Immunophenotypic Analysis of Platelets

Lori A. Krueger1, Marc R. Barnard1, A.L. Frelinger1, Mark I. Furman1, Alan D. Michelson1

1 University of Massachusetts Medical School, Worcester, Massachusetts
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.10
DOI:  10.1002/0471142956.cy0610s19
Online Posting Date:  February, 2002
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Abstract

Platelets are the smallest cellular component in the peripheral circulation. Their primary role is maintenance of hemostasis. The evaluation of platelets by flow cytometry has proven beneficial in the investigation of many disease states, including inherited defects, cardiovascular disease, stroke, and many other inflammatory processes. In the absence of an added exogenous platelet agonist, the activation state of in vivo circulating platelets can be measured. Inclusion of an exogenous agonist in the assay enables analysis of the reactivity of circulating platelets in vitro. This unit describes protocols to evaluate platelet surface glycoproteins, platelet activation status, and platelet responsiveness to agonist. Detailed discussion of appropriate sample handling, reagent preparation, flow cytometric set‐up, and data interpretation are provided for three independent assays.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Immunophenotyping of Platelet Surface Receptors
  • Alternate Protocol 1: Immunophenotyping of Platelet Surface Receptors
  • Basic Protocol 2: Determination of Platelet Activation Using P‐Selectin or PAC1 Expression
  • Basic Protocol 3: Determination of Procoagulant Platelets Using Annexin V Binding or Monoclonal Antibodies Specific for Coagulation Factor V/Va or X/Xa
  • Support Protocol 1: Preparation of Isolated Platelets
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Immunophenotyping of Platelet Surface Receptors

  Materials
  • Whole blood (WB) containing anticoagulant (see ) or isolated platelets (see protocol 5)
  • Modified HT buffer (see recipe)
  • Platelet‐specific antibody cocktail titrated in modified HT buffer (minimum two antibody specificities each conjugated to a different fluorochrome): Specific platelet identifier: monoclonal anti‐CD41, ‐CD61, ‐CD42a, or ‐CD42b Marker of platelet activation: monoclonal anti‐CD62P or PAC1
  • Negative control: antibody isotype‐, concentration‐, fluorochrome‐, and F:P ratio‐matched to the activation marker, or blocking agent inhibiting platelet‐specific marker binding
  • 1% formalin fixative (see recipe)

Alternate Protocol 1: Immunophenotyping of Platelet Surface Receptors

  • 2% formalin fixative (see recipe)

Basic Protocol 2: Determination of Platelet Activation Using P‐Selectin or PAC1 Expression

  Materials
  • Whole blood (WB) containing anticoagulant (see ) or isolated platelets (see protocol 5)
  • Modified HT buffer (see recipe)
  • Platelet‐specific antibody cocktail titrated in modified HT buffer (minimum two antibody specificities each conjugated to a different fluorochrome): Specific platelet identifier: e.g., monoclonal anti‐CD41, ‐CD61, ‐CD42a, ‐CD42b Specific marker of platelet activation: e.g., monoclonal anti‐CD62P or ‐PAC1
  • Platelet agonist (see recipe): e.g., ADP, epinephrine, human α‐thrombin, thrombin receptor‐activating peptide (TRAP)
  • Negative controls: antibody isotype‐, concentration‐, fluorochrome‐, and F:P ratio‐matched to the specific activation marker, or blocking agent that inhibits the platelet‐specific marker binding
  • 10 mM GPRP (see recipe)
  • 1% formalin fixative (see recipe)

Basic Protocol 3: Determination of Procoagulant Platelets Using Annexin V Binding or Monoclonal Antibodies Specific for Coagulation Factor V/Va or X/Xa

  Materials
  • Whole blood (WB) containing anticoagulant (see ) or isolated platelets (see protocol 5)
  • Modified HT buffer (see recipe) containing 5 mM GPRP (see recipe)
  • Coagulation factor V/Va or X/Xa
  • Platelet agonist (see recipe) supplemented with 6 mM CaCl 2: e.g., collagen, combined thrombin/collagen mixture, or calcium ionophore A23183
  • Modified HT buffer
  • Platelet‐specific antibody cocktail titrated in modified HT buffer—minimum two antibodies, or one identifier and annexin V—each conjugated to a different fluorochrome:
  •  Specific platelet identifier: e.g., anti‐CD41, anti‐CD61, anti‐CD42a, or anti‐CD42b
  •  Marker of platelet procoagulant activity: e.g., annexin V, monoclonal anti‐coagulation factor V/Va or X/Xa
  • 1% formalin fixative (see recipe)

Support Protocol 1: Preparation of Isolated Platelets

  Materials
  • Anticoagulated whole blood collected in 5‐ml vacutainer tubes or 15‐ml conical tubes if drawn by syringe
  • Sepharose 2B beads
  • Modified HT buffer (see recipe)
  • Citrate wash buffer (see recipe)
  • Benchtop centrifuge with rotors for 5‐ml Vacutainer tubes and 15‐ml conical tubes
  • Polypropylene or siliconized glass test tube
  • 10‐ml syringe column
  • 15‐ml conical tubes
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Figures

