Cardiovascular diseases are the leading cause of mortality worldwide, with platelets playing a key role through their involvement in hemostasis. Antiplatelet drugs are currently the cornerstone of antithrombotic therapy. However, their effectiveness remains limited, with a 3–5% recurrence risk despite treatment, possibly because they mainly target aggregation without affecting procoagulant activity. Combining a low-dose anticoagulant (rivaroxaban) with aspirin reduces cardiovascular events but increases bleeding risk. Identifying patients with the best risk/benefit ratio for this dual therapy remains a challenge. Notably, there is high interindividual variability in procoagulant platelet levels among patients, suggesting this parameter may associate with bleeding or ischemic risk. Identifying platelet function regulators may help develop new biomarkers to personalize antithrombotic treatment.
Platelet-derived microRNAs (miRNAs), released upon platelet activation, could serve as indicators of platelet function. Since most circulating miRNAs originate from platelets, they represent promising biomarkers for diagnosing or predicting high on-treatment platelet reactivity (HTPR) and cardiovascular event recurrence. MiR-223-3p, the most abundant miRNA in megakaryocytes (MKs) and platelets, has been associated with platelet reactivity, drug response, and cardiovascular recurrence. However, its role in platelet function remains poorly understood.
The aim of this thesis is to investigate the role of miR-223-3p in regulating megakaryopoiesis, thrombopoiesis, and platelet reactivity, as well as to identify the underlying mechanisms related to the potential observed modulations. Using an in vitro model of platelets derived from human hematopoietic stem cells (HSCs), we employed two complementary approaches: upregulating miR-223-3p expression using miRNA mimics and downregulating it using CRISPR/Cas9 technology.
Downregulation of miR-223-3p significantly reduced proplatelet formation, platelet production, and were associated with an increase in STMN1 mRNA expression, a direct target of miR-223-3p and cytoskeletonregulator. While miR-223-3p modulation did not impact GPIIb/IIIa activation, P-selectin expression was significantly increased upon stimulation when miR-223-3p levels were lowered. Investigations into platelet procoagulant activity revealed that miR-223-3p levels inversely correlated with the proportion of procoagulant platelets, while thrombin generation was slightly decreased with miR-223-3p upregulation. Basal calcium flux was influenced by miR-223-3p modulation but did not lead to significant changes in the proportion of procoagulant platelets, suggesting that calcium regulation is unlikely to explain the observed differences in procoagulant platelet generation. Considering that the scramblase TMEM16F is responsible for the translocation of PS to the cell surface upon platelet activation, we explored whether miR-223-3p was involved in its regulation. MiR-223-3p modulation led to significant regulation of TMEM16F mRNA levels, and a luciferase reporter gene assay validated TMEM16F mRNA as a direct target of miR-223-3p.
In summary, miR-223-3p modulates platelet production in vitro, as well as the generation of procoagulant platelets through, at least in part, the regulation of TMEM16F, a newly identified target of miR-223-3p in humans.
