In Vitro Binding of [3H]PSB‐0413 to P2Y12 Receptors

Arnaud Dupuis1, Véronique Heim1, Philippe Ohlmann1, Christian Gachet1

1 UMRS949, INSERM, Université de Strasbourg, Etablissement Français du Sang–Alsace, F‐67065 Strasbourg
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.35
DOI:  10.1002/0471141755.ph0135s71
Online Posting Date:  December, 2015
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Abstract

The P2Y12 /ADP receptor plays a central role in platelet activation. Characterization of this receptor is mandatory for studying disorders associated with a P2Y12 receptor defect and for evaluating P2Y12 receptor agonists and antagonists. In the absence of suitable anti‐P2Y12 antibodies, radioligand binding assays are the only way to conduct such studies. While various radioligands were employed in the past for this purpose, none were found to be suitable for routine use. Described in this unit are protocols for quantitatively and qualitatively assessing P2Y12 receptors with [3H]PSB‐0413, a selective antagonist for this site. The saturation and competition assays described herein make possible the determination of P2Y12 receptor density on cells, as well as the potencies and affinities of test agents at this site. © 2015 by John Wiley & Sons, Inc.

Keywords: P2Y12; binding; blood platelets; radioligand; ADP

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Saturation Binding Assay to Determine the Dissociation Constant (Kd) and Number of Binding Sites in the Cell Sample
  • Basic Protocol 2: Competitive Binding Assays to Determine the Affinity (Ki or IC50) of Non‐Radiolabeled Ligands for the P2Y12 Receptor
  • Support Protocol 1: Preparation of Washed Blood Platelets
  • Support Protocol 2: Preparation of a Cell Suspension to Characterize P2Y12 Receptors on the Cell Surface
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Saturation Binding Assay to Determine the Dissociation Constant (Kd) and Number of Binding Sites in the Cell Sample

  Materials
  • Washed platelets resuspended in Tyrode's buffer containing 0.1% human albumin ( protocol 3) or cells resuspended in CMF‐DPBS/0.1% bovine serum albumin ( protocol 4)
  • Dulbecco's phosphate‐buffered saline without Ca or Mg (CMF‐DPBS; Life Technologies, cat. no. 14190‐094)
  • 10 mM or 20 mM ADP (see recipe)
  • [3H]PSB‐0413 dilutions (see recipe and Tables 1.35.1 and 1.35.2)Liquid scintillation cocktail (GE Healthcare, cat. no. NBCS 104)
  • Whatman glass microfiber filters, binder‐free, Grade GF/C (Sigma‐Aldrich, cat. no. 1822915)
  • Large petri dishes
  • Reaction vials (Novolab, cat. no. A11088)
  • Pump and filtration system (example: BioVac 321B, Rocker Scientific) see Figure for a full description of the filtration system
  • SnapCap Bio‐Vials (Beckman Coulter, cat. no. 566353)
  • β scintillation counter (e.g., Tri‐carb 2810TR liquid scintillation analyzer, PerkinElmer,)
  • GraphPad Prism software (http://www.graphpad.com)
Table 1.5.1   Materials[3H]PSB‐0413 Dilution Table for a Saturation Binding Experiment on Platelets (60 Vials Including Total and Nonspecific Binding Vials)[3H]PSB‐0413 Dilution Table for a Saturation Binding Experiment on Cells (60 Vials Including Total and Nonspecific Binding Vials)

