Analysis of factor V in zebrafish demonstrates minimal levels needed for hemostasis and risk stratifies human variants

Abstract

Text word count: 3731 Abstract word count: 246 Number of figures and tables: 7 (2 tables, 5 figures) Number of references:57 Scientific category: Thrombosis and Hemostasis Running Title: Analysis of human and zebrafish factor V 2 Key Points F5 mutant fish embryos tolerate symptoms lethal in mammals but succumb to bleeding in adulthood  Analysis of human variants demonstrate that all have some residual function and that minimal F5 activity is required to restore hemostasis ABSTRACTIn humans, coagulation factor V (F5) deficiency is a rare, clinically heterogeneous bleeding disorder, suggesting that genetic modifiers may contribute to disease expressivity. Complete loss of mouse F5 results in early lethality. Zebrafish possess many distinct advantages including high fecundity, optical clarity, external development, and homology with the mammalian hemostatic system, features that make it ideal for genetic studies. Our aim was to study the role of F5 in zebrafish through targeted mutagenesis, and apply the model to the study of human F5 variants.CRISPR-mediated genome editing of the zebrafish f5 locus was performed, generating mutants homozygous for a 49 base pair deletion in exon 4. Thrombus formation secondary to vascular endothelial injury was absent in f5 -/mutant embryos and larvae. Despite this severe hemostatic defect, homozygous mutants survived before succumbing to severe hemorrhage in adulthood.Human F5 variants of uncertain significance from patients with F5 deficiency were evaluated, and the causative mutations identified and stratified by their ability to restore thrombus formation in larvae. Analysis of these novel mutations demonstrates variable residual F5 function, with minimal activity being required to restore hemostasis. This in vivo evaluation may be beneficial for patients whose factor activity levels lack correlation with bleeding symptomatology. Furthermore, homozygous mutant embryos tolerate what is a severe and lethal defect in mammals, suggesting the possibility of species-specific factors enabling survival, and allowing further study not possible in the mouse. Identification of these factors or other genetic modifiers could lead to novel therapeutic modalities.

Disruption of the kringle 1 domain of prothrombin leads to late onset mortality in zebrafish

“…In contrast to mammals, in which a number of coagulation factors are necessary for embryonic and/or neonatal viability, zebrafish are able to survive the loss of many aspects of the canonical cascade at least until early adulthood. Loss of antithrombin, fibrinogen, FV, and FX are all compatible with development to adulthood 22,[26][27][28]37 . Targeting of f10 in fish results in the absence of larval hemostasis, but is accompanied by extended survival 28 .…”

Disruption of the kringle 1 domain of prothrombin leads to late onset mortality in zebrafish

“…In contrast to mammals, in which a number of coagulation factors are necessary for embryonic and/or neonatal viability, zebrafish are able to survive the loss of many aspects of the canonical cascade at least until early adulthood. Loss of antithrombin, fibrinogen, FV, and FX are all compatible with development to adulthood 22,[26][27][28]45 . Targeting of f10 in fish results in the absence of larval hemostasis, but is accompanied by extended survival 28 .…”

“…demonstrates a resistance to severe coagulopathies (loss of antithrombin, prothrombin, FV, FX)22,28,45 that cause embryonic and/or neonatal lethality in mammals. This may be due to a combination of influences, including the absence of birthing trauma, limited hemostatic challenges in an aqueous laboratory environment, a relatively low systolic blood pressure, and possible species-specific genetic differences.…”