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A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos
Published on: November 8, 2014
The zebrafish model tackles anti-P2Y12 variability in humans: a translational approach
Paulina Ciepla1, Richard J Fish2, Séverine Nolli3
1Faculty of Medicine, University of Geneva, Geneve, Switzerland.
Abstract:
Antiplatelet drugs are a pillar in the treatment strategy to prevent ischemic events in cardiovascular patients. However, the action of existing therapies is nonuniform, and the causative mechanism for this variability remains poorly understood. The differences between the microRNA profiles of individuals have been suggested to impact platelet reactivity and treatment outcomes. microRNA-150 (miR-150) has been previously associated, in several clinical reports, with platelet function variability and cardiovascular events. Therefore, we initiated our analysis by examining the mechanistic role of miR‑150 in platelet function. We employed transgenic zebrafish larvae designed to specifically increase miR‑150 expression in thrombocytes. Laser-induced caudal vein injury in these animals resulted in a smaller thrombus and decreased thrombocyte accumulation. RNA sequencing of miR-150-overexpressing thrombocytes identified a downregulated transcript encoding microtubule associated serine/threonine kinase-like (mastl), a key regulator of P2Y12 receptor downstream signaling. Overnight treatment of the transgenic fish with clopidogrel, a P2Y12 inhibitor, showed decreased thrombus formation in the control, but not in miR-150-overexpressing animals. This phenotype was reversed by overexpressing mastl. Next, we utilized a VASP phosphorylation assay to show that MASTL deficiency partially protects human platelets from P2Y12 inhibition. Finally, using miRNA:mRNA interaction predictors and experimental assays, we identified MASTL-targeting miRNAs in humans - miR-17-‑5p and miR-106a-5p - which were significantly upregulated in a population of clopidogrel-resistant patients. Our studies therefore support a model in which miR-150 in zebrafish, and miR-17‑5p or miR-106a-5p in humans, modulates MASTL expression, which in turn alters VASP phosphorylation and platelet sensitivity to P2Y12 inhibition.
Insights
MicroRNAs like miR-150 influence platelet function and response to antiplatelet drugs by regulating MASTL. This finding may explain clopidogrel resistance in cardiovascular patients.
Area of Science:
- Cardiovascular Biology
- Molecular Medicine
- Platelet Physiology
Background:
- Antiplatelet drugs are crucial for preventing cardiovascular events, but patient responses vary unpredictably.
- MicroRNA (miRNA) profiles are implicated in platelet reactivity and drug efficacy.
- microRNA-150 (miR-150) has been linked to platelet function variability and cardiovascular events.
Purpose of the Study:
- To investigate the mechanistic role of miR-150 in platelet function and its impact on antiplatelet therapy.
- To identify the molecular targets of miR-150 involved in platelet signaling.
- To explore the relevance of these findings in human patients, particularly those resistant to clopidogrel.
Main Methods:
- Utilized transgenic zebrafish overexpressing miR-150 in thrombocytes to study thrombus formation after injury.
- Performed RNA sequencing on thrombocytes to identify downstream targets of miR-150.
- Conducted VASP phosphorylation assays on human platelets to assess P2Y12 inhibition sensitivity.
- Employed miRNA:mRNA interaction predictors and experimental validation to identify human MASTL-targeting miRNAs.
Main Results:
- miR-150 overexpression in zebrafish reduced thrombus size and thrombocyte accumulation.
- RNA sequencing revealed downregulation of microtubule associated serine/threonine kinase-like (MASTL) in miR-150-overexpressing thrombocytes.
- miR-150 overexpression conferred resistance to clopidogrel in zebrafish, which was reversed by MASTL overexpression.
- MASTL deficiency partially protected human platelets from P2Y12 inhibition.
- miR-17-5p and miR-106a-5p were upregulated in clopidogrel-resistant patients and target MASTL.
Conclusions:
- miR-150 in zebrafish, and miR-17-5p/miR-106a-5p in humans, modulate MASTL expression.
- Altered MASTL expression impacts VASP phosphorylation, affecting platelet sensitivity to P2Y12 inhibitors like clopidogrel.
- These miRNAs represent potential biomarkers for predicting antiplatelet drug response and therapeutic targets for managing cardiovascular events.
