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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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  • References

  • 1 Franco AT, Corken A, Ware J. Platelets at the interface of thrombosis, inflammation, and cancer. Blood 2015; 126 (05) 582-588
    CrossrefPubMedGoogle Scholar
  • 2 Semple JW, Italiano Jr JE, Freedman J. Platelets and the immune continuum. Nat Rev Immunol 2011; 11 (04) 264-274
    CrossrefPubMedGoogle Scholar
  • 3 Mallouk N, Labruyère C, Reny JL. et al. Prevalence of poor biological response to clopidogrel: a systematic review. Thromb Haemost 2012; 107 (03) 494-506
    Article in Thieme ConnectPubMedGoogle Scholar
  • 4 O'Donnell CJ, Larson MG, Feng D. et al; Framingham Heart Study. Genetic and environmental contributions to platelet aggregation: the Framingham heart study. Circulation 2001; 103 (25) 3051-3056
    CrossrefPubMedGoogle Scholar
  • 5 Fontana P, Roffi M, Reny JL. Platelet function test use for patients with coronary artery disease in the early 2020s. J Clin Med 2020; 9 (01) 9
    CrossrefPubMedGoogle Scholar
  • 6 Plé H, Landry P, Benham A, Coarfa C, Gunaratne PH, Provost P. The repertoire and features of human platelet microRNAs. PLoS One 2012; 7 (12) e50746
    CrossrefPubMedGoogle Scholar
  • 7 Sunderland N, Skroblin P, Barwari T. et al. MicroRNA biomarkers and platelet reactivity: the clot thickens. Circ Res 2017; 120 (02) 418-435
    CrossrefPubMedGoogle Scholar
  • 8 Landry P, Plante I, Ouellet DL, Perron MP, Rousseau G, Provost P. Existence of a microRNA pathway in anucleate platelets. Nat Struct Mol Biol 2009; 16 (09) 961-966
    CrossrefPubMedGoogle Scholar
  • 9 Nagalla S, Shaw C, Kong X. et al. Platelet microRNA-mRNA coexpression profiles correlate with platelet reactivity. Blood 2011; 117 (19) 5189-5197
    CrossrefPubMedGoogle Scholar
  • 10 Kondkar AA, Bray MS, Leal SM. et al. VAMP8/endobrevin is overexpressed in hyperreactive human platelets: suggested role for platelet microRNA. J Thromb Haemost 2010; 8 (02) 369-378
    CrossrefPubMedGoogle Scholar
  • 11 Kaudewitz D, Skroblin P, Bender LH. et al. Association of microRNAs and YRNAs with platelet function. Circ Res 2016; 118 (03) 420-432
    CrossrefPubMedGoogle Scholar
  • 12 Garcia A, Dunoyer-Geindre S, Zapilko V, Nolli S, Reny JL, Fontana P. Functional validation of microRNA-126-3p as a platelet reactivity regulator using human haematopoietic stem cells. Thromb Haemost 2019; 119 (02) 254-263
    Article in Thieme ConnectPubMedGoogle Scholar
  • 13 Zufferey A, Ibberson M, Reny JL. et al. New molecular insights into modulation of platelet reactivity in aspirin-treated patients using a network-based approach. Hum Genet 2016; 135 (04) 403-414
    CrossrefPubMedGoogle Scholar
  • 14 Ding T, Zeng X, Cheng B. et al. Platelets in acute coronary syndrome patients with high platelet reactivity after dual antiplatelet therapy exhibit upregulation of miR-204-5p. Ann Clin Lab Sci 2019; 49 (05) 619-631
    PubMedGoogle Scholar
  • 15 Ma L, Deng X, Wu M, Zhang G, Huang J. Down-regulation of miRNA-204 by LMP-1 enhances CDC42 activity and facilitates invasion of EBV-associated nasopharyngeal carcinoma cells. FEBS Lett 2014; 588 (09) 1562-1570
    CrossrefPubMedGoogle Scholar
