Pairwise agonist scanning-flow cytometry (PAS-FC) measures inside-out signaling and patient-specific response to combinatorial platelet agonists

Biotechniques. 2013 May;54(5):271-7. doi: 10.2144/000114027.

Abstract

Understanding the response of cells to multiple stimuli is vital for predicting donor specific responses and better understanding the signaling pathways involved. This is of particular importance in platelets because exposure of phosphatidylserine (PS) occurs upon costimulation but not with a single agonist. Here, we describe a multiplexed pairwise agonist scanning-flow cytometry (PAS-FC) method of measuring platelet inside-out responses to all pairs of six platelet agonists (convulxin, SFLLRN, AYPGKF, ADP, U46619, and PGE(2)) used at their EC(50) concentrations. These agonists allowed exploration of platelet signaling downstream of GPVI, PAR-1, PAR-4, P2Y(1), P2Y(12), TP, and IP receptors. The three-color flow cytometry method simultaneously measured integrin α(IIb)β(3) activation with PAC-1 antibody, P-selectin exposure (via α granule release) with anti-P-selectin, and PS exposure with annexin V. These responses were consistent across a healthy male donor pool. In duplicate measurements with each donor, 4 of the 10 donors had a sufficiently unique 45-parameter (15 pairs × 3 colors) phenotype to self-cluster (P < 0.001). This method has the potential for efficiently scanning for patient specific responses across a broad agonist-receptor space.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / pharmacology
  • Adenosine Diphosphate / pharmacology
  • Annexin A5 / chemistry
  • Annexin A5 / metabolism
  • Blood Donors
  • Blood Platelets / chemistry
  • Blood Platelets / cytology
  • Blood Platelets / drug effects*
  • Blood Platelets / metabolism
  • Cluster Analysis
  • Crotalid Venoms / pharmacology
  • Dinoprostone / pharmacology
  • Flow Cytometry / methods*
  • Humans
  • Lectins, C-Type
  • Male
  • Oligopeptides / pharmacology
  • P-Selectin / chemistry
  • P-Selectin / metabolism
  • Peptide Fragments / pharmacology
  • Phenotype
  • Phosphatidylserines
  • Precision Medicine
  • Signal Transduction

Substances

  • Annexin A5
  • Crotalid Venoms
  • Lectins, C-Type
  • Oligopeptides
  • P-Selectin
  • Peptide Fragments
  • Phosphatidylserines
  • alanyl-tyrosyl-prolyl-glycyl-lysyl-phenylalanine
  • thrombin receptor peptide (42-47)
  • convulxin
  • Adenosine Diphosphate
  • 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid
  • Dinoprostone