Isolation of Platelet Granules

Juliane Nießen1, Gabriele Jedlitschky1, Andreas Greinacher2, Heyo K. Kroemer1

1 Department of Pharmacology, Research Center of Pharmacology and Experimental Therapeutics, Ernst‐Moritz‐Arndt‐University, Greifswald, Germany, 2 Department of Immunology and Transfusion Medicine, Ernst‐Moritz‐Arndt‐University, Greifswald, Germany
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.35
DOI:  10.1002/0471143030.cb0335s46
Online Posting Date:  March, 2010
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Abstract

Functional analysis of platelet intracellular structures requires isolation and purification of these cellular compartments. With regard to the function of platelets, both, dense (delta) and alpha granules are relevant target structures. However, the availability of sufficient purification protocols for these structures is rather limited. This unit describes two protocols for isolation and purification of platelet granule structures. The Basic Protocol describes a new technique based on immunolabeling with target-specific antibodies followed by magnetic sorting, whereas the Alternate Protocol describes the more traditional procedure based on differential centrifugation and density-based sedimentation. For both methods, the degree of granule purification can be most easily determined by immunoblotting using various antibodies that recognize structure-specific proteins. The immunomagnetic sorting method is especially good for studies requiring highly purified material (e.g., for the identification of specific transporters and receptors). Curr. Protoc. Cell Biol. 46:3.35.1-3.35.14. © 2010 by John Wiley & Sons, Inc.

Keywords: human platelets; subcellular fractionation; magnetic sorting; sucrose density gradient; granule-structures; immunopurification

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol: Isolation of Platelet Granules by Immunomagnetic Sorting
  • Elute Target Protein from the Magnetic Bead-Complex
  • Support Protocol 1: Elute Target Protein from the Magnetic Bead Complex by Denaturing Elution
  • Support Protocol 2: Elute Target Protein from the Magnetic Bead Complex by Mild Elution with Citric Acid
  • Alternate Protocol: Isolation of Platelet Granules using a Density Gradient
  • Support Protocol 3: Characterization of Platelet Subcellular Fractions by Immunoblot Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Isolation of Platelet Granules by Immunomagnetic Sorting

 Materials
  • Adenine-citrate-dextrose-anticoagulant (ACD-A; code RDB8652, Baxter Healthcare)
  • Platelet-rich plasma (PRP)
  • Washing buffer A (see recipe)
  • Apyrase (Sigma)
  • Hirudin (Pharmion)
  • Tyrode buffer (see recipe)
  • Washing buffer B (see recipe)
  • Homogenization buffer (see recipe)
  • 1000× protease inhibitors (stock solution, see recipe) or Boehringer Complete protease inhibitor (Roche Applied Science)
  • Liquid nitrogen
  • Tris/sucrose buffer (see recipe)
  • Target-specific primary antibody (see Table 3.35.1)
  • Dynabeads M-280 Sheep anti-rabbit IgG (Invitrogen)
  • Dynabeads Pan anti-mouse IgG (Invitrogen)
  • Phosphate-buffered saline (PBS; appendix 2A)
  • Centrifuge
  • 10-ml polystyrol centrifuge tubes
  • 37°C incubator
  • Refrigerated Hettich Rotixa 50 RS Centrifuge (Hettich) with 4256 swing-out rotor (or equivalent)
  • Loose-fitting Dounce homogenizer, 15-ml capacity (Wheaton)
  • Beckman ultracentrifuge with 70.1Ti rotor and corresponding polycarbonate ultracentrifuge tubes (12-ml or equivalent)
  • Tight-fitting Dounce homogenizer, 15-ml capacity (Wheaton)
  • Teflon pestle glass homogenizer (Glas-Col)
  • DynaMag-2 magnet (Invitrogen)
     
    Table 3.35.1 Antibodies Used for Immunomagnetic Isolation and Immunoblotting of Platelet Granulesa
    Performance with
    AntigenName and isotypeReactivityImmunomagnetic isolationImmunoblottingSupplier
    MRP4SNG rabbitHuman(X)XNC
    M4I-10 ratHumanNTXAxxora
    GPIbGi27 mouseHumanX(X)NC
    GPIIb/IIIaGi5 mouseHumanX(X)NC
    P-selectinCD62P mouseHumanX(X)Beckman Coulter
    LAMP2MouseHuman(X)XSanta Cruz
    GPIbCD42I goatHumanNTXSanta Cruz
    vWFGoatHumanNTXSanta Cruz
    PDIMouseHuman(X)XABCAM
    P-selectinGoatHuman(X)XSanta Cruz
     aAbbreviations: X, best performance; (X), satisfactory performance; NT, not tested; NC, not for sale; PDI, protein disulfide isomerase.