Videos

Literature Cited

Literature Cited
   Faraday, N., Goldschmidt‐Clermont, P., Dise, K., and Bray, P.F. 1994. Quantitation of soluble fibrinogen binding to platelets by fluorescence‐activated flow cytometry. J. Lab. Clin. Med. 123:728‐740.
   Frelinger, A.L., Lam, S.C., Plow, E.F., Smith, M.A., Loftus, J.C., and Ginsberg, M.H. 1988. Occupancy of an adhesive glycoprotein receptor modulates expression of an antigenic site involved in cell adhesion. J. Biol. Chem. 263:12397‐12402.
   Furman, M.I., Krueger, L.A., Frelinger, A.L. III., Barnard, M.R., Mascelli, M.A., Nakada, M.T., and Michelson, A.D. 2000. GPIIb‐IIIa antagonist‐induced reduction in platelet surface factor V/Va binding and phosphatidylserine expression in whole blood. Thromb. Haemost. 84:492‐498.
   Gilbert, G.E., Sims, P.J., Wiedmer, T., Furie, B., Furie, B.C., and Shattil, S.J. 1991. Platelet‐derived microparticles express high affinity receptors for factor VIII. J. Biol. Chem. 266:17261‐17268.
   Henn, V., Slupsky, J.R., Grafe, M., Anagnostopoulos, I., Forster, R., Muller‐Berghaus, G., and Kroczek, R.A. 1998. CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature 391:591‐594.
   Holme, P.A., Brosstad, F., and Solum, N.O. 1995. Platelet‐derived microvesicles and activated platelets express factor Xa activity. Blood Coagul. Fibrinolysis 6:302‐310.
   Jennings, L.K., Ashmun, R.A., Wang, W.C., and Dockter, M.E. 1986. Analysis of human platelet glycoproteins IIb‐IIIa and Glanzmann's thrombasthenia in whole blood by flow cytometry. Blood 68:173‐179.
   McEver, R.P. 2001. P‐selectin/PSGL‐1 and other interactions between platelets, leukocytes, and endothelium. In Platelets (A.D. Michelson, ed.). Academic Press, New York. In press.
   Michelson, A.D. 1987. Flow cytometric analysis of platelet surface glycoproteins: Phenotypically distinct subpopulations of platelets in children with chronic myeloid leukemia. J. Lab. Clin. Med. 110:346‐354.
   Michelson, A.D. 1994. Platelet activation by thrombin can be directly measured in whole blood through the use of the peptide GPRP and flow cytometry: Methods and clinical studies. Blood Coagul. Fibrinolysis 5:121‐131.
   Michelson, A.D., Barnard, M.R., Benoit, S.E., Mitchell, J., Knowles, C., and Ault, K.A. 1995. Characterization of platelet binding of blind panel mAb. In Leucocyte Typing V (S.F. Schlossman, L. Boumsell, W. Gilks, J.W. Harlan, T. Kishimoto, C. Morimoto, J. Ritz, S. Shaw, R.L. Silverstein, T.A. Springer, T.F. Tedder, and R.F. Todd, eds.) pp. 1207‐1210. Oxford University Press, Oxford.
   Michelson, A.D., Benoit, S.E., Furman, M.I., Barnard, M.R., Nurden, P., and Nurden, A.T. 1996a. The platelet surface expression of glycoprotein V is regulated by two independent mechanisms: Proteolysis and a reversible cytoskeletal‐mediated redistribution to the surface‐connected canalicular system. Blood 87:1396‐1408.
   Michelson, A.D., Barnard, M.R., Hechtman, H.B., MacGregor, H., Connolly, R.J., Loscalzo, J., and Valeri, C.R. 1996b. In vivo tracking of platelets: Circulating degranulated platelets rapidly lose surface P‐selectin but continue to circulate and function. Proc. Natl. Acad. Sci. U.S.A. 93:11877‐11882.
   Michelson, A.D., Barnard, M.R., Krueger, L.A., Frelinger, A.L., and Furman, M.I. 2001. Flow cytometry. In Platelets (A.D. Michelson, ed.). Academic Press, New York. In press.
   Rajasekhar, D., Barnard, M.R., Bednarek, F.J., Benoit, S.E., and Michelson, A.D. 1993. Procoagulant activity of platelet‐derived microparticles in whole blood: Differences between neonates and adults. Blood 82:63a‐163a.
   Ruf, A. and Patscheke, H. 1995. Flow cytometric detection of activated platelets: Comparison of determining shape change, fibrinogen binding, and P‐selectin expression. Semin. Thromb. Hemost. 21:146‐151.
   Schmitz, G., Rothe, G., Ruf, A., Barlage, S., Tschöpe, D., Clemetson, K.J., Goodall, A.H., Michelson, A.D., Nurden, A.T., Shankey, T.V., for the European Working Group on Clinical Cell Analysis. 1998. European Working Group on Clinical Cell Analysis: Consensus protocol for the flow cytometric characterisation of platelet function. Thromb. Haemost. 79:885‐896.
   Shattil, S.J., Hoxie, J.A., Cunningham, M., and Brass, L.F. 1985. Changes in the platelet membrane glycoprotein IIb‐IIIa complex during platelet activation. J. Biol. Chem. 260:11107‐11114.
   Vu, T.‐K.H., Hung, D.T., Wheaton, V.I., and Coughlin, S.R. 1991. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64:1057‐1068.
   Zamarron, C., Ginsberg, M.H., and Plow, E.F. 1990. Monoclonal antibodies specific for a conformationally altered state of fibrinogen. Thromb. Haemost. 64:41‐46.
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