Transcriptome Analysis of Reticulated Platelets Reveals a Prothrombotic Profile

Dario Bongiovanni; Gianluca Santamaria; Melissa Klug; Donato Santovito; Arianna Felicetta; Michael Hristov; Moritz von Scheidt; Maria Aslani; Javier Cibella; Christian Weber; Alessandra Moretti; Karl-Ludwig Laugwitz; Clelia Peano; Isabell Bernlochner

doi:10.1055/s-0039-1695009

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2019; 119(11): 1795-1806
DOI: 10.1055/s-0039-1695009

Cellular Haemostasis and Platelets

Georg Thieme Verlag KG Stuttgart · New York

Transcriptome Analysis of Reticulated Platelets Reveals a Prothrombotic Profile

Dario Bongiovanni‡^*

¹Medical Department 1, Cardiology, Klinikum rechts der Isar, Technische Universität, Munich, Germany

²DZHK (German Center for Cardiovascular Research), Partner site Munich Heart Alliance, Munich, Germany

,

Gianluca Santamaria ‡^*

¹Medical Department 1, Cardiology, Klinikum rechts der Isar, Technische Universität, Munich, Germany

,

Melissa Klug

¹Medical Department 1, Cardiology, Klinikum rechts der Isar, Technische Universität, Munich, Germany

,

Donato Santovito

³Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians-University Munich, Germany

,

Arianna Felicetta

⁴Department of Cardiovascular Medicine, Humanitas Research Hospital, Humanitas University, Milan, Italy

,

Michael Hristov

³Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians-University Munich, Germany

,

Moritz von Scheidt

²DZHK (German Center for Cardiovascular Research), Partner site Munich Heart Alliance, Munich, Germany

⁵Department of Cardiology, Deutsches Herzzentrum München, Technische Universität, Munich, Germany

,

Maria Aslani

³Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians-University Munich, Germany

,

Javier Cibella

⁶Genomic Unit, Humanitas Clinical and Research Center, Rozzano, Milan, Italy

,

Christian Weber

²DZHK (German Center for Cardiovascular Research), Partner site Munich Heart Alliance, Munich, Germany

³Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians-University Munich, Germany

,

Alessandra Moretti

¹Medical Department 1, Cardiology, Klinikum rechts der Isar, Technische Universität, Munich, Germany

²DZHK (German Center for Cardiovascular Research), Partner site Munich Heart Alliance, Munich, Germany

,

Karl-Ludwig Laugwitz

¹Medical Department 1, Cardiology, Klinikum rechts der Isar, Technische Universität, Munich, Germany

²DZHK (German Center for Cardiovascular Research), Partner site Munich Heart Alliance, Munich, Germany

,

Clelia Peano^**

⁶Genomic Unit, Humanitas Clinical and Research Center, Rozzano, Milan, Italy

⁷Institute of Genetic and Biomedical Research, National Research Council, UoS Milan, Italy

,

Isabell Bernlochner^**

¹Medical Department 1, Cardiology, Klinikum rechts der Isar, Technische Universität, Munich, Germany

²DZHK (German Center for Cardiovascular Research), Partner site Munich Heart Alliance, Munich, Germany

› Author Affiliations

Abstract

Reticulated platelets (RPs) are larger, hyperreactive platelets that contain significantly more ribonucleic acid (RNA) compared with mature platelets (MPs). High levels of RPs in peripheral blood are predictors of an insufficient response to dual antiplatelet therapy in cardiovascular patients and of adverse cardiovascular events. However, the mechanisms underlying these correlations remain widely unknown and the biology of RPs has not been investigated yet. Here, we compared for the first time the transcriptomic profiles of RPs and MPs isolated from peripheral blood of healthy donors. Total RNA sequencing revealed 1,744 differentially expressed genes (670 downregulated, 1,074 upregulated) in RPs compared with MPs. In particular, transcripts for the collagen receptor GP6, thromboxane receptor A2 (TBXA2R), thrombin receptor PAR4 (F2RL3), and adenosine triphosphate receptors P2RX1, ORAI2, and STIM1 (both involved in calcium signaling) were significantly upregulated in RPs, whereas several RNA regulators as the ribonuclease PARN, the RISC-component TNRC6A, and the splicing factor LUC7L3 were downregulated in RPs. Gene ontology analysis revealed an enrichment of relevant biological categories in RPs including platelet activation and blood coagulation. Gene Set Enrichment Analysis showed an overrepresentation of several platelet activation pathways like thrombin, thromboxane, and glycoprotein IIb/IIIa signaling in RPs. Small-RNA sequencing reported 9 micro-RNAs significantly downregulated in RPs with targets involved in platelet reactivity. Our data show for the first time an enrichment of several prothrombotic transcripts in RPs providing a first biological explanation for their hyperreactive phenotype.

