Zebrafish microRNA-150 controls thrombocyte function and clopidogrel response through the regulation of mastl protein

Introduction: Thrombosis is an underlying pathology of many cardiovascular diseases. Antiplatelet drugs are a primary treatment to prevent its reoccurrence. However, the action of existing therapies is nonuniform amongst patients, leading to the development of bleeding events or reoccurrence of thrombosis. This results from our poor understanding of factors regulating thrombocyte function. The microRNA-150 (miR-150) has been associated with platelet reactivity in multiple clinical studies, making it a promising candidate for biomarkers of cardiovascular events. However, the mechanism of miR-150-mediated regulation of the thrombocyte function remains enigmatic.

Method: To functionally validate miR-150 as a potential biomarker of platelet function, we developed a zebrafish model based on the real-time, in vivo monitoring of thrombus formation upon laser-induced caudal vein injury in the presence or absence of antiplatelet drugs. We also generated a zebrafish transgenic line overexpressing this miRNA in thrombocytes. Finally, to investigate the impact of miR-150 and its target mRNAs on platelet reactivity in humans, we modulated their level/function in platelets derived from whole blood or hematopoietic stem cells (CD34+) to perform functional analyses.

Results: Our miRNA overexpression strategy yielded a 30.8±2.8-fold increase in the miR-150 level in zebrafish thrombocytes. This manipulation resulted in a smaller thrombus (29.8±11.0% lower) with fewer thrombocytes accumulated within (39.7±7.8%) after the injury, as compared to the control. While the overnight treatment of fish with a P2Y₁₂ inhibitor (clopidogrel) decreased thrombus formation in control fish, it had no effect in fish overexpressing miR-150 in their thrombocytes. To unravel the pathway involved in this phenotype, RNAseq of miR-150-overexpressing or control thrombocytes was performed. Among the effected transcripts, we identify a downregulated, predicted target of miR-150: microtubule associated serine/threonine kinase-like (mastl), a protein involved in regulating the phosphorylation of the VASP protein. Now, we are investigating whether the knockdown of MASTL can influence platelet reactivity and their resistance to antiplatelet therapy in human.

Conclusion: The increased level of miR-150 in zebrafish thrombocytes resulted in their reduced aggregation at the injury site and resistance to P2Y₁₂ inhibitor treatment. We identified the downregulation of the mastl transcript as a potential biological pathway responsible for these observations. We are currently investigating whether a decrease in MASTL in human platelets can recapitulate the zebrafish phenotype. Interestingly, several miRNAs that are predicted to target MASTL mRNA have previously been linked to clopidogrel resistance in patients.

Publikationsverlauf

Artikel online veröffentlicht:
13. Februar 2025