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CC BY-NC-ND 4.0 · Thromb Haemost 2021; 121(09): 1206-1219
DOI: 10.1055/a-1497-9649
Cellular Haemostasis and Platelets

miR-204-5p and Platelet Function Regulation: Insight into a Mechanism Mediated by CDC42 and GPIIbIIIa

Alix Garcia
1   Geneva Platelet Group, Faculty of Medicine, University of Geneva, Geneva, Switzerland
,
Sylvie Dunoyer-Geindre
1   Geneva Platelet Group, Faculty of Medicine, University of Geneva, Geneva, Switzerland
,
Séverine Nolli
1   Geneva Platelet Group, Faculty of Medicine, University of Geneva, Geneva, Switzerland
,
Catherine Strassel
2   Unité Mixte de Recherche S1255, INSERM, Strasbourg, France
,
Jean-Luc Reny
1   Geneva Platelet Group, Faculty of Medicine, University of Geneva, Geneva, Switzerland
3   Division of General Internal Medicine, Geneva University Hospitals, Geneva, Switzerland
,
Pierre Fontana 
1   Geneva Platelet Group, Faculty of Medicine, University of Geneva, Geneva, Switzerland
4   Division of Angiology and Haemostasis, Geneva University Hospitals, Geneva, Switzerland
› Author Affiliations Funding This work was supported by the Private Foundation of the University Hospitals of Geneva (grant RC04-05).
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Abstract

Background Several platelet-derived microRNAs are associated with platelet reactivity (PR) and clinical outcome in cardiovascular patients. We previously showed an association between miR-204-5p and PR in stable cardiovascular patients, but data on functional mechanisms are lacking.

Aims To validate miR-204-5p as a regulator of PR in platelet-like structures (PLS) derived from human megakaryocytes and to address mechanistic issues.

Methods Human hematopoietic stem cells were differentiated into megakaryocytes, enabling the transfection of miR-204-5p and the recovery of subsequent PLS. The morphology of transfected megakaryocytes and PLS was characterized using flow cytometry and microscopy. The functional impact of miR-204-5p was assessed using a flow assay, the quantification of the activated form of the GPIIbIIIa receptor, and a fibrinogen-binding assay. Quantitative polymerase chain reaction and western blot were used to evaluate the impact of miR-204-5p on a validated target, CDC42. The impact of CDC42 modulation was investigated using a silencing strategy.

Results miR-204-5p transfection induced cytoskeletal changes in megakaryocytes associated with the retracted protrusion of proPLS, but it had no impact on the number of PLS released. Functional assays showed that the PLS produced by megakaryocytes transfected with miR-204-5p were more reactive than controls. This phenotype is mediated by the regulation of GPIIbIIIa expression, a key contributor in platelet–fibrinogen interaction. Similar results were obtained after CDC42 silencing, suggesting that miR-204-5p regulates PR, at least in part, via CDC42 downregulation.

Conclusion We functionally validated miR-204-5p as a regulator of the PR that occurs through CDC42 downregulation and regulation of fibrinogen receptor expression.

Keywords

miRNAs - CDC42 - GPIIbIIIa - platelet reactivity - biomarkers

Author Contribution

A.G., S.D-.G., C.S., J.-L.R., and P.F. designed the study and analyzed the data. A.G., S.D-G., and S. N. performed the experiments and analyzed the data. A.G., S.D-.G., and P.F. wrote the first draft of the manuscript, and all authors revised the intellectual content and approved the final version.


Supplementary Material



Publication History

Received: 02 November 2020

Accepted: 29 April 2021

Accepted Manuscript online:
03 May 2021

Article published online:
18 June 2021

© 2021. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/)

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