Keywords

reticulated platelet - immature platelet - platelet transcriptome - platelet physiology

Full Text

References

References
1 Clancy L, Beaulieu LM, Tanriverdi K, Freedman JE. The role of RNA uptake in platelet heterogeneity. Thromb Haemost 2017; 117 (05) 948-961
2 Leslie M. Cell biology. Beyond clotting: the powers of platelets. Science 2010; 328 (5978): 562-564
3 Semple JW, Italiano Jr JE, Freedman J. Platelets and the immune continuum. Nat Rev Immunol 2011; 11 (04) 264-274
4 Kaushansky K. Lineage-specific hematopoietic growth factors. N Engl J Med 2006; 354 (19) 2034-2045
5 Thomas MR, Storey RF. The role of platelets in inflammation. Thromb Haemost 2015; 114 (03) 449-458
6 Chen S, Du C, Shen M. , et al. Sympathetic stimulation facilitates thrombopoiesis by promoting megakaryocyte adhesion, migration, and proplatelet formation. Blood 2016; 127 (08) 1024-1035
7 Boilard E, Blanco P, Nigrovic PA. Platelets: active players in the pathogenesis of arthritis and SLE. Nat Rev Rheumatol 2012; 8 (09) 534-542
8 Lakkis N, Dokainish H, Abuzahra M. , et al. Reticulated platelets in acute coronary syndrome: a marker of platelet activity. J Am Coll Cardiol 2004; 44 (10) 2091-2093
9 Grove EL, Hvas AM, Mortensen SB, Larsen SB, Kristensen SD. Effect of platelet turnover on whole blood platelet aggregation in patients with coronary artery disease. J Thromb Haemost 2011; 9 (01) 185-191
10 Michael JV, Wurtzel JGT, Mao GF. , et al. Platelet microparticles infiltrating solid tumors transfer miRNAs that suppress tumor growth. Blood 2017; 130 (05) 567-580
11 Risitano A, Beaulieu LM, Vitseva O, Freedman JE. Platelets and platelet-like particles mediate intercellular RNA transfer. Blood 2012; 119 (26) 6288-6295
12 Best MG, Sol N, Kooi I. , et al. RNA-seq of tumor-educated platelets enables blood-based pan-cancer, multiclass, and molecular pathway cancer diagnostics. Cancer Cell 2015; 28 (05) 666-676
13 Abe Y, Wada H, Tomatsu H. , et al. A simple technique to determine thrombopoiesis level using immature platelet fraction (IPF). Thromb Res 2006; 118 (04) 463-469
14 van der Meijden PEJ. Platelet biology and functions: new concepts and clinical perspectives. Nat Rev Cardiol 2019; 16 (03) 166-179
15 Mills EW, Green R, Ingolia NT. Slowed decay of mRNAs enhances platelet specific translation. Blood 2017; 129 (17) e38-e48
16 Thon JN, Devine DV. Translation of glycoprotein IIIa in stored blood platelets. Transfusion 2007; 47 (12) 2260-2270
17 Zimmerman GA, Weyrich AS. Signal-dependent protein synthesis by activated platelets: new pathways to altered phenotype and function. Arterioscler Thromb Vasc Biol 2008; 28 (03) s17-s24
18 Warshaw AL, Laster L, Shulman NR. The stimulation by thrombin of glucose oxidation in human platelets. J Clin Invest 1966; 45 (12) 1923-1934
19 Zimmerman GA, Weyrich AS. Signal-dependent protein synthesis by activated platelets: new pathways to altered phenotype and function. Arterioscler Thromb Vasc Biol 2008; 28 (03) s17-s24
20 Weyrich AS, Schwertz H, Kraiss LW, Zimmerman GA. Protein synthesis by platelets: historical and new perspectives. J Thromb Haemost 2009; 7 (02) 241-246
21 Cecchetti L, Tolley ND, Michetti N, Bury L, Weyrich AS, Gresele P. Megakaryocytes differentially sort mRNAs for matrix metalloproteinases and their inhibitors into platelets: a mechanism for regulating synthetic events. Blood 2011; 118 (07) 1903-1911
22 Denis MM, Tolley ND, Bunting M. , et al. Escaping the nuclear confines: signal-dependent pre-mRNA splicing in anucleate platelets. Cell 2005; 122 (03) 379-391