  • 16 Pleines I, Cherpokova D, Bender M. Rho GTPases and their downstream effectors in megakaryocyte biology. Platelets 2019; 30 (01) 9-16
    CrossrefPubMedGoogle Scholar
  • 17 Antkowiak A, Viaud J, Severin S. et al. Cdc42-dependent F-actin dynamics drive structuration of the demarcation membrane system in megakaryocytes. J Thromb Haemost 2016; 14 (06) 1268-1284
    CrossrefPubMedGoogle Scholar
  • 18 Aslan JE, McCarty OJ. Rac and Cdc42 team up for platelets. Blood 2013; 122 (18) 3096-3097
    CrossrefPubMedGoogle Scholar
  • 19 Strassel C, Brouard N, Mallo L. et al. Aryl hydrocarbon receptor-dependent enrichment of a megakaryocytic precursor with a high potential to produce proplatelets. Blood 2016; 127 (18) 2231-2240
    CrossrefPubMedGoogle Scholar
  • 20 Kok MG, Halliani A, Moerland PD, Meijers JC, Creemers EE, Pinto-Sietsma SJ. Normalization panels for the reliable quantification of circulating microRNAs by RT-qPCR. FASEB J 2015; 29 (09) 3853-3862
    CrossrefPubMedGoogle Scholar
  • 21 Vandesompele J, De Preter K, Pattyn F. et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002; 3 (07) H0034
    CrossrefPubMedGoogle Scholar
  • 22 Dütting S, Gaits-Iacovoni F, Stegner D. et al. A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis. Nat Commun 2017; 8: 15838
    CrossrefPubMedGoogle Scholar
  • 23 Eckly A, Heijnen H, Pertuy F. et al. Biogenesis of the demarcation membrane system (DMS) in megakaryocytes. Blood 2014; 123 (06) 921-930
    CrossrefPubMedGoogle Scholar
  • 24 Garcia A, Dunoyer-Geindre S, Fish RJ. et al. Methods to Investigate miRNA function: focus on platelet reactivity. Thromb Haemost 2021; 121 (04) 409-421
    Article in Thieme ConnectPubMedGoogle Scholar
  • 25 Six KR, Sicot G, Devloo R, Feys HB, Baruch D, Compernolle V. A comparison of haematopoietic stem cells from umbilical cord blood and peripheral blood for platelet production in a microfluidic device. Vox Sang 2019; 114 (04) 330-339
    CrossrefPubMedGoogle Scholar
  • 26 Schulze H, Korpal M, Hurov J. et al. Characterization of the megakaryocyte demarcation membrane system and its role in thrombopoiesis. Blood 2006; 107 (10) 3868-3875
    CrossrefPubMedGoogle Scholar
  • 27 Strassel C, Eckly A, Léon C. et al. Intrinsic impaired proplatelet formation and microtubule coil assembly of megakaryocytes in a mouse model of Bernard-Soulier syndrome. Haematologica 2009; 94 (06) 800-810
    CrossrefPubMedGoogle Scholar
  • 28 Eckly A, Strassel C, Freund M. et al. Abnormal megakaryocyte morphology and proplatelet formation in mice with megakaryocyte-restricted MYH9 inactivation. Blood 2009; 113 (14) 3182-3189
    CrossrefPubMedGoogle Scholar
  • 29 Mountford JK, Petitjean C, Putra HW. et al. The class II PI 3-kinase, PI3KC2α, links platelet internal membrane structure to shear-dependent adhesive function. Nat Commun 2015; 6: 6535
    CrossrefPubMedGoogle Scholar
  • 30 Barkalow KL, Italiano Jr JE, Chou DE, Matsuoka Y, Bennett V, Hartwig JH. Alpha-adducin dissociates from F-actin and spectrin during platelet activation. J Cell Biol 2003; 161 (03) 557-570
    CrossrefPubMedGoogle Scholar
  • 31 Hartwig JH, Italiano Jr JE. Cytoskeletal mechanisms for platelet production. Blood Cells Mol Dis 2006; 36 (02) 99-103
    CrossrefPubMedGoogle Scholar
