FLUORESCEIN DERIVATIZATION OF FIBRINOGEN FOR FLOW CYTOMETRIC ANALYSISOF FIBRINOGEN BINDING TO PLATELETS

Dog and human fibrinogen were derivatized with N-hydroxysuccinimido-fl uorescein and utilized for flow cytometric estimation of fibrinogen bi nding to activated platelets. Fluorescein-fibrinogen binding fulfilled the criteria for specific binding to platelets; the binding was satur able, dependent on agonist activation, and inhibited by unlabeled fibr inogen. In addition, EDTA and barbourin, a KGD-containing peptide, wer e found to inhibit the binding of fluorescein-fibrinogen. Fluorescein- fibrinogen bound to dog platelets with an apparent affinity of 0.31 mu M after stimulation with either adenosine-5'-diphosphate (ADP) or pla teletactivating factor. The labeled fibrinogen was also used to study the fibrinogen binding capacity of aged, biotinylated platelets. Aged platelets were indistinguishable from young platelets with regard to f ibrinogen binding in response to ADP. These studies document that dire ct derivatization of fibrinogen with fluorescein generates a useful pr obe for analyzing fibrinogen binding to platelets with flow cytometry. (C) 1994 Wiley-Liss, Inc.