Abstract
To better understand hypercoagulability as an underlying cause for thrombosis, the leading cause of death in the Western world, new assays to study ex vivo coagulation are essential. The zebrafish is generally accepted as a good model for human hemostasis and thrombosis, as the hemostatic system proved to be similar to that in man. Their small size however, has been a hurdle for more widespread use in hemostasis related research. In this study we developed a method that enables the measurement of thrombin generation in a single drop of non-anticoagulated zebrafish blood. Pre-treatment of the fish with inhibitors of FXa and thrombin, resulted in a dose dependent diminishing of thrombin generation, demonstrating the validity of the assay. In order to establish the relationship between whole blood thrombin generation and fibrin formation, we visualized the resulting fibrin network by scanning electron microscopy. Taken together, in this study we developed a fast and reliable method to measure thrombin generation in whole blood collected from a single zebrafish. Given the similarities between coagulation pathways of zebrafish and mammals, zebrafish may be an ideal animal model to determine the effect of novel therapeutics on thrombin generation. Additionally, because of the ease with which gene functions can be silenced, zebrafish may serve as a model organism for mechanistical research in thrombosis and hemostasis.
Highlights
Thrombosis remains a leading cause of death in the western world
In a first set of experiments, thrombin generation was determined in 5 μl whole blood collected from single zebrafish, using a method comparable to whole blood calibrated automated thrombography [12]
Since calibrator measurements performed on the blood of 28 different fish in 5 independent experiments demonstrated an acceptable variation, the average calibrator slope per experiment was used for thrombin generation calculations
Summary
Aside from mortality, significant morbidity occurs from thrombotic events. The causes of this hypercoagulability are becoming more and more clear with an enhanced knowledge of hemostasis and the development of new coagulation assays. Most of this knowledge results from extensive in vitro biochemical characterization of blood coagulation, whereas studies investigating blood coagulation in vivo are limited. The zebrafish is generally accepted as a good model for mammalian hemostasis and thrombosis due to the presence of coagulation factors, platelet receptors and its response to anti-coagulant drugs commonly used in clinical treatment [1]. The use of PLOS ONE | DOI:10.1371/journal.pone.0149135 February 12, 2016
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