23 Thompson CB, Eaton KA, Princiotta SM, Rushin CA, Valeri CR. Size dependent platelet subpopulations: relationship of platelet volume to ultrastructure, enzymatic activity, and function. Br J Haematol 1982; 50 (03) 509-519
24 Blair TA, Michelson AD, Frelinger III AL. Mass cytometry reveals distinct platelet subtypes in healthy subjects and novel alterations in surface glycoproteins in Glanzmann thrombasthenia. Sci Rep 2018; 8 (01) 10300
25 Bendall SC, Nolan GP, Roederer M, Chattopadhyay PK. A deep profiler's guide to cytometry. Trends Immunol 2012; 33 (07) 323-332
26 Bigalke B, Lindemann S, Ehlers R. , et al. Expression of platelet collagen receptor glycoprotein VI is associated with acute coronary syndrome. Eur Heart J 2006; 27 (18) 2165-2169
27 Italiano Jr JE, Richardson JL, Patel-Hett S. , et al. Angiogenesis is regulated by a novel mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha granules and differentially released. Blood 2008; 111 (03) 1227-1233
28 van Nispen tot Pannerden H, de Haas F, Geerts W, Posthuma G, van Dijk S, Heijnen HF. The platelet interior revisited: electron tomography reveals tubular alpha-granule subtypes. Blood 2010; 116 (07) 1147-1156
29 Karpatkin S. Heterogeneity of human platelets. II. Functional evidence suggestive of young and old platelets. J Clin Invest 1969; 48 (06) 1083-1087
30 Bernlochner I, Goedel A, Plischke C. , et al. Impact of immature platelets on platelet response to ticagrelor and prasugrel in patients with acute coronary syndrome. Eur Heart J 2015; 36 (45) 3202-3210
31 Guthikonda S, Alviar CL, Vaduganathan M. , et al. Role of reticulated platelets and platelet size heterogeneity on platelet activity after dual antiplatelet therapy with aspirin and clopidogrel in patients with stable coronary artery disease. J Am Coll Cardiol 2008; 52 (09) 743-749
32 McBane II RD, Gonzalez C, Hodge DO, Wysokinski WE. Propensity for young reticulated platelet recruitment into arterial thrombi. J Thromb Thrombolysis 2014; 37 (02) 148-154
33 Cesari F, Marcucci R, Gori AM. , et al. Reticulated platelets predict cardiovascular death in acute coronary syndrome patients. Insights from the AMI-Florence 2 Study. Thromb Haemost 2013; 109 (05) 846-853
34 Ibrahim H, Nadipalli S, DeLao T, Guthikonda S, Kleiman NS. Immature platelet fraction (IPF) determined with an automated method predicts clopidogrel hyporesponsiveness. J Thromb Thrombolysis 2012; 33 (02) 137-142
35 Stratz C, Bömicke T, Younas I. , et al. Comparison of immature platelet count to established predictors of platelet reactivity during thienopyridine therapy. J Am Coll Cardiol 2016; 68 (03) 286-293
36 Guthikonda S, Lev EI, Patel R. , et al. Reticulated platelets and uninhibited COX-1 and COX-2 decrease the antiplatelet effects of aspirin. J Thromb Haemost 2007; 5 (03) 490-496
37 Ibrahim H, Schutt RC, Hannawi B, DeLao T, Barker CM, Kleiman NS. Association of immature platelets with adverse cardiovascular outcomes. J Am Coll Cardiol 2014; 64 (20) 2122-2129
38 Muronoi T, Koyama K, Nunomiya S. , et al. Immature platelet fraction predicts coagulopathy-related platelet consumption and mortality in patients with sepsis. Thromb Res 2016; 144: 169-175
39 Anetsberger A, Blobner M, Haller B. , et al. Immature platelets as a novel biomarker for adverse cardiovascular events in patients after non-cardiac surgery. Thromb Haemost 2017; 117 (10) 1887-1895
40 Siess W, Grünberg B. Phosphorylation of rap1B by protein kinase A is not involved in platelet inhibition by cyclic AMP. Cell Signal 1993; 5 (02) 209-214
