Studies of Fibrinogen Binding to Platelets by Flow Cytometry: An Improved Method for Studies of Platelet Activation

Abstract

SummaryPlatelet function is dependent upon membrane receptors and their interaction with other proteins. Platelet activation appears to cause a structural change of the glycoprotein IIb/IIIa complex that exposes the fibrinogen binding site, which subsequently binds fibrinogen. Fluorescence-activated flow cytometry (FACS) is an efficient method for studying membrane proteins. Flow cytometry gives single-cell data, allowing the detection of only a small proportion of labelled platelets in whole blood without any washing steps. One problem with this method is that the labelled antibodies and the antigen, if present in plasma, form an immune complex, which may cause false positive reactions due to interaction between mammalian IgG and Fcγ receptors on the platelets.We show that immune complexes with chicken IgG do not activate human platelets. We have developed a method for measuring platelet-bound fibrinogen in whole blood and platelet-rich plasma utilising fluorescein isothiocyanate (FITC)-conjugated chicken antibodies directed towards human fibrinogen. As low as 1% activated platelets could be detected without interference from Fc-interactions.