Biology subjects

Richter, C. E.

Publications and source records attributed to Richter, C. E..

3 recordsLinked to original sources

bioRxiv2026

A sensitized model of thrombosis validates known multigenic relationships and suggests novel modifiers of hemostasis

BackgroundVenous thromboembolism is a major cause of morbidity and mortality. Despite identification of risk factors, not all individuals with thrombophilia develop thrombosis. Understanding the multigenic factors modifying this incomplete penetrance would help guide patient care. MethodsThe zebrafish has a conserved hemostatic system and is amenable to large genetic studies. Loss of antithrombin III (At3) in zebrafish leads to an early consumptive coagulopathy and lethality in adulthood. Using this genetic background as a sensitized model we performed a dominant unbiased genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen followed by whole genome sequencing (WGS). We used survival studies, laser-mediated endothelial injury, and ex vivo protein assays to validate hits. ResultsENU-treated at3+/- males were crossed with at3+/- females to produce 4,030 total offspring (1.5x genome coverage). Four permanent lines transmitting a survival benefit beyond 7 months were identified and sequenced. A candidate screen of 63 known coagulation-related loci revealed a missense mutation, C504F, in a highly conserved residue of the prothrombin (F2) heavy chain, which was validated through genetic and biochemical studies. Evaluation of UK Biobank electronic health record (EHR) data was underpowered to detect interactions between F2 and AT3 due to minmal deleterious mutations. Mutations produced through genome editing revealed that heterozygosity for factor X and plasminogen also modified at3-/-, resulting in reduced lethality. The three remaining lines had no coagulation-related variants segregating with survival, suggesting the presence of novel modifier loci. ConclusionsUnbiased genome-wide screening identified a modifier of thrombosis. This demonstrated that re-balancing of hemostasis to mitigate thrombosis is conserved in zebrafish, including an unexpected role for fibrinolysis. This interaction was not detected even in a large human dataset, establishing the continued benefit of the zebrafish model. Finally, we found evidence for novel loci outside of the canonical coagulation cascade that may be new targets for diagnosis or treatment.

cell biology↗

bioRxiv2024

Loss of factor VIII in zebrafish rebalances antithrombin deficiency but has a limited bleeding diathesis

Deficiencies in coagulation factor VIII (FVIII, F8) result in the bleeding disorder hemophilia A. An emerging novel therapeutic strategy for bleeding disorders is to enhance hemostasis by limiting natural anticoagulants, such as antithrombin (AT3). To study pro/anticoagulant hemostatic balance in an in vivo model, we used genome editing to create null alleles for f8 and von Willebrand factor (vwf) in zebrafish, a model organism with a high degree of homology to the mammalian hemostatic system and unique attributes, including external development and optical transparency. f8 homozygous mutant larvae surprisingly formed normal thrombi when subjected to laser-mediated endothelial injury, had no overt signs of hemorrhage, but had a modest increase in mortality. We have previously shown that at3-/- larvae develop disseminated intravascular coagulation (DIC), with spontaneous thrombosis and fibrinogen consumption, resulting in bleeding phenotype marked by secondary lack of induced thrombus formation upon endothelial injury. We found that with loss of FVIII (f8-/-;at3-/-), larvae no longer developed spontaneous fibrin thrombi and did produce clots in response to endothelial injury. However, homozygous loss of zebrafish Vwf failed to rescue the at3 DIC phenotype. These studies demonstrate an altered balance of natural anticoagulants that mitigates FVIII deficiency in zebrafish, similar to human clinical pipeline products. The data also suggest that zebrafish FVIII might circulate independently of Vwf. Further study of this unique balance could provide new insights for management of hemophilia A and von Willebrand disease. Key Points{square} Zebrafish demonstrate a unique balance of natural anticoagulants that mitigates severe FVIII deficiency. {square}Zebrafish FVIII appears to circulate independently of Vwf, which could have implications for management of hemophilia A.

genetics↗

bioRxiv2019

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

In 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.\n\nKey PointsO_LIF5 mutant fish embryos tolerate symptoms lethal in mammals but succumb to bleeding in adulthood\nC_LIO_LIAnalysis of human variants demonstrate that all have some residual function and that minimal F5 activity is required to restore hemostasis\nC_LI

genetics↗