An innovative flow cytometric approach for small-size platelet microparticles: Influence of calcium

Summary

Microparticles (MPs) are small submicron membrane-derived vesicles shed from a variety of cells and they have been implicated in different disorders. Accordingly, understanding of physiological characteristics of MPs and improvement of methods of their quantification are important for further advance in the field. Although flow cytometry is the most widely applied technique for MP analysis, it is limited by lack of adequate standardisation. Annexin V (AnV), which binds surface phos-phatidylserine (PS) with high affinity, has been long regarded as a marker of MPs, but AnV binding is Ca²⁺-dependent and it is unclear how [Ca²⁺] concentrations could affect AnV binding to MPs and its enumeration. MPs from citrated and heparinised plasma were labelled with AnV, anti-CD42b and quantified using an Apogee A50 flow cytometer. The small-size MP gate was defined with the use of size beads (from 0.1 to 0.5 μm) and confirmed with an in vitro assessment of platelet stimulation. Biotinylated anti-CD42b antibodies were then bound to streptavidin conjugated with different fluorochromes, leading to an amplified signal of platelet MPs (PMPs). Moderate increase of [Ca²⁺] concentrations in the annexin V staining buffer allows initial plasma recalcification and more accurate MP quantification in citrated plasma. Thrombin stimulation of platelet-free plasma containing only MPs did not produce any changes in the concentration of AnV⁺ MPs, but decreased the anti-CD42b binding. The results also indicate that prolonged storage and thrombin induce the release of AnV⁺ MPs whereas PS exposure in pre-existent MPs is not affected by thrombin. In conclusion, we present a sensitive protocol for the analysis of circulating and in vitro induced small-size PMPs that might contribute to future cardiovascular and clinical research.

Note: The editorial process for this article was fully handled by Prof. Christian Weber, Editor-in-Chief.

Keywords

Small-size microparticles - annexin V - phosphatidylserine - calcium - streptavidin - flow cytometry

