Activating stimuli induce platelet microRNA modulation and proteome reorganisation

Giovanni Cimmino; Roberta Tarallo; Giovanni Nassa; Maria Rosaria De Filippo; Giorgio Giurato; Maria Ravo; Francesca Rizzo; Stefano Conte; Grazia Pellegrino; Plinio Cirillo; Paolo Calabrò; Tiina Öhman; Tuula A. Nyman; Alessandro Weisz; Paolo Golino

doi:10.1160/TH14-09-0726

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2015; 114(01): 96-108
DOI: 10.1160/TH14-09-0726

Cellular Haemostasis and Platelets

Schattauer GmbH

Activating stimuli induce platelet microRNA modulation and proteome reorganisation

Giovanni Cimmino^*

¹Department of Cardiothoracic and Respiratory Sciences, Second University of Naples, Naples, Italy

,

Roberta Tarallo^*

²Laboratory of Molecular Medicine and Genomics, Department of Medicine and Surgery, University of Salerno, Baronissi (SA), Italy

,

Giovanni Nassa

²Laboratory of Molecular Medicine and Genomics, Department of Medicine and Surgery, University of Salerno, Baronissi (SA), Italy

,

Maria Rosaria De Filippo

²Laboratory of Molecular Medicine and Genomics, Department of Medicine and Surgery, University of Salerno, Baronissi (SA), Italy

³Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, USA

,

Giorgio Giurato

²Laboratory of Molecular Medicine and Genomics, Department of Medicine and Surgery, University of Salerno, Baronissi (SA), Italy

,

Maria Ravo

²Laboratory of Molecular Medicine and Genomics, Department of Medicine and Surgery, University of Salerno, Baronissi (SA), Italy

,

Francesca Rizzo

²Laboratory of Molecular Medicine and Genomics, Department of Medicine and Surgery, University of Salerno, Baronissi (SA), Italy

,

Stefano Conte

¹Department of Cardiothoracic and Respiratory Sciences, Second University of Naples, Naples, Italy

,

Grazia Pellegrino

⁴Department of Advanced Biomedical Sciences, University of Naples Federico II, Naples, Italy

,

Plinio Cirillo

⁴Department of Advanced Biomedical Sciences, University of Naples Federico II, Naples, Italy

,

Paolo Calabrò

¹Department of Cardiothoracic and Respiratory Sciences, Second University of Naples, Naples, Italy

,

Tiina Öhman

⁵Institute of Biotechnology, University of Helsinki, Helsinki, Finland

,

Tuula A. Nyman

¹Department of Cardiothoracic and Respiratory Sciences, Second University of Naples, Naples, Italy

,

Alessandro Weisz

²Laboratory of Molecular Medicine and Genomics, Department of Medicine and Surgery, University of Salerno, Baronissi (SA), Italy

⁶Molecular Pathology and Medical Genomics Unit, ‘SS. Giovanni di Dio e Ruggi d’Aragona – Schola Medica Salernitana’ University Hospital, Salerno, Italy

,

Paolo Golino

¹Department of Cardiothoracic and Respiratory Sciences, Second University of Naples, Naples, Italy

› Author Affiliations

Abstract

Summary

Platelets carry megakaryocyte-derived mRNAs whose translation efficiency before and during activation is not known, although this can greatly affect platelet functions, both under basal conditions and in response to physiological and pathological stimuli, such as those involved in acute coronary syndromes. Aim of the present study was to determine whether changes in microRNA (miRNA) expression occur in response to activating stimuli and whether this affects activity and composition of platelet transcriptome and proteome. Purified platelet-rich plasmas from healthy volunteers were collected and activated with ADP, collagen, or thrombin receptor activating peptide. Transcriptome analysis by RNA-Seq revealed that platelet transcriptome remained largely unaffected within the first 2 hours of stimulation. In contrast, quantitative proteomics showed that almost half of > 700 proteins quantified were modulated under the same conditions. Global miRNA analysis indicated that reorganisation of platelet proteome occurring during activation reflected changes in mature miRNA expression, which therefore, appears to be the main driver of the observed discrepancy between transcriptome and proteome changes. Platelet functions significantly affected by modulated miRNAs include, among others, the integrin/cytoskeletal, coagulation and inflammatory-immune response pathways. These results demonstrate a significant reprogramming of the platelet miRNome during activation, with consequent significant changes in platelet proteome and provide for the first time substantial evidence that fine-tuning of resident mRNA translation by miRNAs is a key event in platelet pathophysiology.

