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CC BY 4.0 · TH Open 2018; 02(03): e250-e260
DOI: 10.1055/s-0038-1667204
Original Article
Georg Thieme Verlag KG Stuttgart · New York

Sensitive and Specific Detection of Platelet-Derived and Tissue Factor–Positive Extracellular Vesicles in Plasma Using Solid-Phase Proximity Ligation Assay

Åsa Thulin
1   Department of Medical Sciences, Clinical Chemistry and Science for Life Laboratory, Uppsala University, Uppsala, Sweden
,
Junhong Yan
2   Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden
3   Department of Biomedical Engineering, Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, The Netherlands
,
Mikael Åberg
1   Department of Medical Sciences, Clinical Chemistry and Science for Life Laboratory, Uppsala University, Uppsala, Sweden
,
Christina Christersson
4   Department of Medical Sciences, Cardiology, Uppsala University, Uppsala, Sweden
,
Masood Kamali-Moghaddam
2   Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden
,
Agneta Siegbahn
1   Department of Medical Sciences, Clinical Chemistry and Science for Life Laboratory, Uppsala University, Uppsala, Sweden
› Author Affiliations Funding The study was supported by the Swedish Research Council (K2013–65X-11568–18–5 and K2011–67X-13096–13–4), the Swedish Heart-Lung foundation, the Swedish Foundation for Strategic Research, and the Science for Life Laboratory, Uppsala University.
Further Information

Publication History

14 February 2018

13 June 2018

Publication Date:
27 July 2018 (online)

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Abstract

Extracellular vesicles (EVs) derived from blood cells are promising biomarkers for various diseases. However, they are difficult to measure accurately in plasma due to their small size. Here, we demonstrate that platelet-derived EVs in plasma can be measured using solid-phase proximity ligation assay with high sensitivity and specificity using very small sample volume of biological materials. The results correlate well with high-sensitivity flow cytometry with the difference that the smallest EVs are detected. Briefly, the EVs are first captured on a solid phase, using lactadherin binding, and detection requires recognition with two antibodies followed by qPCR. The assay, using cholera toxin subunit-B or lactadherin as capture agents, also allowed detection of the more rare population of tissue factor (TF)-positive EVs at a concentration similar to sensitive TF activity assays. Thus, this assay can detect different types of EVs with high specificity and sensitivity, and has the potential to be an attractive alternative to flow cytometric analysis of preclinical and clinical samples. Improved techniques for measuring EVs in plasma will hopefully contribute to the understanding of their role in several diseases.

Keywords

microvesicles - myocardial infarction - cardiovascular diseases - vascular homeostasis

Authors' Contributions

A.S. conceived the research; M.K-M. and J.Y. participated in the initial discussions and provided the technical protocols for SP-PLA with antibodies as catchers and antibody conjugations with oligonucleotides; J.Y. performed preliminary experiments. A.S. and Å.T. designed the SP-PLA using cholera toxin subunit B and lactadherin to capture EVs; and A.S., Å.T., M.Å., and C.C. designed the research. Å.T. performed the experiments. C.C. provided plasma samples from patients with myocardial infarction. Å.T. and A.S. analyzed the results and Å.T. wrote the first draft of the manuscript. All authors read and commented on the manuscript and approved for submission.


 

