Download PDF
CC BY-NC-ND 4.0 · Thromb Haemost 2022; 122(05): 726-738
DOI: 10.1055/s-0041-1735972
Cellular Haemostasis and Platelets

Effects of Platelet Agonists and Priming on the Formation of Platelet Populations

Alicia Veninga
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands
,
Constance C. F. M. J. Baaten
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands
2   Institute for Molecular Cardiovascular Research, University Hospital Aachen, RWTH Aachen University, Germany
,
Ilaria De Simone
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands
3   Institute for Cardiovascular and Metabolic Research, University of Reading, Reading, United Kingdom
,
Bibian M. E. Tullemans
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands
,
Marijke J. E. Kuijpers 
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands
4   Thrombosis Expertise Center, Heart and Vascular Center, Maastricht University Medical Center, Maastricht, The Netherlands
,
Johan W. M. Heemskerk 
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands
,
Paola E. J. van der Meijden
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands
4   Thrombosis Expertise Center, Heart and Vascular Center, Maastricht University Medical Center, Maastricht, The Netherlands
› Author Affiliations Funding This work is supported by the Landsteiner Foundation for Blood Transfusion Research Grant No. 1711. C.C.F.M.J.B. received funding from The Dutch Heart Foundation (2020T020) and the START-Program of the Faculty of Medicine at the RWTH Aachen University (105/20). I.D.S. is supported by a joint PhD scholarship of Maastricht and Reading Universities, and by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 766118.
› Further Information
    Permissions and Reprints

Abstract

Platelets from healthy donors display heterogeneity in responsiveness to agonists. The response thresholds of platelets are controlled by multiple bioactive molecules, acting as negatively or positively priming substances. Higher circulating levels of priming substances adenosine and succinate, as well as the occurrence of hypercoagulability, have been described for patients with ischaemic heart disease. Here, we present an improved methodology of flow cytometric analyses of platelet activation and the characterisation of platelet populations following activation and priming by automated clustering analysis.

Platelets were treated with adenosine, succinate, or coagulated plasma before stimulation with CRP-XL, 2-MeSADP, or TRAP6 and labelled for activated integrin αIIbβ3 (PAC1), CD62P, TLT1, CD63, and GPIX. The Super-Enhanced Dmax subtraction algorithm and 2% marker (quadrant) setting were applied to identify populations, which were further defined by state-of-the-art clustering techniques (tSNE, FlowSOM).

Following activation, five platelet populations were identified: resting, aggregating (PAC1 + ), secreting (α- and dense-granules; CD62P + , TLT1 + , CD63 + ), aggregating plus α-granule secreting (PAC1 + , CD62P + , TLT1 + ), and fully active platelet populations. The type of agonist determined the distribution of platelet populations. Adenosine in a dose-dependent way suppressed the fraction of fully activated platelets (TRAP6 > 2-MeSADP > CRP-XL), whereas succinate and coagulated plasma increased this fraction (CRP-XL > TRAP6 > 2-MeSADP). Interestingly, a subset of platelets showed a constant response (aggregating, secreting, or aggregating plus α-granule secreting), which was hardly affected by the stimulus strength or priming substances.

Keywords

platelets - populations - priming - activation markers - flow cytometric analysis

Supplementary Material



Publication History

Received: 14 December 2020

Accepted: 12 July 2021

Article published online:
24 October 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/)

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

Comment to this article:

Functional Diversity of Primed Platelets—Powered by Machine LearningThromb Haemost 2022; 122(05): 661-661
DOI: 10.1055/a-1690-8971

 