• References

• 1 Shcherbina A, Remold-O'Donnell E. Role of caspase in a subset of human platelet activation responses. Blood 1999; 93: 4222-4231.
• 2 Owens 3rd AP, Mackman N. Microparticles in haemostasis and thrombosis. Circ Res 2011; 108: 1284-1297.
• 3 Tsimerman G, Roguin A, Bachar A. et al. Involvement of microparticles in diabetic vascular complications. Thromb Haemost 2011; 106: 310-321.
• 4 Shantsila E, Kamphuisen PW, Lip GY. Circulating microparticles in cardiovascular disease: implications for atherogenesis and atherothrombosis. J Thromb Haemost 2010; 08: 2358-2368.
• 5 Trappenburg MC, van Schilfgaarde M, Marchetti M. et al. Elevated procoagulant microparticles expressing endothelial and platelet markers in essential thrombocythemia. Haematologica 2009; 94: 911-918.
• 6 Jimenez JJ, Jy W, Mauro LM. et al. Endothelial microparticles released in thrombotic thrombocytopenic purpura express von Willebrand factor and markers of endothelial activation. Br J Haematol 2003; 123: 896-902.
• 7 Horstman LL, Jy W, Jimenez JJ. et al. Endothelial microparticles as markers of en-dothelial dysfunction. Front Biosci 2004; 09: 1118-1135.
• 8 Jayachandran M, Litwiller RD, Owen WG. et al. Characterization of blood borne microparticles as markers of premature coronary calcification in newly menopausal women. Am J Physiol Heart Circ Physiol 2008; 295: H931-H938.
• 9 Lacroix R, Robert S, Poncelet P. et al. Standardization of platelet-derived microparticle enumeration by flow cytometry with calibrated beads: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop. J Thromb Haemost 2010; 08: 2571-2574.
• 10 Robert S, Poncelet P, Lacroix R. et al. Standardization of platelet-derived microparticle counting using calibrated beads and a Cytomics FC500 routine flow cytometer: a first step towards multicenter studies?. J Thromb Haemost 2009; 07: 190-197.
• 11 Orozco AF, Lewis DE. Flow cytometric analysis of circulating microparticles in plasma. Cytometry A 2010; 77: 502-514.
• 12 Dragovic RA, Gardiner C, Brooks AS. et al. Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis. Nanomedicine 2011; 07: 780-788.
• 13 Yuana Y, Oosterkamp TH, Bahatyrova S. et al. Atomic force microscopy: a novel approach to the detection of nanosized blood microparticles. J Thromb Haemost 2010; 08: 315-323.
• 14 Ayers L, Kohler M, Harrison P. et al. Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay. Thromb Res 2011; 127: 370-377.
• 15 Chandler WL, Yeung W, Tait JF. A new microparticle size calibration standard for use in measuring smaller microparticles using a new flow cytometer. J Thromb Haemost 2011; 09: 1216-1224.
• 16 Lacroix R, Robert S, Poncelet P. et al. Overcoming limitations of microparticle measurement by flow cytometry. Semin Thromb Hemost 2010; 36: 807-818.
• 17 van Tits LJ, van Heerde WL, Landburg PP. et al. Plasma annexin A5 and microparticle phosphatidylserine levels are elevated in sickle cell disease and increase further during painful crisis. Biochem Biophys Res Commun 2009; 390: 161-164.
• 18 Connor DE, Exner T, Ma DD. et al. The majority of circulating platelet-derived microparticles fail to bind annexin V, lack phospholipid-dependent procoagulant activity and demonstrate greater expression of glycoprotein Ib. Thromb Haemost 2010; 103: 1044-1052.
• 19 Fathi F, Altiraihi T, Mowla SJ. et al. Formation of embryoid bodies from mouse embryonic stem cells cultured on silicon-coated surfaces. Cytotechnology 2009; 59: 11-16.
• 20 Plaza EM, Lozano ML, Sánchez Guiu I. et al. Evaluation of platelet function during extended storage in additive solution, prepared in a new container that allows manual buffy-coat platelet pooling and leucoreduction in the same system. Blood Transfus. 2012 epub ahead of print.
• 21 Bogusiewicz A, Mock NI, Mock DM. Instability of the biotin-protein bond in human plasma. Anal Biochem 2004; 327: 156-1561.
• 22 Yuana Y, Bertina RM, Osanto S. Pre-analytical and analytical issues in the analysis of blood microparticles. Thromb Haemost 2011; 105: 396-408.
• 23 Shah MD, Bergeron AL, Dong J F. et al. Flow cytometric measurement of microparticles: pitfalls and protocol modifications. Platelets 2008; 19: 365-372.
• 24 Richter R P, Him JL, Tessier B. et al. On the kinetics of adsorption and two-dimensional self-assembly of annexin A5 on supported lipid bilayers. Biophys J 2005; 89: 3372-3385.
• 25 Tsai WJ, Chen JC, Wang CT. Changes in both calcium pool size and morphology of human platelets incubated in various concentrations of calcium ion. Calcium-specific bleb formation on platelet-membrane surface. Biochim Biophys Acta 1988; 940: 105-120.
• 26 Lentz BR. Exposure of platelet membrane phosphatidylserine regulates blood coagulation. Prog Lipid Res 2003; 42: 423-438.
• 27 Rober t S, Lacroix R, Poncelet P. et al. High-sensitivity flow cytometry provides access to standardized measurement of small-size microparticles. Arterioscler Thromb Vasc Biol. 2012 Epub ahead of print.
• 28 Li CQ, Vindigni A, Sadler JE. et al. Platelet glycoprotein Ib alpha binds to thrombin anion-binding exosite II inducing allosteric changes in the activity of thrombin. J Biol Chem 2001; 276: 6161-6168.
• 29 Hourdille P, Heilmann E, Combrie R. et al. Thrombin induces a rapid redistribution of glycoprotein Ib-IX complexes within the membrane systems of activated human platelets. Blood 1990; 76: 1503-1513.
• 30 György B, Módos K, Pállinger E. et al. Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters. Blood 2011; 117: e39-48.
• 31 Dasgupta SK, Guchhait P, Thiagarajan P. Lactadherin binding and phosphatidylserine expression on cell surface-comparison with annexin A5. Transl Res 2006; 148: 19-25.
• 32 Shi J, Gilbert GE. Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid-binding sites. Blood 2003; 101: 2628-2636.