Thrombosis is the main consequence of cardiovascular diseases and antithrombotics, such as antiplatelet drugs, are a cornerstone for the prevention of the recurrence of these ischemic events. However, the biological impact of antiplatelet drugs on platelet reactivity is variable among patients, leading to the possibility of developing thrombotic or bleeding events. This unfortunate reality results from a poor understanding of the factors regulating thrombocyte function. Several microRNAs (miRNAs) have been associated with platelet reactivity, making them promising candidates for biomarkers of cardiovascular events. However, the mechanism of miRNA-mediated regulation of thrombocyte function remains mostly enigmatic.

To functionally validate miRNAs as potential biomarkers of platelet function, we developed a zebrafish model based on the real-time, in vivo monitoring of thrombus formation upon laser-induced blood vessel injury in larvae. We focused our attention on miR-96, miR-150 and miR-223, some of the most abundant miRNAs in human platelets, that are associated with platelet reactivity in multiple clinical and/or animal studies. Multiple transgenic zebrafish lines specifically overexpressing these miRNAs in thrombocytes were generated.

The miR-96 and miR-150 manipulations resulted in a decreased number of thrombocyte attachment in the forming thrombus after the venous laser injury. Similar results were also observed in the artery but only in the miR-96-overexpressing fish. While our miR-223 manipulations did not significantly affect the size of the venous thrombus after laser injury, they did however lead to a decrease in the time to first cell adhesion to the injured tissue. Concomitantly, the forming thrombus was less stable, resulting in more cells leaving this site following initial attachment.

The elevated level of each of these miRNAs led to widely different characteristic of the forming thrombus in our assay, in line with a major role of miRNAs in platelet reactivity and thrombus formation. The increased level of miR-96, miR-150 and miR-223 in zebrafish thrombocytes resulted in unique changes in the thrombocytes’ properties. Currently, we are working on determining the specific genes and biological pathways targeted by these miRNAs in thrombocytes that leads to the observed phenotypic changes in cellular behavior.