  • References

  • 1 Baaten CCFMJ, Ten Cate H, van der Meijden PEJ, Heemskerk JWM. Platelet populations and priming in hematological diseases. Blood Rev 2017; 31 (06) 389-399
    CrossrefPubMedGoogle Scholar
  • 2 van der Meijden PEJ, Heemskerk JWM. Platelet biology and functions: new concepts and clinical perspectives. Nat Rev Cardiol 2019; 16 (03) 166-179
    Google Scholar
  • 3 Swieringa F, Kuijpers MJ, Heemskerk JW, van der Meijden PE. Targeting platelet receptor function in thrombus formation: the risk of bleeding. Blood Rev 2014; 28 (01) 9-21
    CrossrefPubMedGoogle Scholar
  • 4 Gariboldi V, Vairo D, Guieu R. et al. Expressions of adenosine A2A receptors in coronary arteries and peripheral blood mononuclear cells are correlated in coronary artery disease patients. Int J Cardiol 2017; 230: 427-431
    CrossrefPubMedGoogle Scholar
  • 5 Marlinge M, Vairo D, Marolda V. et al. Rapid measurement of adenosine concentration in human blood using FixedPotential amperometry: comparison with mass spectrometry and high-performance liquid chromatography. J Anal Bioanal Tech 2017; 2017: 1-5
    PubMedGoogle Scholar
  • 6 Funaya H, Kitakaze M, Node K, Minamino T, Komamura K, Hori M. Plasma adenosine levels increase in patients with chronic heart failure. Circulation 1997; 95 (06) 1363-1365
    CrossrefPubMedGoogle Scholar
  • 7 Aguiar CJ, Rocha-Franco JA, Sousa PA. et al. Succinate causes pathological cardiomyocyte hypertrophy through GPR91 activation. Cell Commun Signal 2014; 12: 78
    CrossrefPubMedGoogle Scholar
  • 8 Eltzschig HK, Ibla JC, Furuta GT. et al. Coordinated adenine nucleotide phosphohydrolysis and nucleoside signaling in posthypoxic endothelium: role of ectonucleotidases and adenosine A2B receptors. J Exp Med 2003; 198 (05) 783-796
    CrossrefPubMedGoogle Scholar
  • 9 Eckle T, Krahn T, Grenz A. et al. Cardioprotection by ecto-5′-nucleotidase (CD73) and A2B adenosine receptors. Circulation 2007; 115 (12) 1581-1590
    CrossrefPubMedGoogle Scholar
  • 10 Paganelli F, Gaudry M, Ruf J. et al. Recent advances in the role of the adenosinergic system in coronary artery disease. Cardiovasc Res 2021; 117 (05) 1284-1294
    CrossrefPubMedGoogle Scholar
  • 11 Johnston-Cox HA, Ravid K. Adenosine and blood platelets. Purinergic Signal 2011; 7 (03) 357-365
    CrossrefPubMedGoogle Scholar
  • 12 Fuentes E, Pereira J, Mezzano D, Alarcón M, Caballero J, Palomo I. Inhibition of platelet activation and thrombus formation by adenosine and inosine: studies on their relative contribution and molecular modeling. PLoS One 2014; 9 (11) e112741
    CrossrefPubMedGoogle Scholar
  • 13 Löfgren L, Pehrsson S, Hägglund G, Tjellström H, Nylander S. Accurate measurement of endogenous adenosine in human blood. PLoS One 2018; 13 (10) e0205707
    CrossrefPubMedGoogle Scholar
  • 14 Martin C, Leone M, Viviand X, Ayem ML, Guieu R. High adenosine plasma concentration as a prognostic index for outcome in patients with septic shock. Crit Care Med 2000; 28 (09) 3198-3202
    CrossrefPubMedGoogle Scholar
  • 15 Gaudry M, Vairo D, Marlinge M. et al. Adenosine and its receptors: an expected tool for the diagnosis and treatment of coronary artery and ischemic heart diseases. Int J Mol Sci 2020; 21 (15) E5321
    CrossrefPubMedGoogle Scholar
