The zebrafish model tackles anti-P2Y12 variability in humans: a translational approach

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 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 where an increase in miR-150 in zebrafish and miR-17-5p or miR-106a-5p in humans results in MASTL downregulation, which decreases VASP phosphorylation in platelets and thus makes them resistant to P2Y12 inhibition. Key pointsO_LImicroRNA-150 downregulates mastl expression in zebrafish larvae, leading to decreased thrombocyte adhesion upon vascular endothelial injury and resistance to P2Y12 inhibition. C_LIO_LIIn humans, downregulation of MASTL results in a decreased response to P2Y12 antagonists. C_LIO_LIMASTL targeting microRNAs are upregulated in cardiovascular patients with poor on-treatment response to clopidogrel. C_LI