Department of Genetic Medicine and Development, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland.
Int J Mol Sci. 2021 Jan 11;22(2):655. doi: 10.3390/ijms22020655.
Venous thrombosis occurs in patients with quantitative and qualitative fibrinogen disorders. Injury-induced thrombosis in zebrafish larvae has been used to model human coagulopathies. We aimed to determine whether zebrafish models of afibrinogenemia and dysfibrinogenemia have different thrombotic phenotypes. Laser injuries were used to induce venous thrombosis and the time-to-occlusion (TTO) and the binding and aggregation of fluorescent thrombocytes measured. The larvae failed to support occlusive venous thrombosis and showed reduced thrombocyte binding and aggregation at injury sites. The larvae were largely unaffected. When genome editing zebrafish to produce fibrinogen Aα R28C, equivalent to the human Aα R35C dysfibrinogenemia mutation, we detected in-frame skipping of exon 2 in the fga mRNA, thereby encoding Aα. This mutation is similar to Fibrinogen Montpellier II which causes hypodysfibrinogenemia. Aα fish had prolonged TTO and reduced thrombocyte activity, a dominant effect of the mutation. Finally, we used transgenic expression of fga R28C cDNA in fga knock-down or mutants to model thrombosis in dysfibrinogenemia. Aα R28C expression had similar effects on TTO and thrombocyte activity as Aα. We conclude that thrombosis assays in larval zebrafish can distinguish between quantitative and qualitative fibrinogen disorder models and may assist in anticipating a thrombotic phenotype of novel fibrinogen mutations.
静脉血栓形成发生在纤维蛋白原数量和质量异常的患者中。斑马鱼幼鱼的损伤诱导性血栓形成已被用于模拟人类凝血疾病。我们旨在确定无纤维蛋白原血症和纤维蛋白原血症的斑马鱼模型是否具有不同的血栓形成表型。使用激光损伤诱导静脉血栓形成，并测量闭塞时间 (TTO) 和荧光血小板的结合和聚集。 幼虫不能支持闭塞性静脉血栓形成，并且在损伤部位显示出减少的血小板结合和聚集。 幼虫基本不受影响。当对斑马鱼进行基因组编辑以产生纤维蛋白原 Aα R28C 时，相当于人类 Aα R35C 纤维蛋白原血症突变，我们在 fga mRNA 中检测到外显子 2 的框内跳跃，从而编码 Aα。该突变类似于导致低纤维蛋白原血症的纤维蛋白原蒙彼利埃 II。Aα 鱼的 TTO 延长，血小板活性降低，这是该突变的显性效应。最后，我们使用 fga R28C cDNA 的转基因表达在 fga 敲低或 突变体中模拟纤维蛋白原血症中的血栓形成。Aα R28C 表达对 TTO 和血小板活性的影响与 Aα 相似。我们得出结论，幼鱼斑马鱼中的血栓形成测定可以区分定量和定性纤维蛋白原紊乱模型，并可能有助于预测新型纤维蛋白原突变的血栓形成表型。
