Abstract：
BACKGROUND: Pituitary adenylate cyclase-activating polypeptide is a neuropeptide with diverse roles in biological processes. Its involvement in the blood coagulation cascade is unclear.
OBJECTIVE: This study unraveled adcyap1b\'s role in blood coagulation using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 in zebrafish. Effects were validated via adcyap1b knockdown. Gene expression changes in adcyap1b mutants were explored, linking them to clotting disorders. An analysis of proca gene splicing illuminated its role in adcyap1b-related anticoagulation deficiencies.
METHODS: Zebrafish were genetically modified using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 to induce adcyap1b knockout. Morpholino-mediated gene knockdown was employed for validation. Expression levels of coagulation factors, anticoagulant proteins, and fibrinolytic system genes were assessed in adcyap1b mutant zebrafish. Alternative splicing of proca gene was analyzed.
RESULTS: Adcyap1b mutant zebrafish exhibited severe hemorrhage, clotting disorders, and disrupted blood coagulation. Morpholino-mediated knockdown replicated observed phenotypes. Downregulation in transcripts related to coagulation factors V and IX, anticoagulation protein C, and plasminogen was observed. Abnormal alternative splicing of the proca gene was identified, providing a mechanistic explanation for anticoagulation system deficiencies.
CONCLUSIONS: Adcyap1b plays a crucial role in maintaining zebrafish blood coagulation and hemostasis. Its influence extends to the regulation of procoagulant and anticoagulant pathways, with abnormal alternative splicing contributing to observed deficiencies. These findings unveil a novel aspect of adcyap1b function, offering potential insights into similar processes in mammalian systems.
OBJECTIVE: This study unraveled adcyap1b\'s role in blood coagulation using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 in zebrafish. Effects were validated via adcyap1b knockdown. Gene expression changes in adcyap1b mutants were explored, linking them to clotting disorders. An analysis of proca gene splicing illuminated its role in adcyap1b-related anticoagulation deficiencies.
METHODS: Zebrafish were genetically modified using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 to induce adcyap1b knockout. Morpholino-mediated gene knockdown was employed for validation. Expression levels of coagulation factors, anticoagulant proteins, and fibrinolytic system genes were assessed in adcyap1b mutant zebrafish. Alternative splicing of proca gene was analyzed.
RESULTS: Adcyap1b mutant zebrafish exhibited severe hemorrhage, clotting disorders, and disrupted blood coagulation. Morpholino-mediated knockdown replicated observed phenotypes. Downregulation in transcripts related to coagulation factors V and IX, anticoagulation protein C, and plasminogen was observed. Abnormal alternative splicing of the proca gene was identified, providing a mechanistic explanation for anticoagulation system deficiencies.
CONCLUSIONS: Adcyap1b plays a crucial role in maintaining zebrafish blood coagulation and hemostasis. Its influence extends to the regulation of procoagulant and anticoagulant pathways, with abnormal alternative splicing contributing to observed deficiencies. These findings unveil a novel aspect of adcyap1b function, offering potential insights into similar processes in mammalian systems.
摘要：
背景：垂体腺苷酸环化酶激活多肽（PACAP）是一种在生物过程中具有多种作用的神经肽。其参与凝血级联反应尚不清楚。
目的：本研究在斑马鱼中使用CRISPR/Cas9揭示了adcyap1b在凝血中的作用。通过adcyap1b敲低验证效果。探索了adcyap1b突变体的基因表达变化，将它们与凝血障碍联系起来。对proca基因剪接的分析揭示了其在adcyap1b相关抗凝缺陷中的作用。
方法：使用CRISPR/Cas9对斑马鱼进行遗传修饰以诱导adcyap1b基因敲除。使用吗啉代介导的基因敲低进行验证。凝血因子的表达水平,抗凝蛋白,在adcyap1b突变斑马鱼中评估了纤溶系统基因。分析了proca基因的选择性剪接。
结果：Adcyap1b突变斑马鱼表现出严重出血，凝血障碍,并破坏了血液凝固。吗啉代介导的敲低复制了观察到的表型。与凝血因子V和IX相关的转录物下调，抗凝蛋白C,并观察到纤溶酶原。鉴定了proca基因的异常可变剪接，为抗凝系统缺陷提供机制解释。
结论：Adcyap1b在维持斑马鱼凝血和止血中起着至关重要的作用。它的影响延伸到促凝血和抗凝血途径的调节，异常的选择性剪接导致观察到的缺陷。这些发现揭示了adcyap1b功能的一个新方面，提供对哺乳动物系统中类似过程的潜在见解。
目的：本研究在斑马鱼中使用CRISPR/Cas9揭示了adcyap1b在凝血中的作用。通过adcyap1b敲低验证效果。探索了adcyap1b突变体的基因表达变化，将它们与凝血障碍联系起来。对proca基因剪接的分析揭示了其在adcyap1b相关抗凝缺陷中的作用。
方法：使用CRISPR/Cas9对斑马鱼进行遗传修饰以诱导adcyap1b基因敲除。使用吗啉代介导的基因敲低进行验证。凝血因子的表达水平,抗凝蛋白,在adcyap1b突变斑马鱼中评估了纤溶系统基因。分析了proca基因的选择性剪接。
结果：Adcyap1b突变斑马鱼表现出严重出血，凝血障碍,并破坏了血液凝固。吗啉代介导的敲低复制了观察到的表型。与凝血因子V和IX相关的转录物下调，抗凝蛋白C,并观察到纤溶酶原。鉴定了proca基因的异常可变剪接，为抗凝系统缺陷提供机制解释。
结论：Adcyap1b在维持斑马鱼凝血和止血中起着至关重要的作用。它的影响延伸到促凝血和抗凝血途径的调节，异常的选择性剪接导致观察到的缺陷。这些发现揭示了adcyap1b功能的一个新方面，提供对哺乳动物系统中类似过程的潜在见解。
