Quantitation of soluble fibrinogen binding to platelets by fluorescence-activated flow cytometry.

N. Faraday, Pascal Goldschmidt-Clermont, K. Dise, P. F. Bray

Research output: Contribution to journalArticle

68 Citations (Scopus)

Abstract

Soluble fibrinogen binding to agonist-stimulated blood platelets is the essential physiologic function of the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor. We describe a method of quantifying this receptor-ligand interaction by using flow cytometry to detect the binding of fluorescein-labeled fibrinogen to activated platelets. Fibrinogen conjugated with fluorescein isothiocyanate (FITC-FGN) was structurally and functionally indistinguishable from native fibrinogen when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thrombin clottability, and receptor affinity studies. Platelet samples, at a concentration of 2 x 10(7) ml, were incubated with FITC-FGN and activated with adenosine diphosphate (ADP) before cytometric acquisition of fluorescence and scatter data. ADP-induced binding of soluble FITC-FGN to platelet GPIIb-IIIa was specific, time dependent, and saturable. Cytometric analysis of FITC calibration beads allowed generation of standard curves relating bead fluorescence intensity to the number of fluorescein equivalents per bead. With this information, platelet fluorescence intensity was converted into the number of FITC-FGN molecules bound per platelet. Such quantitative analysis of fibrinogen binding yielded a dissociation constant of 2.48 +/- 0.5 x 10(-7) mol/L and a maximum fibrinogen binding capacity of 42, 124 +/- 5, 628 molecules per platelet (mean +/- SEM), which are comparable to published results with radioligand assays. The simplicity, sensitivity, and quantifiability of this method may make it a useful technique for basic and clinical research involving GPIIb-IIIa function.

Original languageEnglish
Pages (from-to)728-740
Number of pages13
JournalJournal of Laboratory and Clinical Medicine
Volume123
Issue number5
StatePublished - May 1 1994
Externally publishedYes

Fingerprint

Flow cytometry
Platelets
Fibrinogen
Flow Cytometry
Fluorescein-5-isothiocyanate
Blood Platelets
Fluorescence
Fluorescein
Platelet Glycoprotein GPIIb-IIIa Complex
Adenosine Diphosphate
Thrombin Receptors
Radioligand Assay
Molecules
Electrophoresis
Sodium Dodecyl Sulfate
Calibration
Polyacrylamide Gel Electrophoresis
Assays
Blood
Ligands

ASJC Scopus subject areas

  • Medicine(all)
  • Pathology and Forensic Medicine

Cite this

Quantitation of soluble fibrinogen binding to platelets by fluorescence-activated flow cytometry. / Faraday, N.; Goldschmidt-Clermont, Pascal; Dise, K.; Bray, P. F.

In: Journal of Laboratory and Clinical Medicine, Vol. 123, No. 5, 01.05.1994, p. 728-740.

Research output: Contribution to journalArticle

Faraday, N, Goldschmidt-Clermont, P, Dise, K & Bray, PF 1994, 'Quantitation of soluble fibrinogen binding to platelets by fluorescence-activated flow cytometry.', Journal of Laboratory and Clinical Medicine, vol. 123, no. 5, pp. 728-740.
Faraday, N. ; Goldschmidt-Clermont, Pascal ; Dise, K. ; Bray, P. F. / Quantitation of soluble fibrinogen binding to platelets by fluorescence-activated flow cytometry. In: Journal of Laboratory and Clinical Medicine. 1994 ; Vol. 123, No. 5. pp. 728-740.
@article{fd4649c7ecfe4c43a52eeba70caa6ba8,
title = "Quantitation of soluble fibrinogen binding to platelets by fluorescence-activated flow cytometry.",
abstract = "Soluble fibrinogen binding to agonist-stimulated blood platelets is the essential physiologic function of the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor. We describe a method of quantifying this receptor-ligand interaction by using flow cytometry to detect the binding of fluorescein-labeled fibrinogen to activated platelets. Fibrinogen conjugated with fluorescein isothiocyanate (FITC-FGN) was structurally and functionally indistinguishable from native fibrinogen when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thrombin clottability, and receptor affinity studies. Platelet samples, at a concentration of 2 x 10(7) ml, were incubated with FITC-FGN and activated with adenosine diphosphate (ADP) before cytometric acquisition of fluorescence and scatter data. ADP-induced binding of soluble FITC-FGN to platelet GPIIb-IIIa was specific, time dependent, and saturable. Cytometric analysis of FITC calibration beads allowed generation of standard curves relating bead fluorescence intensity to the number of fluorescein equivalents per bead. With this information, platelet fluorescence intensity was converted into the number of FITC-FGN molecules bound per platelet. Such quantitative analysis of fibrinogen binding yielded a dissociation constant of 2.48 +/- 0.5 x 10(-7) mol/L and a maximum fibrinogen binding capacity of 42, 124 +/- 5, 628 molecules per platelet (mean +/- SEM), which are comparable to published results with radioligand assays. The simplicity, sensitivity, and quantifiability of this method may make it a useful technique for basic and clinical research involving GPIIb-IIIa function.",
author = "N. Faraday and Pascal Goldschmidt-Clermont and K. Dise and Bray, {P. F.}",
year = "1994",
month = "5",
day = "1",
language = "English",
volume = "123",
pages = "728--740",
journal = "Translational Research",
issn = "1931-5244",
publisher = "Mosby Inc.",
number = "5",

