PMID: 8611461Apr 1, 1996Paper

Optimally functional fluorescein isothiocyanate-labelled fibrinogen for quantitative studies of binding to activated platelets and platelet aggregation

British Journal of Haematology
Z XiaM Frojmovic

Abstract

Dynamic and quantitative studies of the binding of fibrinogen (Fg) to its receptor, GPIIb-IIIa, on activated platelets, leading to platelet aggregation, are best studied with fluorescently-labelled Fg by flow cytometry. Due to conflicting reports on the functionality of FITC-labelled Fg, we have developed a reproducible and 'mild' labelling of fibrinogen with FITC-celite at pH 7.4-8.5 for direct and dynamic studies of specific Fg binding to activated platelets evaluated for native platelet-rich plasma, for washed platelets, and for activated, fixed platelets. We have demonstrated the equivalence of FITC-labelled and unlabelled Fg for binding to activated GPIIb-IIIa receptors, and in the rate and extent of mediating platelet aggregation. We found that FITC-Fg labelled at pH > or = 9 had reduced to absent specific binding to activated platelets, whether using soluble FITC or FITC-celite. The FITC-labelled Fg must be diluted 3-fold with unlabelled Fg when evaluating maximal Fg binding to activated platelets in order to prevent autoquenching of the FITC-Fg which leads to underestimation of Fg levels. The dissociation constant (KD) of Fg on stable preparations of activated, fixed platelets, determined with FITC-Fg binding to platele...Continue Reading

Citations

Jun 25, 2008·Journal of Thrombosis and Thrombolysis·Friederike K Keating, David J Schneider
Feb 28, 2002·The American Journal of Cardiology·Samer S KabbaniDavid J Schneider
Sep 11, 2010·Biochemistry·Roy R HantganSusan T Lord
Mar 23, 2004·Nephrology, Dialysis, Transplantation : Official Publication of the European Dialysis and Transplant Association - European Renal Association·Atul AggarwalDavid J Schneider
Feb 9, 2010·Arteriosclerosis, Thrombosis, and Vascular Biology·Ahmed KlinkZahi A Fayad
Sep 11, 2007·Analytical Biochemistry·Celine HoffmannEmmanuel Pauthe
Sep 1, 2005·Journal of Thrombosis and Haemostasis : JTH·A KoukiD C Tsoukatos
Sep 15, 2015·Journal of Medicinal Chemistry·Pavel G PolishchukAlexandre Varnek
Sep 20, 2005·Toxicon : Official Journal of the International Society on Toxinology·Brent JenningsEnid Shephard
Mar 23, 2005·Experimental Hematology·Oscar BerlangaJon Frampton
Aug 12, 2008·Cell Host & Microbe·Kalyan SrivastavaCraig N Morrell
May 29, 2000·Biophysical Journal·A BonnefoyM M Frojmovic
Sep 20, 2016·Langmuir : the ACS Journal of Surfaces and Colloids·Moritz Beck-Broichsitter
Oct 27, 2017·Transfusion Medicine and Hemotherapy : Offizielles Organ Der Deutschen Gesellschaft Fur̈ Transfusionsmedizin Und Immunham̈atologie·Anne BertlingBeate E Kehrel
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May 26, 2018·Platelets·Andrew L Frelinger
Nov 27, 2020·International Journal of Molecular Sciences·Claudia PielstickerBeate E Kehrel

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