Keywords

Platelet activation - microRNA - RNA-Seq - quantitative proteomics - platelet system biology

Full Text

References

References
1 Fuster V, Badimon L, Badimon JJ. et al. The pathogenesis of coronary artery disease and the acute coronary syndromes (1). N Engl J Med 1992; 326: 242-250.
2 Bennett JS, Mousa S. Platelet function inhibitors in the Year 2000. Thromb Hae-most 2001; 85: 395-400.
3 Trip MD, Cats VM, van Capelle FJ. et al. Platelet hyperreactivity and prognosis in survivors of myocardial infarction. N Engl J Med 1990; 322: 1549-1554.
4 Newman PJ, Gorski J, White GC. 2nd, et al. Enzymatic amplification of platelet-specific messenger RNA using the polymerase chain reaction. J Clin Invest 1988; 82: 739-743.
5 Gnatenko DV, Dunn JJ, Schwedes J. et al. Transcript profiling of human platelets using microarray and serial analysis of gene expression (SAGE). Meth Mol Biol 2009; 496: 245-272.
6 Freedman JE. A platelet transcriptome revolution. Blood 2011; 118: 3760-3761.
7 Ple H, Maltais M, Corduan A. et al. Alteration of the platelet transcriptome in chronic kidney disease. Thromb Haemost 2012; 108: 605-615.
8 Bray PF, McKenzie SE, Edelstein LC. et al. The complex transcriptional landscape of the anucleate human platelet. BMC Genom 2013; 14: 1.
9 Kieffer N, Guichard J, Farcet JP. et al. Biosynthesis of major platelet proteins in human blood platelets. Eur J Biochem 1987; 164: 189-195.
10 Weyrich AS, Lindemann S, Tolley ND. et al. Change in protein phenotype without a nucleus: translational control in platelets. Semin Thromb Hemost 2004; 30: 491-498.
11 Denis MM, Tolley ND, Bunting M. et al. Escaping the nuclear confines: signal-dependent pre-mRNA splicing in anucleate platelets. Cell 2005; 122: 379-391.
12 Schwertz H, Tolley ND, Foulks JM. et al. Signal-dependent splicing of tissue factor pre-mRNA modulates the thrombogenicity of human platelets. J Exp Med 2006; 203: 2433-2440.
13 Baek D, Villen J, Shin C. et al. The impact of microRNAs on protein output. Nature 2008; 455: 64-71.
14 Landry P, Plante I, Ouellet DL. et al. Existence of a microRNA pathway in anucleate platelets. Nature Struct Mol Biol 2009; 16: 961-966.
15 Ple H, Landry P, Benham A. et al. The repertoire and features of human platelet microRNAs. PloS one 2012; 07: e50746.
16 Simon LM, Edelstein LC, Nagalla S. et al. Human platelet microRNA-mRNA networks associated with age and gender revealed by integrated plateletomics. Blood. 2014 Epub ahead of print.
17 Brambilla M, Camera M, Colnago D. et al. Tissue factor in patients with acute coronary syndromes: expression in platelets, leukocytes, and platelet-leukocyte aggregates. Arterioscl Thromb Vasc Biol 2008; 28: 947-953.
18 Amisten S. A rapid and efficient platelet purification protocol for platelet gene expression studies. Meth Mol Biol 2012; 788: 155-172.
19 Sener A, Egemen G, Cevik O. et al. In vitro effects of nitric oxide donors on apoptosis and oxidative/nitrative protein modifications in ADP-activated platelets. Human Exp Toxicol 2013; 32: 225-235.
20 Cirillo P, Golino P, Ragni M. et al. Activated platelets and leucocytes cooperatively stimulate smooth muscle cell proliferation and proto-oncogene expression via release of soluble growth factors. Cardiovasc Res 1999; 43: 210-218.
21 Mangalpally KK, Siqueiros-Garcia A, Vaduganathan M. et al. Platelet activation patterns in platelet size sub-populations: differential responses to aspirin in vitro. J Thromb Thrombol 2010; 30: 251-262.
22 Nassa G, Tarallo R, Giurato G. et al. Post-transcriptional regulation of human breast cancer cell proteome by unliganded estrogen receptor beta via microRNAs. Mol Cell Proteom 2014; 13: 1076-1090.
23 Shilov IV, Seymour SL, Patel AA. et al. The Paragon Algorithm, a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra. Mol Cell Proteom 2007; 06: 1638-1655.
24 Vizcaino JA, Cote RG, Csordas A. et al. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013. Nucl Acids Res 2013; 41 Database issue D1063-1069.
25 D’Andrea D, Grassi L, Mazzapioda M. et al. FIDEA: a server for the functional interpretation of differential expression analysis. Nucl Acids Res 2013; 41 Web Server issue W84-88.
