Download PDF
CC BY 4.0 · TH Open 2018; 02(02): e147-e157
DOI: 10.1055/s-0038-1642021
Original Article
Georg Thieme Verlag KG Stuttgart · New York

Postprandial Increase in Blood Plasma Levels of Tissue Factor–Bearing (and Other) Microvesicles Measured by Flow Cytometry: Fact or Artifact?

Morten Mørk
1   Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark
2   Aalborg AF Study Group, Aalborg University Hospital, Aalborg, Denmark
3   EVsearch.dk, Aalborg, Denmark
,
Morten H. Nielsen
1   Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark
3   EVsearch.dk, Aalborg, Denmark
,
Rikke Bæk
3   EVsearch.dk, Aalborg, Denmark
4   Department of Clinical Immunology, Aalborg University Hospital, Aalborg, Denmark
,
Malene M. Jørgensen
3   EVsearch.dk, Aalborg, Denmark
4   Department of Clinical Immunology, Aalborg University Hospital, Aalborg, Denmark
,
Shona Pedersen
1   Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark
3   EVsearch.dk, Aalborg, Denmark
5   Department of Clinical Medicine, Aalborg University, Aalborg, Denmark
,
Søren R. Kristensen
1   Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark
2   Aalborg AF Study Group, Aalborg University Hospital, Aalborg, Denmark
3   EVsearch.dk, Aalborg, Denmark
5   Department of Clinical Medicine, Aalborg University, Aalborg, Denmark
› Author Affiliations Funding The study was supported by research grants from the Danish Council for Strategic Research.
Further Information

Publication History

15 November 2017

01 March 2018

Publication Date:
16 April 2018 (online)

Also available at
   Permissions and Reprints

Abstract

Tissue factor (TF)–bearing microvesicles (MVs) and exosomes may play a role in hemostasis and thrombosis. MVs may be quantified by flow cytometry (FC)–based detection of phosphatidylserine (PS)-positive submicron particles carrying specific antigens, although interference from lipoproteins complicates this approach. In this study, we evaluated the effect of food intake on blood levels of TF-bearing particles measured by FC and small extracellular vesicles (EVs) measured by a protein microarray–based test termed EV Array. Platelet-free plasma (PFP) was obtained from 20 healthy persons in the fasting state and 75 minutes after consumption of a meal. Postprandial changes in the concentration of PS-positive particles, including subgroups binding labeled antibodies against TF, CD41, CD146, and CD62E, respectively (FC), small EVs (EV Array), and TF antigen and procoagulant phospholipids (PPLs) were measured. Furthermore, we tested the effect on FC results of in vitro addition of lipoproteins to fasting PFP. We found significantly increased plasma concentrations of PS-positive particles and all examined subgroups postprandially, while no changes in small EVs, PPL, or TF antigen levels were found. Levels of all types of particles measured by FC were also elevated by lipoprotein spiking. In conclusion, meal consumption as well as in vitro addition of lipoproteins to fasting plasma induces increased levels of PS-positive particles as measured by FC, including TF-positive subtypes and subtypes exposing other antigens. While the observed postprandial increase may to some extent reflect elevated MV levels, our results indicate a substantial interference from lipoproteins.

Keywords

extracellular vesicles - tissue factor - phosphatidylserine - lipoproteins - flow cytometry
 

