The transfer of miRNA from stimulated platelets to the plasma150 suggests that circulating platelet-derived miRNAs may reflect platelet activity in real time. Our translational approach combining human-derived cells model and the observation of the association between circulating miRNA level and several facets of platelet reactivity, including aggregation in response to a set of agonists and in vivo thrombin generation markers, unveils that one miRNA may govern one or several aspects of platelet reactivity.
In this work, the mechanism of action of two distinct miRNAs was investigated (126-3p and miR-204-5p). miR-126-3p is one of the most widely expressed miRNA in platelets.106 miR-204-5p was shown for the first time, in a previous study of our group, as associated with platelet reactivity in stable cardiovascular patients,117 and latter an independent study showed that miR-204-5p was positively associated to platelet aggregation.120
3.2.1
MIR-126-3PA large number of studies have shown that circulating miR-126-3p correlates with platelet reactivity and predict cardiovascular complications.109, 116, 149 The first investigation dedicated to unveil the underlying mechanism was performed in mice.12 In this model, the systemic inhibition of miR-126-3p induced a significant decrease in platelet aggregation after AA and ADP stimulation but did not affect significantly collagen-induced platelet aggregation. This indicates that miR-126-3p may regulate several platelet activation pathways. However, the possible non-conservation of seed sequences during the evolution suggests that results from animal model may differ from those obtained in human cells, further supporting our approach to use human-derived cells.
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Due to the low concentration of PLS harvested after differentiation of human hematopoietic stem cells, assessment of PLS function cannot be performed using conventional methods such as light transmission aggregometry (LTA). Therefore, we have developed original assays to monitor the effect of miR-126-3p on PLS function. In Section 2.1140 a flow chamber assay was developed where both PLS and megakaryocytes were perfused onto fibrinogen-coated slides. It should be noted that the flow chamber assay only partially mimics the vessels since the matrix does not mirror the sub-endothelial components and the engineering of the channel does not mimic turbulence at the site of atherosclerosis plaque. In addition, no red blood cells were added to the cell suspension to promote margination of PLS that allows the circulation of platelets at the periphery of the blood vessel and enhances the contact between the platelets and the vessel wall. However, the perfusion of megakaryocytes may overcome, at least in part, this issue.
Our model allowed us to go further in the understanding of the mechanisms governing miR-126-3p-incuded modification of platelet reactivity since silencing of one its validated target recapitulated the phenotype. This suggests that the regulation of platelet reactivity by miR-126-3p is, at least in part, mediated by direct regulation of PLXNB2. miR-126-3p is predicted to regulate a high number of mRNAs.
Among them, ADAM9, a protein involved in collagen adhesion113 has already been validated by a reporter gene assay.112
In the present study, miR-126-3p overexpression was associated with P-selectin secretion, which was also observed in an independent cohort.12 The secretion of P-selectin is observed in both activated151 and procoagulant platelet152 that is consistent with the results of our clinical study (Section 2.3 and Appendix 2) where miR-126-3p was positively associated to both platelet aggregation and in vivo thrombin generation markers. Although several other studies showed an association between miR-126-3p and platelet aggregation,12 the impact of miR-126-3p on platelet-supported thrombin generation is new. Indeed, we showed in a zebrafish model, that miR-126-3p overexpression in thrombocytes induces a thrombus sensitive to an anti-thrombin treatment, but not aspirin, and that miR-126-3p overexpression in PLS induces a procoagulant phenotype.145 Of note, the gene network described in section 2.3 pointed miR-126-3p as putative regulator of AKAP13, a protein known to modulate calcium release153-155 which is closely linked to the platelet procoagulant activity.63 Although this predicted interaction may explained the impact of miR-126-3p on thrombin generation, AKAP13 remains unvalidated as a target of miR-126-3p yet. In addition, miR-126-3p is predicted to target hundreds of mRNA targets, and we cannot exclude that other mechanisms are involved.
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3.2.2
MIR-204-5PPrevious omic’s and in silico investigations predicted that miR-204-5p was associated with platelet reactivity in stable cardiovascular patients by regulation of a set of actin binding proteins.117 Among them, CDC42, a Rho GTPase protein has already been biologically validated as a target of miR-204-5p.156 In addition, several studies described the impact of CDC42 on the modulation of platelet biogenesis and platelet function, further supporting that the pair miR-204-5p/CDC42 is of interest to be functionally validated using our model.23, 157, 158
We showed that miR-204-5p regulates CDC42 that in turn induces megakaryocyte cytoskeleton defects and leads to the alteration of PLS biogenesis. Moreover, miR-204-5p overexpression and CDC42 silencing increase both the activation of the GPIIbIIIa receptor after PLS stimulation and the PLS adhesion to immobilized and soluble fibrinogen. In addition, in a cohort of cardiovascular patients under aspirin treatment, miR-204-5p is positively correlated with the aggregation to AA, ADP and collagen. Taken together, these results suggest that miR-204-5p may regulate platelet aggregation via the regulation of fibrinogen binding through a CDC42-dependent mechanism. Although the role of Rho GTPase protein in the regulation of actin cytoskeleton has been largely investigated, its impact on platelet function is poorly understood. The Rho GTPase protein function is mediated by a rapid activation or inactivation characterized by GTP and GDP-binding, respectively.159 The Rho GTPase activation is closely linked to guanine nucleotide exchange factors (GEFs) while the GTPase-activating proteins (GAPs) mediate the Rho GTPase inactivation. There are more than 80 GEF and 70 GAP, suggesting that the regulation of Rho GTPase family activity is highly complex.160 In addition, the rapid switch between activated and non-activated form as well as some technical issues makes difficult the investigation of the protein activity status. This is even more true in the case of platelets for which Rho GTPase proteins activation is closely linked to the platelet activation status.161 The Rho GTPase proteins mediate cytoskeleton remodelling via various and distinct downstream effectors. CDC42, RHOA and RAC1 are known to regulate the formation of filopodia, lamellipodia and stress fibers, respectively.35 In the Section 2.2, we observed a modulation of stress fibers formation in megakaryocytes transfected with miR-204-5p as well as after silencing of CDC42 suggesting a regulation of RHOA activity via down regulation of CDC42. The mechanism has not been entirely elucidated. However, Dutting and colleagues23 showed a functional link between CDC42 and RHOA during platelet biogenesis. They proposed a model of “Stop and Go” signal showing that a balance between CDC42 and RHOA activation may exist in downstream of GPIbα. A decrease of CDC42 may be associated to an increase of RHOA mediating the “Stop” signal characterized by an alteration of DMS polarization and platelet formation.
In contrast, the “Go” signal is associated with an increase of CDC42 and a reduced level of RHOA that
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mediates megakaryocytes transmigration into sinusoidal vessels.162 Although the “Stop and Go” signal has been investigated in platelet biogenesis, a similar mechanism could be involved in platelet activation and could explain the impact of miR-204-5p via CDC42 regulation on platelet reactivity. In addition, actin remodelling by RhoGTPase proteins may govern the GPIIbIIIa inside-out and outside-in signalling that contribute to enable GPIIbIIIa in its active form that dramatically increases its affinity for fibrinogen and activation of GPIIbIIIa‘s downstream effectors triggering platelet spreading, aggregation and clot retraction.31 Moreover, miR-204-5p overexpression and silencing of CDC42 increase PLS adhesion to fibrinogen that suggests the existence of a miR-204-5p/CDC42/GPIIbIIIa axis.
Taken together, these data suggest that miR-204-5p may govern outside-in and/or inside-out signalling mediating platelet aggregation.