NOTE: Carry out all procedures at 4°C using solutions prechilled to that temperature.
Support Protocol 1:  Elute Target Protein from the Magnetic Bead Complex by Denaturing Elution
 Materials
  • Platelet fraction–Dynabead complex (see the Basic Protocol)
  • 4% (w/v) SDS (appendix 2A)
  • 95°C heating apparatus
  • DynaMag-2 magnet
  • –80°C freezer
  • Additional reagents and equipment for SDS-PAGE (unit 6.1)
Support Protocol 2:  Elute Target Protein from the Magnetic Bead Complex by Mild Elution with Citric Acid
 Materials
  • Target protein–Dynabead complex (see the Basic Protocol)
  • 0.1 M citric acid
  • 1 M NaOH
  • DynaMag-2 magnet
  • –80°C freezer
Support Protocol 2:  Elute Target Protein from the Magnetic Bead Complex by Mild Elution with Citric Acid
 Materials
  • Platelet-rich plasma (PRP)
  • Washing buffer B (see recipe)
  • Homogenization buffer (see recipe)
  • 1000× protease inhibitors (stock solution; see recipe) or Boehringer Complete protease inhibitor (Roche Applied Science)
  • Tris/sucrose buffer (see recipe)
  • 60% (w/v) sucrose solution in 5 mM EDTA, pH 7.4
  • Refrigerated Hettich Rotixa 50 RS centrifuge (Hettich) with 4256 swing out rotor (or equivalent)
  • Ultrasonic processor UW60 (Bandelin Electronic)
  • Loose-fitting Dounce homogenizer, 15-ml capacity (Wheaton)
  • 15-ml centrifuge tubes
  • Ultracentrifuge (Beckmann) with 70.1Ti fixed-angle rotor and a swing-out rotor (Beckmann SW40 or Sorvall TH-641) and corresponding polycarbonate ultracentrifuge tubes (or equivalent)
  • Tight-fitting homogenizer Dounce, 15-ml capacity (Wheaton)
  • Teflon pestle glass homogenizer (Glas-Col)

NOTE: Carry out all procedures at 4°C using solutions prechilled to that temperature, except when noted otherwise.
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Figures

  •  FigureFigure 3.35.1 Scheme of the cellular compartments in a platelet and the marker proteins in the respective membrane. , , L: alpha, dense, and lysosomal granules; MTS: microtubular system; DTS: dense tubular system.
    View Image
  •  FigureFigure 3.35.2 Biophysical principles of the two separation methods. (A) Immunomagnetic sorting: Platelet lysate including the granules (G) is incubated with the primary antibody (1. Ab). This binds to a marker protein on the target granule. Dynabeads (DB) coated with the secondary antibody (2.Ab) are added to form the Dynabead-antibody-organelle complex. This complex is extracted by a magnet. Separation of the Dynabead-antibody-organelle-complex is achieved by SDS (Support Protocol 1) or mild citrate elution (Support Protocol 2). (B) Sucrose density gradient separation (modified scheme according to Broekman, 1992). Nine zones are indicated. Visible bands/zones can be isolated and are enriched in cytosol (1), plasma membrane (2,3), mitochondria and lysosomes (4), alpha granules (7) and dense granules (9).
    View Image
  •  FigureFigure 3.35.3 An example of analysis of platelet subcellular fractions. Platelet subcellular fractions are separated by sucrose density gradient or by immunomagnetic sorting. Equal protein amounts of each fraction are separated by SDS-PAGE and transferred to a PVDF membrane. The blots are probed with antibodies against fraction-specific markers: anti-P-selectin antibody for the alpha granules, anti-LAMP2 and anti-MRP4 for the dense granules, and an antibody against the GPIb receptor for the outer plasma membrane (PM). The results are quantified by densitometric analysis and the immunodetection in each fraction is here expressed as percentage of the sum of the optical densities of the respective proteins in all fractions (relative optical density). For original data and blots, see Jedlitschky et al. (2004) and Niessen et al. (2007).
    View Image

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Literature Cited

Literature Cited
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