  • References

  • 1 Owens III AP, Mackman N. Microparticles in hemostasis and thrombosis. Circ Res 2011; 108 (10) 1284-1297
    CrossrefPubMedSearch in Google Scholar
  • 2 Zwicker JI, Trenor III CC, Furie BC, Furie B. Tissue factor-bearing microparticles and thrombus formation. Arterioscler Thromb Vasc Biol 2011; 31 (04) 728-733
    CrossrefPubMedSearch in Google Scholar
  • 3 Bogdanov VY, Balasubramanian V, Hathcock J, Vele O, Lieb M, Nemerson Y. Alternatively spliced human tissue factor: a circulating, soluble, thrombogenic protein. Nat Med 2003; 9 (04) 458-462
    CrossrefPubMedSearch in Google Scholar
  • 4 Morel O, Toti F, Hugel B. , et al. Procoagulant microparticles: disrupting the vascular homeostasis equation?. Arterioscler Thromb Vasc Biol 2006; 26 (12) 2594-2604
    CrossrefPubMedSearch in Google Scholar
  • 5 Davila M, Robles-Carrillo L, Unruh D. , et al. Microparticle association and heterogeneity of tumor-derived tissue factor in plasma: is it important for coagulation activation?. J Thromb Haemost 2014; 12 (02) 186-196
    CrossrefPubMedSearch in Google Scholar
  • 6 Giesen PL, 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
    CrossrefPubMedSearch in Google Scholar
  • 7 Furie B, Furie BC. Mechanisms of thrombus formation. N Engl J Med 2008; 359 (09) 938-949
    CrossrefPubMedSearch in Google Scholar
  • 8 Gould SJ, Raposo G. As we wait: coping with an imperfect nomenclature for extracellular vesicles. J Extracell Vesicles 2013; 2: 2013
    PubMedSearch in Google Scholar
  • 9 Kalra H, Drummen GP, Mathivanan S. Focus on extracellular vesicles: introducing the next small big thing. Int J Mol Sci 2016; 17 (02) 170
    CrossrefPubMedSearch in Google Scholar
  • 10 Théry C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 2009; 9 (08) 581-593
    CrossrefPubMedSearch in Google Scholar
  • 11 Heijnen HF, Schiel AE, Fijnheer R, Geuze HJ, Sixma JJ. Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. Blood 1999; 94 (11) 3791-3799
    CrossrefPubMedSearch in Google Scholar
  • 12 Mørk M, Pedersen S, Botha J, Lund SM, Kristensen SR. Preanalytical, analytical, and biological variation of blood plasma submicron particle levels measured with nanoparticle tracking analysis and tunable resistive pulse sensing. Scand J Clin Lab Invest 2016; 76 (05) 349-360
    CrossrefPubMedSearch in Google Scholar
  • 13 Sódar BW, Kittel Á, Pálóczi K. , et al. Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection. Sci Rep 2016; 6: 24316
    CrossrefPubMedSearch in Google Scholar
  • 14 Wojczynski MK, Glasser SP, Oberman A. , et al. High-fat meal effect on LDL, HDL, and VLDL particle size and number in the Genetics of Lipid-Lowering Drugs and Diet Network (GOLDN): an interventional study. Lipids Health Dis 2011; 10: 181
    CrossrefPubMedSearch in Google Scholar
  • 15 Tushuizen ME, Nieuwland R, Scheffer PG, Sturk A, Heine RJ, Diamant M. Two consecutive high-fat meals affect endothelial-dependent vasodilation, oxidative stress and cellular microparticles in healthy men. J Thromb Haemost 2006; 4 (05) 1003-1010
    CrossrefPubMedSearch in Google Scholar
  • 16 Tushuizen ME, Diamant M, Peypers EG. , et al. Postprandial changes in the phospholipid composition of circulating microparticles are not associated with coagulation activation. Thromb Res 2012; 130 (01) 115-121
    CrossrefPubMedSearch in Google Scholar
  • 17 Suštar V, Bedina-Zavec A, Stukelj R. , et al. Post-prandial rise of microvesicles in peripheral blood of healthy human donors. Lipids Health Dis 2011; 10: 47
    CrossrefPubMedSearch in Google Scholar