41 Ault KA, Rinder HM, Mitchell J. The Significance of Platelets with Increased RNA Content (Reticulated Platelets): A Measure of the Rate of Thrombopoiesis. Am J Clin Pathol 1992; 98 (06) 637-646
42 Dale GL, Friese P, Hynes LA, Burstein SA. Demonstration that thiazole-orange-positive platelets in the dog are less than 24 hours old. Blood 1995; 85 (07) 1822-1825
43 Angénieux C, Maître B, Eckly A, Lanza F, Gachet C. Time-dependent decay of mRNA and ribosomal RNA during platelet aging and its correlation with translation activity. PLOS ONE 2016; 11 (01) e0148064
44 Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 2014; 30 (15) 2114-2120
45 Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 2014; 15 (12) 550
46 Huang W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 2009; 4 (01) 44-57
47 Zhang X-O, Wang H-B, Zhang Y, Lu X, Chen LL, Yang L. Complementary sequence-mediated exon circularization. Cell 2014; 159 (01) 134-147
48 Chou C-H, Shrestha S, Yang C-D. , et al. miRTarBase update 2018: a resource for experimentally validated microRNA-target interactions. Nucleic Acids Res 2018; 46 (D1): D296-D302
49 Krzywinski M, Schein J, Birol I. , et al. Circos: an information aesthetic for comparative genomics. Genome Res 2009; 19 (09) 1639-1645
50 Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008; 3 (06) 1101-1108
51 Virtanen A, Henriksson N, Nilsson P, Nissbeck M. Poly(A)-specific ribonuclease (PARN): an allosterically regulated, processive and mRNA cap-interacting deadenylase. Crit Rev Biochem Mol Biol 2013; 48 (02) 192-209
52 Lee D, Park D, Park JH, Shin C. Poly(A)-specific ribonuclease sculpts the 3′ ends of microRNAs. RNA 2019; 25 (03) 388-405
53 Zhang X, Devany E, Murphy MR, Glazman G, Persaud M, Kleiman FE. PARN deadenylase is involved in miRNA-dependent degradation of TP53 mRNA in mammalian cells. Nucleic Acids Res 2015; 43 (22) 10925-10938
54 Niaz S, Hussain MU. Role of GW182 protein in the cell. Int J Biochem Cell Biol 2018; 101: 29-38
55 Vital SA, Becker F, Holloway PM. , et al. Formyl-peptide receptor 2/3/lipoxin A4 receptor regulates neutrophil-platelet aggregation and attenuates cerebral inflammation: impact for therapy in cardiovascular disease. Circulation 2016; 133 (22) 2169-2179
56 Perl L, Lerman-Shivek H, Rechavia E. , et al. Response to prasugrel and levels of circulating reticulated platelets in patients with ST-segment elevation myocardial infarction. J Am Coll Cardiol 2014; 63 (06) 513-517
57 Armstrong PC, Hoefer T, Knowles RB. , et al. Newly formed reticulated platelets undermine pharmacokinetically short-lived antiplatelet therapies. Arterioscler Thromb Vasc Biol 2017; 37 (05) 949-956
58 Eikelboom JW, Warkentin TE. Immature platelet count: part of the cardiologist's complete blood count?. J Am Coll Cardiol 2014; 64 (20) 2130-2132
59 Stratz C, Nührenberg T, Amann M. , et al. Impact of reticulated platelets on antiplatelet response to thienopyridines is independent of platelet turnover. Thromb Haemost 2016; 116 (05) 941-948
60 Reininger AJ, Bernlochner I, Penz SM. , et al. A 2-step mechanism of arterial thrombus formation induced by human atherosclerotic plaques. J Am Coll Cardiol 2010; 55 (11) 1147-1158
61 Penz S, Reininger AJ, Brandl R. , et al. Human atheromatous plaques stimulate thrombus formation by activating platelet glycoprotein VI. FASEB J 2005; 19 (08) 898-909
