, a tool for high-throughput genetic analysis. Using zinc finger nucleases, we show that disruption of the zebrafish antithrombin III (at3) locus results in spontaneous venous thrombosis in larvae. Although homozygous mutants survive into early adulthood, they eventually succumb to massive intracardiac thrombosis. Characterization of null fish revealed disseminated intravascular coagulation in larvae secondary to unopposed thrombin activity and fibrinogen consumption, which could be rescued by both human and zebrafish at3 complementary DNAs. Mutation of the human AT3-reactive center loop abolished the ability to rescue, but the heparin-binding site was dispensable. These results demonstrate overall conservation of AT3 function in zebrafish, but reveal developmental variances in the ability to tolerate excessive clot formation. The accessibility of early zebrafish development will provide unique methods for dissection of the underlying mechanisms of thrombosis.
Department of Pediatrics and.
Yang
http://orcid.org/0000-0002-0413-5637
Y
Liu
Life Sciences Institute, University of Michigan, Ann Arbor, MI;
Colin A
http://orcid.org/0000-0001-7979-7603
CA
Kretz
Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA;
Morgan L
ML
Maeder
Department of Pediatrics and.
Catherine E
CE
Richter
Department of Pediatrics and.
Philip
P
Tsao
Department of Pediatrics and.
Andy H
AH
Vo
Department of Pediatrics and.
Michael C
MC
Huarng
Department of Pediatrics and.
Thomas
T
Rode
Department of Pediatrics and.
Zhilian
Z
Hu
Department of Pathology, University of Michigan, Ann Arbor, MI;
Rohit
R
Mehra
Center for Molecular Biology of Oral Diseases, University of Illinois, Chicago, IL;
Steven T
ST
Olson
Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA; Department of Pathology, Harvard Medical School, Boston, MA; and.
J Keith
JK
Joung
Department of Pediatrics and Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, MI.
Jordan A
http://orcid.org/0000-0002-2874-4904
JA
Shavit
D000818:Animals; D030801:Animals, Genetically Modified; D000990:Antithrombin III / Q000235:genetics*; D020152:Antithrombin III Deficiency / Q000235:genetics*; D004195:Disease Models, Animal; D004211:Disseminated Intravascular Coagulation / Q000235:genetics*; D006801:Humans; D017403:In Situ Hybridization; D016297:Mutagenesis, Site-Directed; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000235:genetics*
D029961:Zebrafish Proteins; D000990:Antithrombin III