  • 16 Mills E, O'Neill LA. Succinate: a metabolic signal in inflammation. Trends Cell Biol 2014; 24 (05) 313-320
    CrossrefPubMedGoogle Scholar
  • 17 Sanborn T, Gavin W, Berkowitz S, Perille T, Lesch M. Augmented conversion of aspartate and glutamate to succinate during anoxia in rabbit heart. Am J Physiol 1979; 237 (05) H535-H541
    PubMedGoogle Scholar
  • 18 Taegtmeyer H. Metabolic responses to cardiac hypoxia. Increased production of succinate by rabbit papillary muscles. Circ Res 1978; 43 (05) 808-815
    CrossrefPubMedGoogle Scholar
  • 19 Kakinuma Y, Matsubara T, Hashimoto T, Sakamoto N. Myocardial metabolic markers of total ischemia in vitro. Nagoya J Med Sci 1994; 57 (1-4): 35-42
    PubMedGoogle Scholar
  • 20 Hakak Y, Lehmann-Bruinsma K, Phillips S. et al. The role of the GPR91 ligand succinate in hematopoiesis. J Leukoc Biol 2009; 85 (05) 837-843
    CrossrefPubMedGoogle Scholar
  • 21 Grimolizzi F, Arranz L. Multiple faces of succinate beyond metabolism in blood. Haematologica 2018; 103 (10) 1586-1592
    CrossrefPubMedGoogle Scholar
  • 22 Macaulay IC, Tijssen MR, Thijssen-Timmer DC. et al. Comparative gene expression profiling of in vitro differentiated megakaryocytes and erythroblasts identifies novel activatory and inhibitory platelet membrane proteins. Blood 2007; 109 (08) 3260-3269
    CrossrefPubMedGoogle Scholar
  • 23 Loeffen R, Spronk HM, ten Cate H. The impact of blood coagulability on atherosclerosis and cardiovascular disease. J Thromb Haemost 2012; 10 (07) 1207-1216
    CrossrefPubMedGoogle Scholar
  • 24 Gerotziafas GT, Zografos T, Pantos I. et al. Prospective assessment of biomarkers of hypercoagulability for the identification of patients with severe coronary artery disease. The ROADMAP-CAD study. Clin Appl Thromb Hemost 2020; 26: 1076029620964590
    Google Scholar
  • 25 White TC, Berny MA, Tucker EI. et al. Protein C supports platelet binding and activation under flow: role of glycoprotein Ib and apolipoprotein E receptor 2. J Thromb Haemost 2008; 6 (06) 995-1002
    CrossrefPubMedGoogle Scholar
  • 26 Magwenzi SG, Ajjan RA, Standeven KF, Parapia LA, Naseem KM. Factor XIII supports platelet activation and enhances thrombus formation by matrix proteins under flow conditions. J Thromb Haemost 2011; 9 (04) 820-833
    CrossrefPubMedGoogle Scholar
  • 27 Maecker HT, McCoy Jr JP, Amos M. et al; FOCIS Human Immunophenotyping Consortium. A model for harmonizing flow cytometry in clinical trials. Nat Immunol 2010; 11 (11) 975-978
    CrossrefPubMedGoogle Scholar
  • 28 Pitoiset F, Cassard L, El Soufi K. et al. Deep phenotyping of immune cell populations by optimized and standardized flow cytometry analyses. Cytometry A 2018; 93 (08) 793-802
    CrossrefPubMedGoogle Scholar
  • 29 Baaten CCFMJ, Moenen FCJI, Henskens YMC. et al. Impaired mitochondrial activity explains platelet dysfunction in thrombocytopenic cancer patients undergoing chemotherapy. Haematologica 2018; 103 (09) 1557-1567
    CrossrefPubMedGoogle Scholar
  • 30 Södergren AL, Ramström S. Platelet subpopulations remain despite strong dual agonist stimulation and can be characterised using a novel six-colour flow cytometry protocol. Sci Rep 2018; 8 (01) 1441
    CrossrefPubMedGoogle Scholar