26 Rolf N, Knoefler R, Suttorp M. et al. Optimized procedure for platelet RNA profiling from blood samples with limited platelet numbers. Clin Chem 2005; 51: 1078-1080.
27 Rox JM, Bugert P, Muller J. et al. Gene expression analysis in platelets from a single donor: evaluation of a PCR-based amplification technique. Clin Chem 2004; 50: 2271-2278.
28 Pfaff J, Meister G. Argonaute and GW182 proteins: an effective alliance in gene silencing. Biochem Soc Transact 2013; 41: 855-860.
29 Chekulaeva M, Mathys H, Zipprich JT. et al. miRNA repression involves GW182-mediated recruitment of CCR4-NOT through conserved W-containing motifs. Nature Struct Mol Biol 2011; 18: 1218-1226.
30 Stratz C, Nuhrenberg TG, Binder H. et al. Micro-array profiling exhibits remarkable intra-individual stability of human platelet micro-RNA. Thromb Hae-most 2012; 107: 634-641.
31 Osman A, Falker K. Characterisation of human platelet microRNA by quantitative PCR coupled with an annotation network for predicted target genes. Platelets 2011; 22: 433-441.
32 Nagalla S, Shaw C, Kong X. et al. Platelet microRNA-mRNA coexpression profiles correlate with platelet reactivity. Blood 2011; 117: 5189-5197.
33 Giurato G, De Filippo MR, Rinaldi A. et al. iMir: an integrated pipeline for high-throughput analysis of small non-coding RNA data obtained by smallRNA-Seq. BMC Bioinform 2013; 14: 362.
34 Fawcett J, Caberos LP, Littleton JM. The potentiation of platelet aggregation by mechanical stimuli is inhibited by ethanol in vitro. Alcohol Alcoholism 1987; 01: 573-576.
35 Cattaneo M, Tenconi PM, Lecchi A. et al. In vitro effects of picotamide on human platelet aggregation, the release reaction and thromboxane B2 production. Thromb Res 1991; 62: 717-724.
36 Born GV, Cross MJ. The Aggregation of Blood Platelets. J Physiol 1963; 168: 178-195.
37 Cheloufi S, Dos Santos CO, Chong MM. et al. A dicer-independent miRNA bio-genesis pathway that requires Ago catalysis. Nature 2010; 465: 584-589.
38 Merkerova M, Belickova M, Bruchova H. Differential expression of microRNAs in hematopoietic cell lineages. Eur J Haematol 2008; 81: 304-310.
39 Cimmino G, Nassa RT, De Filippo MR. et al. Platelet activation results in modulation of miRNA expression profile: new insights into the pathophysiology of platelet activation. Circulation. 2012 126. Suppl. Abstract.
40 Coughlin SR. Thrombin signalling and protease-activated receptors. Nature 2000; 407: 258-264.
41 Gidlof O, van der Brug M, Ohman J. et al. Platelets activated during myocardial infarction release functional miRNA, which can be taken up by endothelial cells and regulate ICAM1 expression. Blood 2013; 121: 3908-3917 S1–26.
42 Willeit P, Zampetaki A, Dudek K. et al. Circulating microRNAs as novel biomarkers for platelet activation. Circulation Res 2013; 112: 595-600.
43 Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116: 281-297.
44 Hammond SM, Bernstein E, Beach D. et al. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 2000; 404: 293-296.
45 Hutvagner G, Zamore PD. A microRNA in a multiple-turnover RNAi enzyme complex. Science 2002; 297: 2056-2060.
46 Mourelatos Z, Dostie J, Paushkin S. et al. miRNPs: a novel class of ribonucleo-proteins containing numerous microRNAs. Genes Develop 2002; 16: 720-728.
47 Meister G, Landthaler M, Patkaniowska A. et al. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol Cell 2004; 15: 185-197.
48 Rivas FV, Tolia NH, Song JJ. et al. Purified Argonaute2 and an siRNA form recombinant human RISC. Nature Struct Mol Biol 2005; 12: 340-349.
49 Chi SW, Hannon GJ, Darnell RB. An alternative mode of microRNA target recognition. Nature Struct Mol Biol 2012; 19: 321-327.
50 Sattler M, Salgia R, Shrikhande G. et al. Differential signaling after beta1 inte-grin ligation is mediated through binding of CRKL to p120 (CBL) and p110 (HEF1). J Biol Chem 1997; 272: 14320-14326.
51 Hirota T, Morisaki T, Nishiyama Y. et al. Zyxin, a regulator of actin filament assembly, targets the mitotic apparatus by interacting with h-warts/LATS1 tumor suppressor. J Cell Biol 2000; 149: 1073-1086.
52 Verschoor A, Langer HF. Crosstalk between platelets and the complement system in immune protection and disease. Thromb Haemost 2013; 110: 910-919.

Supplementary Material