}

TY - JOUR

T1 - Quantitation of soluble fibrinogen binding to platelets by fluorescence-activated flow cytometry.

AU - Faraday, N.

AU - Goldschmidt-Clermont, Pascal

AU - Dise, K.

AU - Bray, P. F.

PY - 1994/5/1

Y1 - 1994/5/1

N2 - Soluble fibrinogen binding to agonist-stimulated blood platelets is the essential physiologic function of the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor. We describe a method of quantifying this receptor-ligand interaction by using flow cytometry to detect the binding of fluorescein-labeled fibrinogen to activated platelets. Fibrinogen conjugated with fluorescein isothiocyanate (FITC-FGN) was structurally and functionally indistinguishable from native fibrinogen when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thrombin clottability, and receptor affinity studies. Platelet samples, at a concentration of 2 x 10(7) ml, were incubated with FITC-FGN and activated with adenosine diphosphate (ADP) before cytometric acquisition of fluorescence and scatter data. ADP-induced binding of soluble FITC-FGN to platelet GPIIb-IIIa was specific, time dependent, and saturable. Cytometric analysis of FITC calibration beads allowed generation of standard curves relating bead fluorescence intensity to the number of fluorescein equivalents per bead. With this information, platelet fluorescence intensity was converted into the number of FITC-FGN molecules bound per platelet. Such quantitative analysis of fibrinogen binding yielded a dissociation constant of 2.48 +/- 0.5 x 10(-7) mol/L and a maximum fibrinogen binding capacity of 42, 124 +/- 5, 628 molecules per platelet (mean +/- SEM), which are comparable to published results with radioligand assays. The simplicity, sensitivity, and quantifiability of this method may make it a useful technique for basic and clinical research involving GPIIb-IIIa function.

AB - Soluble fibrinogen binding to agonist-stimulated blood platelets is the essential physiologic function of the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor. We describe a method of quantifying this receptor-ligand interaction by using flow cytometry to detect the binding of fluorescein-labeled fibrinogen to activated platelets. Fibrinogen conjugated with fluorescein isothiocyanate (FITC-FGN) was structurally and functionally indistinguishable from native fibrinogen when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thrombin clottability, and receptor affinity studies. Platelet samples, at a concentration of 2 x 10(7) ml, were incubated with FITC-FGN and activated with adenosine diphosphate (ADP) before cytometric acquisition of fluorescence and scatter data. ADP-induced binding of soluble FITC-FGN to platelet GPIIb-IIIa was specific, time dependent, and saturable. Cytometric analysis of FITC calibration beads allowed generation of standard curves relating bead fluorescence intensity to the number of fluorescein equivalents per bead. With this information, platelet fluorescence intensity was converted into the number of FITC-FGN molecules bound per platelet. Such quantitative analysis of fibrinogen binding yielded a dissociation constant of 2.48 +/- 0.5 x 10(-7) mol/L and a maximum fibrinogen binding capacity of 42, 124 +/- 5, 628 molecules per platelet (mean +/- SEM), which are comparable to published results with radioligand assays. The simplicity, sensitivity, and quantifiability of this method may make it a useful technique for basic and clinical research involving GPIIb-IIIa function.

UR - http://www.scopus.com/inward/record.url?scp=0028433323&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028433323&partnerID=8YFLogxK

M3 - Article

C2 - 7515093

AN - SCOPUS:0028433323

VL - 123

SP - 728

EP - 740

JO - Translational Research

JF - Translational Research

SN - 1931-5244

IS - 5

ER -