  • 18 van der Pol E, Böing AN, Harrison P, Sturk A, Nieuwland R. Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev 2012; 64 (03) 676-705
    CrossrefPubMedSearch in Google Scholar
  • 19 Morel O, Jesel L, Freyssinet JM, Toti F. Cellular mechanisms underlying the formation of circulating microparticles. Arterioscler Thromb Vasc Biol 2011; 31 (01) 15-26
    CrossrefPubMedSearch in Google Scholar
  • 20 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
    CrossrefPubMedSearch in Google Scholar
  • 21 Baek R, Jorgensen M. Multiplexed phenotyping of small extracellular vesicles using protein microarray (EV array). In: Hill AF. , ed. Exosomes and Microvesicles: Methods and Protocols, Methods in Molecular Biology. 1st ed. New York, NY: Springer Science + Business Media LLC; 2017: 117-127
    Search in Google Scholar
  • 22 Andreu Z, Yáñez-Mó M. Tetraspanins in extracellular vesicle formation and function. Front Immunol 2014; 5: 442
    PubMedSearch in Google Scholar
  • 23 Lacroix R, Judicone C, Poncelet P. , et al. Impact of pre-analytical parameters on the measurement of circulating microparticles: towards standardization of protocol. J Thromb Haemost 2012; 10 (03) 437-446
    CrossrefPubMedSearch in Google Scholar
  • 24 Nielsen MH, Beck-Nielsen H, Andersen MN, Handberg A. A flow cytometric method for characterization of circulating cell-derived microparticles in plasma. J Extracell Vesicles 2014; 3
    PubMedSearch in Google Scholar
  • 25 Otzen DE, Blans K, Wang H, Gilbert GE, Rasmussen JT. Lactadherin binds to phosphatidylserine-containing vesicles in a two-step mechanism sensitive to vesicle size and composition. Biochim Biophys Acta 2012; 1818 (04) 1019-1027
    CrossrefPubMedSearch in Google Scholar
  • 26 Lacroix R, Robert S, Poncelet P, Dignat-George F. Overcoming limitations of microparticle measurement by flow cytometry. Semin Thromb Hemost 2010; 36 (08) 807-818
    Thieme ConnectPubMedSearch in Google Scholar
  • 27 Takahashi T, Kobayashi S, Fujino N. , et al. Increased circulating endothelial microparticles in COPD patients: a potential biomarker for COPD exacerbation susceptibility. Thorax 2012; 67 (12) 1067-1074
    CrossrefPubMedSearch in Google Scholar
  • 28 Jørgensen MM, Bæk R, Varming K. Potentials and capabilities of the extracellular vesicle (EV) array. J Extracell Vesicles 2015; 4
    PubMedSearch in Google Scholar
  • 29 Jørgensen M, Bæk R, Pedersen S, Søndergaard EK, Kristensen SR, Varming K. Extracellular vesicle (EV) array: microarray capturing of exosomes and other extracellular vesicles for multiplexed phenotyping. J Extracell Vesicles 2013; 2
    PubMedSearch in Google Scholar
  • 30 Savorani F, Kristensen M, Larsen FH, Astrup A, Engelsen SB. High throughput prediction of chylomicron triglycerides in human plasma by nuclear magnetic resonance and chemometrics. Nutr Metab (Lond) 2010; 7: 43
    CrossrefPubMedSearch in Google Scholar
  • 31 Nakajima K, Nakano T, Tokita Y. , et al. Postprandial lipoprotein metabolism: VLDL vs chylomicrons. Clin Chim Acta 2011; 412 (15–16): 1306-1318
    CrossrefPubMedSearch in Google Scholar
  • 32 Schneeman BO, Kotite L, Todd KM, Havel RJ. Relationships between the responses of triglyceride-rich lipoproteins in blood plasma containing apolipoproteins B-48 and B-100 to a fat-containing meal in normolipidemic humans. Proc Natl Acad Sci U S A 1993; 90 (05) 2069-2073
    CrossrefPubMedSearch in Google Scholar
  • 33 van der Pol E, Böing AN, Gool EL, Nieuwland R. Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles. J Thromb Haemost 2016; 14 (01) 48-56
    CrossrefPubMedSearch in Google Scholar