Initial concentration of radioligand (μM) Volume of initial solution (μl) Volume of CMF‐DPBS (μl) Final volume (μl) Target concentration (nM) a
20 b 22 528 550 800
0.8 125 75 200 500
0.8 75 125 200 300
0.8 50 150 200 200
0.8 31.25 218.75 250 100
0.8 12.5 187.5 200 50
0.8 7.5 192.5 200 30
0.8 5 195 200 20
0.8 3 197 200 12
0.8 1 199 200 4
Initial concentration of radioligand (μM) Volume of initial solution (μl) Volume of CMF‐DPBS (μl) Final volume (μl) Target concentration (nM) a
20 b 44 506 550 1600
1.6 125 75 200 1000
1.6 75 125 200 600
1.6 50 150 200 400
1.6 25 175 200 200
1.6 15.6 234.4 250 100
1.6 7.5 192.5 200 60
1.6 5 195 200 40
1.6 3 197 200 24
1.6 1 199 200 8

 aConcentration to use to prepare the vials for the saturation binding assay, called "Initial concentration of radioligand" in Table 1.35.3.
 bCommercial stock solution.
Table 1.5.2   Materials[3H]PSB‐0413 Dilution Table for a Saturation Binding Experiment on Platelets (60 Vials Including Total and Nonspecific Binding Vials)[3H]PSB‐0413 Dilution Table for a Saturation Binding Experiment on Cells (60 Vials Including Total and Nonspecific Binding Vials)

Initial concentration of radioligand (μM) Volume of initial solution (μl) Volume of CMF‐DPBS (μl) Final volume (μl) Target concentration (nM) a
20 b 22 528 550 800
0.8 125 75 200 500
0.8 75 125 200 300
0.8 50 150 200 200
0.8 31.25 218.75 250 100
0.8 12.5 187.5 200 50
0.8 7.5 192.5 200 30
0.8 5 195 200 20
0.8 3 197 200 12
0.8 1 199 200 4
Initial concentration of radioligand (μM) Volume of initial solution (μl) Volume of CMF‐DPBS (μl) Final volume (μl) Target concentration (nM) a
20 b 44 506 550 1600
1.6 125 75 200 1000
1.6 75 125 200 600
1.6 50 150 200 400
1.6 25 175 200 200
1.6 15.6 234.4 250 100
1.6 7.5 192.5 200 60
1.6 5 195 200 40
1.6 3 197 200 24
1.6 1 199 200 8

 aConcentration to use to prepare the vials for the saturation binding assay, called "Initial concentration of radioligand" in Table 1.35.4.
 bCommercial stock solution.

Basic Protocol 2: Competitive Binding Assays to Determine the Affinity (Ki or IC50) of Non‐Radiolabeled Ligands for the P2Y12 Receptor

  Materials
  • Tyrode's buffer containing 0.35% HSA (see recipe)
  • Healthy blood donor or patient
  • Acid‐citrate‐dextrose anticoagulant
  • 5 U/μl heparin (Cerp Rhin Rhone Mediterrannée, cat. no. ANM 3048450)
  • 10−3 M prostaglandin I 2 (PGI 2; Sigma, cat. no. P6188)
  • Apyrase (ATP diphosphohydrolase 3.6.1.5; Sigma, cat. no. A6535)
  • 15‐ and 50‐ml conical centrifuge tubes (e.g., Sarstedt or Corning Falcon)
  • Centrifuge (e.g., Sorvall RC3BP)

Support Protocol 1: Preparation of Washed Blood Platelets

  Materials
  • Adherent cell line: e.g., 13.21N1 astrocytoma cells (human brain astrocytoma; Public Health England, cat. no. 86030402) cultured in 175‐cm2 flasks
  • Dulbecco's phosphate‐buffered saline without Ca or Mg (CMF‐DPBS; Life Technologies, cat. no. 14190‐094)
  • TrypLE Express (Life Technologies, cat. no. 12605‐028)DMEM medium (Life Technologies, cat. no. 424230‐025) containing 10% fetal bovine serum (FBS)Bovine serum albumin (BSA), fatty acid–free (Sigma‐Aldrich, cat. no. A1887)
  • 50‐ml conical centrifuge tubes (Sarstedt, cat. no. 62547254,)
  • Cell counter or hemacytometer
  • Centrifuge
NOTE: Begin this protocol at step 5 when using a cultured cell line in suspension.
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Figures

Videos

Literature Cited

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Internet Resources
  http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=52
  IUPHAR. Guide to pharmacology: P2Y receptor.
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