Fluorescein derivatization of fibrinogen for flow cytometric analysis of fibrinogen binding to platelets

Dog and human fibrinogen were derivatized with N‐hydroxysuccinimidofluorescein and utilized for flow cytometric estimation of fibrinogen binding to activated platelets. Fluorescein‐fibrinogen binding fulfilled the criteria for specific binding to platelets; the binding was saturable, dependent on agonist activation, and inhibited by unlabeled fibrinogen. In addition, EDTA and barbourin, a KGD‐containing peptide, were found to inhibit the binding of fluorescein‐fibrinogen. Fluorescein‐fibrinogen bound to dog platelets with an apparent affinity of 0.31 μM after stimulation with either adenosine‐5′‐diphosphate (ADP) or plateletactivating 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 fibrinogen binding in response to ADP. These studies document that direct derivatization of fibrinogen with fluorescein generates a useful probe for analyzing fibrinogen binding to platelets with flow cytometry. © 1994 Wiley‐Liss, Inc.