62 Jamasbi J, Megens RTA, Bianchini M. , et al. Differential inhibition of human atherosclerotic plaque-induced platelet activation by dimeric GPVI-Fc and anti-GPVI antibodies: functional and imaging studies. J Am Coll Cardiol 2015; 65 (22) 2404-2415
63 Wong PC, Seiffert D, Bird JE. , et al. Blockade of protease-activated receptor-4 (PAR4) provides robust antithrombotic activity with low bleeding. Sci Transl Med 2017; 9 (371) eaaf5294
64 Li R, Emsley J. The organizing principle of the platelet glycoprotein Ib-IX-V complex. J Thromb Haemost 2013; 11 (04) 605-614
65 Canobbio I, Balduini C, Torti M. Signalling through the platelet glycoprotein Ib-V-IX complex. Cell Signal 2004; 16 (12) 1329-1344
66 Sunderland N, Skroblin P, Barwari T. , et al. MicroRNA biomarkers and platelet reactivity: the clot thickens. Circ Res 2017; 120 (02) 418-435
67 Plé H, Landry P, Benham A, Coarfa C, Gunaratne PH. The repertoire and features of human platelet microRNAs. PLOS ONE 2012; 7 (12) e50746
68 Stakos DA, Gatsiou A, Stamatelopoulos K, Tselepis AD, Stellos K. Platelet microRNAs: from platelet biology to possible disease biomarkers and therapeutic targets. Platelets 2013; 24 (08) 579-589
69 Nagalla S, Shaw C, Kong X. , et al. Platelet microRNA-mRNA coexpression profiles correlate with platelet reactivity. Blood 2011; 117 (19) 5189-5197
70 Kaudewitz D, Skroblin P, Bender LH. , et al. Association of microRNAs and YRNAs with platelet function. Circ Res 2016; 118 (03) 420-432
71 McManus DD, Freedman JE. MicroRNAs in platelet function and cardiovascular disease. Nat Rev Cardiol 2015; 12 (12) 711-717
72 Gao J, Ma X, Zhang Y, Guo M, Shi D. The role of microRNAs in prethrombotic status associated with coronary artery disease. Thromb Haemost 2017; 117 (03) 429-436
73 Stiegler G, Fischer G, Ramanathan G, Bencur P, Weigel G, Mannhalter C. P-selectin mRNA is maintained in platelet concentrates stored at 4 ° C. Transfusion 2009; 49 (05) 921-927
74 Osei-Bimpong A. The effect of storage on the clinical utility of the immature platelet fraction. Hematology 2009; 14 (02) 118-121
75 Schubert S, Weyrich AS, Rowley JW. A tour through the transcriptional landscape of platelets. Blood 2014; 124 (04) 493-502
76 Memczak S, Jens M, Elefsinioti A. , et al. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 2013; 495 (7441): 333-338
77 Glažar P, Papavasileiou P, Rajewsky N. circBase: a database for circular RNAs. RNA 2014; 20 (11) 1666-1670
78 Alhasan AA, Izuogu OG, Al-Balool HH. , et al. Circular RNA enrichment in platelets is a signature of transcriptome degradation. Blood 2016; 127 (09) e1-e11
79 Nührenberg TG, Cederqvist M, Marini F. , et al. Uncontrolled diabetes mellitus has no major influence on the platelet transcriptome. BioMed Res Int 2018; 2018: 8989252
80 Burkhart JM, Gambaryan S, Watson SP. , et al. What can proteomics tell us about platelets?. Circ Res 2014; 114 (07) 1204-1219
81 Stratz C, Nührenberg TG, Binder H. , et al. Micro-array profiling exhibits remarkable intra-individual stability of human platelet micro-RNA. Thromb Haemost 2012; 107 (04) 634-641
82 Rowley JW, Weyrich AS. Coordinate expression of transcripts and proteins in platelets. Blood 2013; 121 (26) 5255-5256
83 Burkhart JM, Vaudel M, Gambaryan S. , et al. The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways. Blood 2012; 120 (15) e73-e82
84 Londin ER, Hatzimichael E, Loher P. , et al. The human platelet: strong transcriptome correlations among individuals associate weakly with the platelet proteome. Biol Direct 2014; 9 (01) 3

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Supplementary Material