Flow Cytometric Analysis of Calcium Mobilization in Whole‐Blood Platelets

Maria‐do‐Céu Monteiro1, Maria‐José Gonçalves1, Filipe Sansonetty2, José‐Enrique O'Connor3

1 Instituto Politécnico de Saüde‐Norte, Paredes, Portugal, 2 Escola de Ciências de Saúde, Universidade do Minho, Braga, Portugal, 3 Centro de Citometría y Citómica, Universidad de Valencia, Valencia, Spain
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.20
DOI:  10.1002/0471142956.cy0920s24
Online Posting Date:  May, 2003
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Abstract

Flow cytometry provides a convenient method to evaluate platelet activation by following the kinetics of intracellular free Ca2+, using sensitive fluorescent indicators that can be loaded into intact cells. Moreover, in the clinical setting, whole‐blood techniques have obvious advantages to avoid artifactual platelet activation and allow the maintenance of near‐physiological conditions. This unit describes a fast and sensitive flow cytometric procedure using the Ca2+‐sensitive dye fluo‐3 AM and the platelet‐specific antibody CD41‐PE to determine the kinetics of intracellular Ca2+ mobilization in whole‐blood platelets with minimal manipulation of the samples. The technique may be applied to reveal fast and transient increases in cytosolic calcium upon platelet stimulation with the agonists ADP and thrombin. This protocol provides a simple and sensitive tool to assess in vitro the time course and intensity of signal‐transduction responses to agonists under near‐physiological conditions, and should be broadly applicable to studies of platelet reactivity.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Rested donor free from antiplatelet treatment
  • 135 mM tribasic sodium citrate, pH 7.4
  • Modified Tyrode's buffer (see recipe)
  • 1 mM fluo‐3 AM (see recipe)
  • CD41‐PE monoclonal antibody (Immunotech)
  • 5 mg/ml glycyl‐L‐prolyl‐L‐arginyl‐L‐proline (GPRP; see recipe); for thrombin activation studies
  • Agonist (e.g., human α‐thrombin, ADP; see recipe)
  • 19‐G needle
  • 12 × 75–mm polypropylene tubes
  • Flow cytometer with 488‐nm excitation and band‐pass filters centered at 525 (green) and 575 nm (orange)
NOTE: Use new or carefully washed polypropylene tubes in all steps to avoid platelet activation on glass or polystyrene surfaces.NOTE: The reader is encouraged to read unit 6.10 for more detail on platelet handling and unit 9.8 for data analysis.
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Figures

Videos

Literature Cited

Literature Cited
   Achyuthan, K.E., Dobson, J.V., and Greenberg, C.S. 1986. Gly‐Pro‐Arg‐Pro modifies the glutamine residues in alpha‐ gamma‐chains of fibrinogen: Inhibition of transglutaminase cross‐linking. Biochem. Biophys. Acta 872:261‐268.
   Bennett, J.S. and Kolodziej, M.A. 1992. Disorders of platelet function. Dis. Mon. 38:577‐631.
   Davies, T.A., Drotts, D., Weil, G.J., and Simons, E.R. 1988. Flow cytometric measurements of cytoplasmic calcium changes in human platelets. Cytometry 9:138‐142.
   Davies, T.A., Drotts, D.L., Weil, G.J., and Simons, E.R. 1989. Cytoplasmatic Ca2+ is necessary for thrombin‐induced platelet activation. J. Biol. Chem. 264:19600‐19606.
   Fijnheer, R., Homburg, C.H.E., Hooibrink, B., Boomgaard, M.N., Korte, D., and Ross, D. 1991. Loss of thrombin‐induced Ca2+ mobilization in a subpopulation of platelets during storage. Thromb. Haemost. 66:350‐354.
   Jennings, L.K., Dockter, M.E., Wall, C.D., Fox, C.F., and Kennedy, D.M. 1989. Calcium mobilization in human platelets using indo‐1 and flow cytometry. Blood 74:2674‐2680.
   Johnson, P.C., Ware, J.A., and Cliveden, P.B. 1985. Measurement of ionized calcium in blood platelets with photoprotein aequorin. Comparison with quin‐2. J. Biol. Chem. 260:2069‐2076.
   Kyle, P.M., Jackson, M.C., Buckley, D.C., Swiet, M., and Redman, C.W.G. 1995. Platelet intracellular free calcium response to arginine vasopressin is similar in preeclampsia and normal pregnancy. Am. J. Obstet. Gynecol. 172:654‐660.
   Larsen, E., Celi, A., Gilbert, G.E., Furie, B.C., Erban, J.K., Bonfanti, R., Wagner, D.D., and Furie, B. 1989. PADGEM protein: A receptor that mediates the interaction of activated platelets with neutrophils and monocytes. Cell 59:305‐312.
   Marcus, A.J., Safier, L.B., Broekman, J.B., Islam, N., Fliesssbach, J.H., Hajjar, C.A., Kaminski, W.E., Jendraschak, E., Silverstein, R.L., and Schacky, C. 1995. Thrombosis and inflammation as multicellular processes: Significance of cell‐cell interactions. Thromb. Haemost. 74:213‐217.
   Merrit, J.E., McCarthy, S.A., Davies, M.P.A., and Moores, K.E. 1990. Use of fluo‐3 to measure cytosolic Ca2+ in platelets and neutrophils. Biochem. J. 269:513‐519.
   Michelson, A.D., Ellis, P.A., Barnard, M.R., Matic, G.B., Viles, A.F., and Kestin, A.S. 1991. Downregulation of platelet surface glycoprotein Ib‐IX complex in whole‐blood stimulated by thrombin, ADP or an in vivo wound. Blood 77:770‐779.
   Modderman, P.W., Huisman, H.G., van Mourik, J.A., von dem Borne, A.E. 1988. A monoclonal antibody to the human platelet glycoprotein IIb/IIIa complex induces platelet activation. Thromb. Haemost. 60:68‐74.
   Monteiro, M.C., Sansonetty, F., Gonçalves, M.J., and O'Connor, J.E. 1999. Flow cytometric kinetic assay of calcium mobilization in whole‐blood platelets using fluo‐3 and CD41. Cytometry 35:302‐310.
   Rink, T., Smith, S., and Tsien, R. 1982. Cytoplasmic free Ca2+ threshold and Ca2+‐independent activation for shape‐change and secretion. FEBS Lett. 148:21‐26.
   Sage, S.O., Merritt, J.E., Hallam, T.J., and Rink, T.J. 1989. Receptor‐mediated calcium entry in fura‐2‐loaded human platelets stimulated with ADP and thrombin. Dual wavelength studies with Mn2+. Biochem. J. 258:923‐926.
   Salzman, E.W. and Ware, J.A. 1989. Ionized calcium as an intracellular messenger in blood platelets. Prog. Haemost. Thromb. 9:177‐202.
   Santos, M.T., Vallés, J., Aznar, J., and Requejo, J.L.P. 1986. Role of red blood cells in early stages of platelet activation by collagen. Thromb. Haemost. 56:376‐381.
   Ware, J.A., Johnson, P.C., Smith, M., and Salzman, E.W. 1996. Effect of common agonists on cytoplasmatic ionized calcium concentration in platelets. J. Clin. Invest. 77:878‐886.
   Weiss, H.J. and Lages, B. 1992. Platelet prothrombinase activity and intracellular calcium response in patients with storage pool deficiency, Glycoprotein IIb‐IIIa deficiency or impaired platelet coagulant activity: A comparison with Scott Syndrome. Blood 89:1599‐1611.
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