Developmental adcyap1b loss leads to hemorrhage, disrupted hemostasis, and a blood coagulation cascade in zebrafish

Xinyan Ma; Ruixian Guo; Huibo Xu; Yuanyuan Ma; Rui Zhang; Xinyan Liu; Jingpu Zhang; Ying Han

doi:10.1016/j.jtha.2023.12.010

Developmental adcyap1b loss leads to hemorrhage, disrupted hemostasis, and a blood coagulation cascade in zebrafish

J Thromb Haemost. 2024 Apr;22(4):951-964. doi: 10.1016/j.jtha.2023.12.010. Epub 2023 Dec 15.

Authors

Xinyan Ma¹, Ruixian Guo², Huibo Xu³, Yuanyuan Ma⁴, Rui Zhang⁴, Xinyan Liu⁴, Jingpu Zhang⁵, Ying Han⁶

Affiliations

¹ Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; School of Pharmacy, Minzu University of China, Beijing, China.
² Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; School of Traditional Chinese Medicine, Capital Medical University, Beijing, China.
³ University of Science and Technology of China, Hefei, China.
⁴ Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
⁵ Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. Electronic address: zhangjingpu@imb.pumc.edu.cn.
⁶ Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. Electronic address: hanying@imb.pumc.edu.cn.

PMID: 38104724
DOI: 10.1016/j.jtha.2023.12.010

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.

Objectives: 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.

Conclusion: 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.

Keywords: CRISPR/Cas9; adcyap1b; blood coagulation; hemostasis; zebrafish.

MeSH terms

Animals
Anticoagulants / metabolism
Blood Coagulation / genetics
CRISPR-Associated Protein 9 / metabolism
Factor V / metabolism
Hemorrhage
Mammals / metabolism
Morpholinos / genetics
Morpholinos / metabolism
Zebrafish Proteins* / genetics
Zebrafish Proteins* / metabolism
Zebrafish* / genetics
Zebrafish* / metabolism

Substances

Zebrafish Proteins
CRISPR-Associated Protein 9
Morpholinos
Factor V
Anticoagulants