  • References

  • 1 Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 2013; 200 (04) 373-383
    CrossrefPubMedGoogle Scholar
  • 2 Vestad B, Llorente A, Neurauter A. , et al. Size and concentration analyses of extracellular vesicles by nanoparticle tracking analysis: a variation study. J Extracell Vesicles 2017; 6 (01) 1344087 ; doi: 10.1080/20013078.2017.1344087
    PubMedGoogle Scholar
  • 3 Ponpuak M, Mandell MA, Kimura T, Chauhan S, Cleyrat C, Deretic V. Secretory autophagy. Curr Opin Cell Biol 2015; 35: 106-116
    CrossrefPubMedGoogle Scholar
  • 4 Hessvik NP, Øverbye A, Brech A. , et al. PIKfyve inhibition increases exosome release and induces secretory autophagy. Cell Mol Life Sci 2016; 73 (24) 4717-4737
    CrossrefPubMedGoogle Scholar
  • 5 De Paoli SH, Tegegn TZ, Elhelu OK. , et al. Dissecting the biochemical architecture and morphological release pathways of the human platelet extracellular vesiculome. Cell Mol Life Sci 2018; DOI: 10.1007/s00018-018-2771-6.
    CrossrefPubMedGoogle Scholar
  • 6 Ayers L, Nieuwland R, Kohler M, Kraenkel N, Ferry B, Leeson P. Dynamic microvesicle release and clearance within the cardiovascular system: triggers and mechanisms. Clin Sci (Lond) 2015; 129 (11) 915-931
    CrossrefPubMedGoogle Scholar
  • 7 Morel O, Toti F, Hugel B, Freyssinet JM. Cellular microparticles: a disseminated storage pool of bioactive vascular effectors. Curr Opin Hematol 2004; 11 (03) 156-164
    CrossrefPubMedGoogle Scholar
  • 8 Amabile N, Rautou P-E, Tedgui A, Boulanger CM. Microparticles: key protagonists in cardiovascular disorders. Semin Thromb Hemost 2010; 36 (08) 907-916
    Article in Thieme ConnectPubMedGoogle Scholar
  • 9 Gidlöf O, van der Brug M, Öhman 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 (19) 3908-3917 , S1–S26
    CrossrefPubMedGoogle Scholar
  • 10 Camussi G, Deregibus MC, Bruno S, Cantaluppi V, Biancone L. Exosomes/microvesicles as a mechanism of cell-to-cell communication. Kidney Int 2010; 78 (09) 838-848
    CrossrefPubMedGoogle Scholar
  • 11 Jansen F, Yang X, Hoyer FF. , et al. Endothelial microparticle uptake in target cells is annexin I/phosphatidylserine receptor dependent and prevents apoptosis. Arterioscler Thromb Vasc Biol 2012; 32 (08) 1925-1935
    CrossrefPubMedGoogle Scholar
  • 12 Thulin Å, Christersson C, Alfredsson J, Siegbahn A. Circulating cell-derived microparticles as biomarkers in cardiovascular disease. Biomarkers Med 2016; 10 (09) 1009-1022
    CrossrefPubMedGoogle Scholar
  • 13 Christersson C, Thulin Å, Siegbahn A. Microparticles during long-term follow-up after acute myocardial infarction. Association to atherosclerotic burden and risk of cardiovascular events. Thromb Haemost 2017; 117 (08) 1571-1581
    Article in Thieme ConnectPubMedGoogle Scholar
  • 14 Tavoosidana G, Ronquist G, Darmanis S. , et al. Multiple recognition assay reveals prostasomes as promising plasma biomarkers for prostate cancer. Proc Natl Acad Sci U S A 2011; 108 (21) 8809-8814
    CrossrefPubMedGoogle Scholar
  • 15 Abid Hussein MN, Böing AN, Sturk A, Hau CM, Nieuwland R. Inhibition of microparticle release triggers endothelial cell apoptosis and detachment. Thromb Haemost 2007; 98 (05) 1096-1107
    Article in Thieme ConnectPubMedGoogle Scholar
  • 16 Taylor Jr FB, Chang A, Ruf W. , et al. Lethal E. coli septic shock is prevented by blocking tissue factor with monoclonal antibody. Circ Shock 1991; 33 (03) 127-134
    PubMedGoogle Scholar
  • 17 Tesselaar ME, Romijn FP, Van Der Linden IK, Prins FA, Bertina RM, Osanto S. Microparticle-associated tissue factor activity: a link between cancer and thrombosis?. J Thromb Haemost 2007; 5 (03) 520-527
    CrossrefPubMedGoogle Scholar
  • 18 Nemerson Y. Tissue factor and hemostasis. Blood 1988; 71 (01) 1-8
    PubMedGoogle Scholar
  • 19 Mackman N. The role of tissue factor and factor VIIa in hemostasis. Anesth Analg 2009; 108 (05) 1447-1452
    CrossrefPubMedGoogle Scholar
