Department of Pediatrics, University of Michigan, Ann Arbor, MI, USA.
Oxford University, Oxford, UK.
Sci Rep. 2020 Mar 4;10(1):4049. doi: 10.1038/s41598-020-60840-7.
The ability to prevent blood loss in response to injury is a conserved function of all vertebrates. Complete deficiency of the central clotting enzyme prothrombin has never been observed in humans and is incompatible with postnatal life in mice, thus limiting the ability to study its role in vivo. Zebrafish are able to tolerate severe hemostatic deficiencies that are lethal in mammals. We have generated a targeted genetic deletion in the kringle 1 domain of zebrafish prothrombin. Homozygous mutant embryos develop normally into the mid-juvenile stage but demonstrate complete mortality by 2 months of age primarily due to internal hemorrhage. Mutants are unable to form occlusive venous and arterial thrombi in response to endothelial injury, a defect that was phenocopied using direct oral anticoagulants. Human prothrombin engineered with the equivalent mutation exhibits a severe reduction in secretion, thrombin generation, and fibrinogen cleavage. Together, these data demonstrate the conserved function of thrombin in zebrafish and provide insight into the role of kringle 1 in prothrombin maturation and activity. Understanding how zebrafish are able to develop normally and survive into early adulthood without thrombin activity will provide important insight into its pleiotropic functions as well as the management of patients with bleeding disorders.
防止出血是所有脊椎动物的一项保守功能。人类从未观察到中央凝血酶原完全缺乏，而且这种情况在老鼠中也无法存活，这限制了对其在体内作用的研究。斑马鱼能够耐受严重的止血缺陷，而这些缺陷在哺乳动物中是致命的。我们在斑马鱼凝血酶原的kringle 1 结构域中产生了靶向基因缺失。纯合突变胚胎正常发育到中期幼体阶段，但在 2 个月大时几乎全部死亡，主要是由于内出血。突变体不能对内皮损伤形成闭塞性静脉和动脉血栓，这一缺陷可以通过直接口服抗凝剂来模拟。具有等效突变的人凝血酶原表现出严重的分泌减少、凝血酶生成和纤维蛋白原裂解。这些数据共同证明了凝血酶在斑马鱼中的保守功能，并为kringle 1 在凝血酶原成熟和活性中的作用提供了深入了解。了解斑马鱼在没有凝血酶活性的情况下如何正常发育并存活到成年早期，将为其多功能性以及出血性疾病患者的管理提供重要的见解。
