Investigating the Genetic Modifiers of Thrombosis Using the Zebrafish Model
Grzegorski, Steven
2022
Abstract
Thrombosis is a leading cause of morbidity and mortality. Although 50-60% of thrombotic risk is estimated to be due to genetics, only 35% of individuals with a thrombotic event carry one of the 20-30 known genetic modifying risk factors. Antithrombin (AT3) is the primary endogenous inhibitor of the coagulation cascade, but deficiency of AT3 leads to an incompletely penetrant risk of thrombosis due to presumed genetic modifiers. The zebrafish is a small aquatic vertebrate with a well-characterized and highly conserved hemostatic system, but previous work from our laboratory suggests species specific factors allow zebrafish to survive severe hemostatic defects. This dissertation focuses on multiple areas: understanding the influence of known factors on thrombosis, developing a framework to identify novel factors and characterizing the conservation of hemostasis in zebrafish. Zebrafish are able to survive into adulthood with a severe genetic deficiency of factor V (f5) or factor X (f10), two factors upstream of prothrombin (f2), raising the possibility of alternative pathways for thrombin generation. To rule out this possibility, a f2 knockout phenocopied the life expectancy of the f5 and f10 knockouts, confirming canonical function. The knockout led to an unexpected deletion in the kringle 1 domain and revealed a potential maturation and functional roles for (pro)thrombin. This model allows the study of the broad in vivo roles of thrombin such as cell signaling and response to inflammation that are impossible to explore in mice due to lethality. Loss of At3 leads to larval consumptive coagulopathy followed by lethal adult thrombosis. Intercrossing knockout alleles of f2, f10 and plasminogen (plg) onto the At3-deficient zebrafish genetic background demonstrated that heterozygosity for each individual mutation improved survival in the thrombotic population. After demonstrating the feasibility of genetically suppressing thrombosis, an unbiased chemical mutagenesis screen was performed to identify novel suppressors. Four lines were established that demonstrated stable improvement in survival. A candidate approach identified frost, a point mutation in prothrombin. Heterozygosity for frost in at3-/- fish rescued both survival and the larval consumptive coagulopathy. Biochemical studies of the frost mutation demonstrated decreased prothrombin production and an inability to cleave fibrinogen. In parallel, the remaining lines were sequenced and a modern genomics pipeline confirmed the mutagenesis protocol was a successful unbiased survey, but did not achieve functional saturation of the genome. This indicates the strong potential of this protocol to identify new thrombosis biology. Finally, loss of tissue factor (TF), a key initiating factor of the coagulation cascade, is incompatible with murine embryonic survival and studies in humans are lacking. Zebrafish have 2 copies of TF (TFa and TFb) with unknown conservation of function, and loss of function alleles for both copies were generated. Loss of both resulted in early lethality, but single copies were sufficient for normal survival. TFa and TFb were shown to have predominant roles in the arterial and venous systems, respectively. Finally, although TFb has lower in vitro procoagulant activity, it may play a role in factor VII mediated activation of the intrinsic pathway. Overall, the work in this dissertation provides a strong foundation for studying and discovering modifiers of hemostasis and thrombosis using the zebrafish. Throughout this study, the utility of zebrafish was confirmed through the discovery of unexpected biology, demonstration of hemostatic rebalancing through thrombotic suppression and identification of promising novel alleles and pathways for informing human biology.Deep Blue DOI
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thrombosis hemostasis genomics zebrafish antithrombin tissue factor
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