  • 31 Blair TA, Moore SF, Hers I. Circulating primers enhance platelet function and induce resistance to antiplatelet therapy. J Thromb Haemost 2015; 13 (08) 1479-1493
    CrossrefPubMedGoogle Scholar
  • 32 Schmitz G, Rothe G, Ruf A. et al. European Working Group on Clinical Cell Analysis: consensus protocol for the flow cytometric characterisation of platelet function. Thromb Haemost 1998; 79 (05) 885-896
    Article in Thieme ConnectPubMedGoogle Scholar
  • 33 Waller AK, Lantos L, Sammut A. et al. Flow cytometry for near-patient testing in premature neonates reveals variation in platelet function: a novel approach to guide platelet transfusion. Pediatr Res 2019; 85 (06) 874-884
    CrossrefPubMedGoogle Scholar
  • 34 Gilio K, Munnix IC, Mangin P. et al. Non-redundant roles of phosphoinositide 3-kinase isoforms alpha and beta in glycoprotein VI-induced platelet signaling and thrombus formation. J Biol Chem 2009; 284 (49) 33750-33762
    CrossrefPubMedGoogle Scholar
  • 35 Bagwell B. Super enhanced D-value method (possed). Accessed September 5, 2021 at: http://www.vsh.com/Publication/SuperEnhancedDValue.pdf
    Google Scholar
  • 36 Monaco G, Chen H, Poidinger M, Chen J, de Magalhães JP, Larbi A. flowAI: automatic and interactive anomaly discerning tools for flow cytometry data. Bioinformatics 2016; 32 (16) 2473-2480
    CrossrefPubMedGoogle Scholar
  • 37 Van Gassen S, Callebaut B, Van Helden MJ. et al. FlowSOM: using self-organizing maps for visualization and interpretation of cytometry data. Cytometry A 2015; 87 (07) 636-645
    CrossrefPubMedGoogle Scholar
  • 38 Mattheij NJ, Gilio K, van Kruchten R. et al. Dual mechanism of integrin αIIbβ3 closure in procoagulant platelets. J Biol Chem 2013; 288 (19) 13325-13336
    CrossrefPubMedGoogle Scholar
  • 39 Eckly A, Rinckel JY, Proamer F. et al. Respective contributions of single and compound granule fusion to secretion by activated platelets. Blood 2016; 128 (21) 2538-2549
    CrossrefPubMedGoogle Scholar
  • 40 Jonnalagadda D, Izu LT, Whiteheart SW. Platelet secretion is kinetically heterogeneous in an agonist-responsive manner. Blood 2012; 120 (26) 5209-5216
    CrossrefPubMedGoogle Scholar
  • 41 Spath B, Hansen A, Bokemeyer C, Langer F. Succinate reverses in-vitro platelet inhibition by acetylsalicylic acid and P2Y receptor antagonists. Platelets 2012; 23 (01) 60-68
    CrossrefPubMedGoogle Scholar
  • 42 Prodan CI, Joseph PM, Vincent AS, Dale GL. Coated-platelets in ischemic stroke: differences between lacunar and cortical stroke. J Thromb Haemost 2008; 6 (04) 609-614
    CrossrefPubMedGoogle Scholar
  • 43 Prodan CI, Stoner JA, Cowan LD, Dale GL. Higher coated-platelet levels are associated with stroke recurrence following nonlacunar brain infarction. J Cereb Blood Flow Metab 2013; 33 (02) 287-292
    CrossrefPubMedGoogle Scholar
  • 44 Kirkpatrick AC, Stoner JA, Dale GL, Prodan CI. Elevated coated-platelets in symptomatic large-artery stenosis patients are associated with early stroke recurrence. Platelets 2014; 25 (02) 93-96
    CrossrefPubMedGoogle Scholar
  • 45 Kirkpatrick AC, Tafur AJ, Vincent AS, Dale GL, Prodan CI. Coated-platelets improve prediction of stroke and transient ischemic attack in asymptomatic internal carotid artery stenosis. Stroke 2014; 45 (10) 2995-3001
    CrossrefPubMedGoogle Scholar