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

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.