Optimally functional fluorescein isothiocyanate-labelled fibrinogen for quantitative studies of binding to activated platelets and platelet aggregation
| Autor: | Qingde Liu, T. Wong, Z. Xia, Mony M. Frojmovic, Ana Kasirer-Friede, Elizabeth Brown |
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| Rok vydání: | 1996 |
| Předmět: |
Blood Platelets
Platelet Aggregation Chemistry Fibrinogen Platelet Glycoprotein GPIIb-IIIa Complex Hematology Flow Cytometry Platelet Activation Dissociation constant chemistry.chemical_compound Biochemistry medicine Humans Platelet Platelet activation Fluorescein Receptor Fluorescein isothiocyanate Fluorescein-5-isothiocyanate Protein Binding medicine.drug |
| Zdroj: | British Journal of Haematology. 93:204-214 |
| ISSN: | 1365-2141 0007-1048 |
| DOI: | 10.1046/j.1365-2141.1996.445980.x |
| Popis: | 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 ≥ 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 (K D ) of Fg on stable preparations of activated, fixed platelets. determined with FITC-Fg binding to platelets by flow cytometry, was in the range reported for 125 I-labelled Fg, 70-255 nM with B max = 10 000-25 000 Fg per platelet (n = 20). The FITC-Fg was used to monitor Fg binding to activated platelets directly in plasma, as well as to evaluate platelet subpopulations which maximally bind Fg according to the concentration of ADP used as activator. It is expected that this 'mildly' labelled FITC-Fg will stimulate further studies of platelet activation directly in native anticoagulated blood/plasma, for both basic and clinical research. |
| Databáze: | OpenAIRE |
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