  • 34 Lacroix R, Robert S, Sabatier F, Dignat-George F. Flow cytometry. In: Harrison P, Gardiner C, Sargent I. , eds. Extracellular Vesicles in Health and Disease. 1st ed. Singapore: Pan Stanford Publishing Pte. Ltd.; 2014: 201-222
    Search in Google Scholar
  • 35 Arraud N, Linares R, Tan S. , et al. Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration. J Thromb Haemost 2014; 12 (05) 614-627
    CrossrefPubMedSearch in Google Scholar
  • 36 Mathivanan S, Ji H, Simpson RJ. Exosomes: extracellular organelles important in intercellular communication. J Proteomics 2010; 73 (10) 1907-1920
    CrossrefPubMedSearch in Google Scholar
  • 37 van der Pol E, van Gemert MJ, Sturk A, Nieuwland R, van Leeuwen TG. Single vs. swarm detection of microparticles and exosomes by flow cytometry. J Thromb Haemost 2012; 10 (05) 919-930
    CrossrefPubMedSearch in Google Scholar
  • 38 Mørk M, Handberg A, Pedersen S. , et al. Prospects and limitations of antibody-mediated clearing of lipoproteins from blood plasma prior to nanoparticle tracking analysis of extracellular vesicles. J Extracell Vesicles 2017; 6 (01)
    PubMedSearch in Google Scholar
  • 39 Nappo F, Esposito K, Cioffi M. , et al. Postprandial endothelial activation in healthy subjects and in type 2 diabetic patients: role of fat and carbohydrate meals. J Am Coll Cardiol 2002; 39 (07) 1145-1150
    CrossrefPubMedSearch in Google Scholar
  • 40 Lundman P, Eriksson MJ, Silveira A. , et al. Relation of hypertriglyceridemia to plasma concentrations of biochemical markers of inflammation and endothelial activation (C-reactive protein, interleukin-6, soluble adhesion molecules, von Willebrand factor, and endothelin-1). Am J Cardiol 2003; 91 (09) 1128-1131
    CrossrefPubMedSearch in Google Scholar
  • 41 Dignat-George F, Boulanger CM. The many faces of endothelial microparticles. Arterioscler Thromb Vasc Biol 2011; 31 (01) 27-33
    CrossrefPubMedSearch in Google Scholar
  • 42 Ferreira AC, Peter AA, Mendez AJ. , et al. Postprandial hypertriglyceridemia increases circulating levels of endothelial cell microparticles. Circulation 2004; 110 (23) 3599-3603
    CrossrefPubMedSearch in Google Scholar
  • 43 Fragopoulou E, Detopoulou P, Nomikos T, Pliakis E, Panagiotakos DB, Antonopoulou S. Mediterranean wild plants reduce postprandial platelet aggregation in patients with metabolic syndrome. Metabolism 2012; 61 (03) 325-334
    CrossrefPubMedSearch in Google Scholar
  • 44 Spectre G, Östenson CG, Li N, Hjemdahl P. Postprandial platelet activation is related to postprandial plasma insulin rather than glucose in patients with type 2 diabetes. Diabetes 2012; 61 (09) 2380-2384
    CrossrefPubMedSearch in Google Scholar
  • 45 Michelsen AE, Notø AT, Brodin E, Mathiesen EB, Brosstad F, Hansen JB. Elevated levels of platelet microparticles in carotid atherosclerosis and during the postprandial state. Thromb Res 2009; 123 (06) 881-886
    CrossrefPubMedSearch in Google Scholar
  • 46 Berckmans RJ, Nieuwland R, Böing AN, Romijn FP, Hack CE, Sturk A. Cell-derived microparticles circulate in healthy humans and support low grade thrombin generation. Thromb Haemost 2001; 85 (04) 639-646
    Thieme ConnectPubMedSearch in Google Scholar
  • 47 Kocatürk B, Versteeg HH. Tissue factor isoforms in cancer and coagulation: may the best isoform win. Thromb Res 2012; 129 (Suppl. 01) S69-S75
    CrossrefPubMedSearch in Google Scholar
  • 48 Motton DD, Mackman N, Tilley RE, Rutledge JC. Postprandial elevation of tissue factor antigen in the blood of healthy adults. Thromb Haemost 2005; 94 (03) 504-509
    Thieme ConnectPubMedSearch in Google Scholar
  • 49 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
    CrossrefPubMedSearch in Google Scholar