  • 20 Giesen PLA, Rauch U, Bohrmann B. , et al. Blood-borne tissue factor: another view of thrombosis. Proc Natl Acad Sci U S A 1999; 96 (05) 2311-2315
    CrossrefPubMedGoogle Scholar
  • 21 Pawlinski R, Pedersen B, Schabbauer G. , et al. Role of tissue factor and protease-activated receptors in a mouse model of endotoxemia. Blood 2004; 103 (04) 1342-1347
    CrossrefPubMedGoogle Scholar
  • 22 Lindmark E, Tenno T, Siegbahn A. Role of platelet P-selectin and CD40 ligand in the induction of monocytic tissue factor expression. Arterioscler Thromb Vasc Biol 2000; 20 (10) 2322-2328
    CrossrefPubMedGoogle Scholar
  • 23 Åberg M, Eriksson O, Siegbahn A. Tissue factor noncoagulant signaling: mechanisms and implications for cell migration and apoptosis. Semin Thromb Hemost 2015; 41 (07) 691-699
    Article in Thieme ConnectPubMedGoogle Scholar
  • 24 Schaffner F, Yokota N, Ruf W. Tissue factor proangiogenic signaling in cancer progression. Thromb Res 2012; 129 (Suppl. 01) S127-S131
    CrossrefPubMedGoogle Scholar
  • 25 Ruf W, Disse J, Carneiro-Lobo TC, Yokota N, Schaffner F. Tissue factor and cell signalling in cancer progression and thrombosis. J Thromb Haemost 2011; 9 (Suppl. 01) 306-315
    CrossrefPubMedGoogle Scholar
  • 26 Mackman N, Tilley RE, Key NS. Role of the extrinsic pathway of blood coagulation in hemostasis and thrombosis. Arterioscler Thromb Vasc Biol 2007; 27 (08) 1687-1693
    CrossrefPubMedGoogle Scholar
  • 27 Lechner D, Weltermann A. Circulating tissue factor-exposing microparticles. Thromb Res 2008; 122 (Suppl. 01) S47-S54
    CrossrefPubMedGoogle Scholar
  • 28 Rao LVM, Pendurthi UR. Regulation of tissue factor coagulant activity on cell surfaces. J Thromb Haemost 2012; 10 (11) 2242-2253
    CrossrefPubMedGoogle Scholar
  • 29 Robert S, Lacroix R, Poncelet P. , et al. High-sensitivity flow cytometry provides access to standardized measurement of small-size microparticles--brief report. Arterioscler Thromb Vasc Biol 2012; 32 (04) 1054-1058
    CrossrefPubMedGoogle Scholar
  • 30 Aass HCD, Øvstebø R, Trøseid A-MS, Kierulf P, Berg JP, Henriksson CE. Fluorescent particles in the antibody solution result in false TF- and CD14-positive microparticles in flow cytometric analysis. Cytometry A 2011; 79 (12) 990-999
    PubMedGoogle Scholar
  • 31 Christersson C, Johnell M, Siegbahn A. Evaluation of microparticles in whole blood by multicolour flow cytometry assay. Scand J Clin Lab Invest 2013; 73 (03) 229-239
    CrossrefPubMedGoogle Scholar
  • 32 Linares R, Tan S, Gounou C. , et al. High-speed centrifugation induces aggregation of extracellular vesicles. J Extracell Vesicles 2015; 4 (01) 29509 ; doi: 10.3402/jev.v4.29509
    PubMedGoogle Scholar
  • 33 Löf L, Ebai T, Dubois L. , et al. Detecting individual extracellular vesicles using a multicolor in situ proximity ligation assay with flow cytometric readout. Sci Rep 2016; 6: 34358
    CrossrefPubMedGoogle Scholar
  • 34 Headland SE, Jones HR, D'Sa ASV, Perretti M, Norling LV. Cutting-edge analysis of extracellular microparticles using ImageStream(X) imaging flow cytometry. Sci Rep 2014; 4: 5237
    PubMedGoogle Scholar
  • 35 Wisgrill L, Lamm C, Hartmann J. , et al. Peripheral blood microvesicles secretion is influenced by storage time, temperature, and anticoagulants. Cytometry A 2016; 89 (07) 663-672
    CrossrefPubMedGoogle Scholar
  • 36 Nong RY, Wu D, Yan J. , et al. Solid-phase proximity ligation assays for individual or parallel protein analyses with readout via real-time PCR or sequencing. Nat Protoc 2013; 8 (06) 1234-1248
    CrossrefPubMedGoogle Scholar
  • 37 Darmanis S, Nong RY, Hammond M. , et al. Sensitive plasma protein analysis by microparticle-based proximity ligation assays. Mol Cell Proteomics 2010; 9 (02) 327-335
    CrossrefPubMedGoogle Scholar
  • 38 Cuatrecasas P. Interaction of Vibrio cholerae enterotoxin with cell membranes. Biochemistry 1973; 12 (18) 3547-3558
    CrossrefPubMedGoogle Scholar