  • 32 Bender M, Eckly A, Hartwig JH. et al. ADF/n-cofilin-dependent actin turnover determines platelet formation and sizing. Blood 2010; 116 (10) 1767-1775
    CrossrefPubMedGoogle Scholar
  • 33 Nayak RC, Chang KH, Vaitinadin NS, Cancelas JA. Rho GTPases control specific cytoskeleton-dependent functions of hematopoietic stem cells. Immunol Rev 2013; 256 (01) 255-268
    CrossrefPubMedGoogle Scholar
  • 34 Palazzo A, Bluteau O, Messaoudi K. et al. The cell division control protein 42-Src family kinase-neural Wiskott-Aldrich syndrome protein pathway regulates human proplatelet formation. J Thromb Haemost 2016; 14 (12) 2524-2535
    CrossrefPubMedGoogle Scholar
  • 35 Aslan JE, McCarty OJ. Rho GTPases in platelet function. J Thromb Haemost 2013; 11 (01) 35-46
    CrossrefPubMedGoogle Scholar
  • 36 Huveneers S, Danen EH. Adhesion signaling - crosstalk between integrins, Src and Rho. J Cell Sci 2009; 122 (Pt 8): 1059-1069
    CrossrefPubMedGoogle Scholar
  • 37 Sanders LC, Matsumura F, Bokoch GM, de Lanerolle P. Inhibition of myosin light chain kinase by p21-activated kinase. Science 1999; 283 (5410): 2083-2085
    CrossrefPubMedGoogle Scholar
  • 38 Sumi T, Matsumoto K, Takai Y, Nakamura T. Cofilin phosphorylation and actin cytoskeletal dynamics regulated by rho- and Cdc42-activated LIM-kinase 2. J Cell Biol 1999; 147 (07) 1519-1532
    CrossrefPubMedGoogle Scholar
  • 39 Pleines I, Eckly A, Elvers M. et al. Multiple alterations of platelet functions dominated by increased secretion in mice lacking Cdc42 in platelets. Blood 2010; 115 (16) 3364-3373
    CrossrefPubMedGoogle Scholar
  • 40 Pleines I, Dütting S, Cherpokova D. et al. Defective tubulin organization and proplatelet formation in murine megakaryocytes lacking Rac1 and Cdc42. Blood 2013; 122 (18) 3178-3187
    CrossrefPubMedGoogle Scholar
  • 41 Weisel JW, Litvinov RI. Red blood cells: the forgotten player in hemostasis and thrombosis. J Thromb Haemost 2019; 17 (02) 271-282
    CrossrefPubMedGoogle Scholar
  • 42 Basak I, Bhatlekar S, Manne BK. et al. miR-15a-5p regulates expression of multiple proteins in the megakaryocyte GPVI signaling pathway. J Thromb Haemost 2019; 17 (03) 511-524
    CrossrefPubMedGoogle Scholar
  • 43 Dupont A, Fontana P, Bachelot-Loza C. et al. An intronic polymorphism in the PAR-1 gene is associated with platelet receptor density and the response to SFLLRN. Blood 2003; 101 (05) 1833-1840
    CrossrefPubMedGoogle Scholar
  • 44 Akbar H, Shang X, Perveen R. et al. Gene targeting implicates Cdc42 GTPase in GPVI and non-GPVI mediated platelet filopodia formation, secretion and aggregation. PLoS One 2011; 6 (07) e22117
    CrossrefPubMedGoogle Scholar
  • 45 Goggs R, Williams CM, Mellor H, Poole AW. Platelet Rho GTPases-a focus on novel players, roles and relationships. Biochem J 2015; 466 (03) 431-442
    CrossrefPubMedGoogle Scholar
  • 46 Bertoni A, Tadokoro S, Eto K. et al. Relationships between Rap1b, affinity modulation of integrin alpha IIbbeta 3, and the actin cytoskeleton. J Biol Chem 2002; 277 (28) 25715-25721
    CrossrefPubMedGoogle Scholar
  • 47 Chrzanowska-Wodnicka M, Smyth SS, Schoenwaelder SM, Fischer TH, White II GC. Rap1b is required for normal platelet function and hemostasis in mice. J Clin Invest 2005; 115 (03) 680-687
    CrossrefPubMedGoogle Scholar