  • 39 Tan KH, Tan SS, Sze SK, Lee WK, Ng MJ, Lim SK. Plasma biomarker discovery in preeclampsia using a novel differential isolation technology for circulating extracellular vesicles. Am J Obstet Gynecol 2014; 211 (04) 380.e1-380.e13
    CrossrefPubMedGoogle Scholar
  • 40 Shi J, Heegaard CW, Rasmussen JT, Gilbert GE. Lactadherin binds selectively to membranes containing phosphatidyl-L-serine and increased curvature. Biochim Biophys Acta 2004; 1667 (01) 82-90
    CrossrefPubMedGoogle Scholar
  • 41 Christersson C, Lindahl B, Berglund L, Siegbahn A, Oldgren J. The utility of coagulation activity for prediction of risk of mortality and cardiovascular events in guideline-treated myocardial infarction patients. Ups J Med Sci 2017; 122 (04) 224-233
    CrossrefPubMedGoogle Scholar
  • 42 Sánchez-López V, Vila-Liante V, Arellano-Orden E. , et al. High correlation between 2 flow cytometry platforms in the microparticles analysis using a new calibrated beads strategy. Transl Res 2015; 166 (06) 733-739
    CrossrefPubMedGoogle Scholar
  • 43 Khorana AA, Francis CW, Menzies KE. , et al. Plasma tissue factor may be predictive of venous thromboembolism in pancreatic cancer. J Thromb Haemost 2008; 6 (11) 1983-1985
    CrossrefPubMedGoogle Scholar
  • 44 Ollivier V, Wang J, Manly D. , et al. Detection of endogenous tissue factor levels in plasma using the calibrated automated thrombogram assay. Thromb Res 2010; 125 (01) 90-96
    CrossrefPubMedGoogle Scholar
  • 45 Suades R, Padró T, Vilahur G. , et al. Growing thrombi release increased levels of CD235a(+) microparticles and decreased levels of activated platelet-derived microparticles. Validation in ST-elevation myocardial infarction patients. J Thromb Haemost 2015; 13 (10) 1776-1786
    CrossrefPubMedGoogle Scholar
  • 46 Ebai T, Kamali-Moghaddam M, Landegren U. Parallel protein detection by solid-phase proximity ligation assay with real-time PCR or sequencing. Curr Protoc Mol Biol 2015; 109: 1-25
    PubMedGoogle Scholar
  • 47 Darmanis S, Nong RY, Vänelid J. , et al. ProteinSeq: high-performance proteomic analyses by proximity ligation and next generation sequencing. PLoS One 2011; 6 (09) e25583
    CrossrefPubMedGoogle Scholar
  • 48 Parton RG. Ultrastructural localization of gangliosides; GM1 is concentrated in caveolae. J Histochem Cytochem 1994; 42 (02) 155-166
    CrossrefPubMedGoogle Scholar
  • 49 Davizon P, Munday AD, López JA. Tissue factor, lipid rafts, and microparticles. Semin Thromb Hemost 2010; 36 (08) 857-864
    Article in Thieme ConnectPubMedGoogle Scholar
  • 50 Del Conde I, Shrimpton CN, Thiagarajan P, López JA. Tissue-factor-bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation. Blood 2005; 106 (05) 1604-1611
    CrossrefPubMedGoogle Scholar
  • 51 Lee RD, Barcel DA, Williams JC. , et al. Pre-analytical and analytical variables affecting the measurement of plasma-derived microparticle tissue factor activity. Thromb Res 2012; 129 (01) 80-85
    CrossrefPubMedGoogle Scholar
  • 52 Hellum M, Øvstebø R, Trøseid A-MS, Berg JP, Brandtzaeg P, Henriksson CE. Microparticle-associated tissue factor activity measured with the Zymuphen MP-TF kit and the calibrated automated thrombogram assay. Blood Coagul Fibrinolysis 2012; 23 (06) 520-526
    CrossrefPubMedGoogle Scholar
  • 53 Breimo ES, Østerud B. Generation of tissue factor-rich microparticles in an ex vivo whole blood model. Blood Coagul Fibrinolysis 2005; 16 (06) 399-405
    CrossrefPubMedGoogle Scholar
  • 54 Chen VM, Hogg PJ. Encryption and decryption of tissue factor. J Thromb Haemost 2013; 11 (Suppl. 01) 277-284
    CrossrefPubMedGoogle Scholar
  • 55 Aras O, Shet A, Bach RR. , et al. Induction of microparticle- and cell-associated intravascular tissue factor in human endotoxemia. Blood 2004; 103 (12) 4545-4553
    CrossrefPubMedGoogle Scholar
  • 56 Shet AS, Aras O, Gupta K. , et al. Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes. Blood 2003; 102 (07) 2678-2683
    CrossrefPubMedGoogle Scholar