QUANTITATIVE DETECTION OF PLATELET GPIIB-IIIA RECEPTOR ANTAGONIST ACTIVITY USING A FLOW CYTOMETRIC METHOD

Platelet-membrane surface receptors are important targets for pharmaco logic intervention in cardiovascular disease. Among these, glycoprotei n (GP) IIb-IIIa is dominant and integrally involved in platelet aggreg ation and thrombus formation. When activated, GPIIb-IIIa binds soluble fibrinogen (Fb) in a key, early step of this process. New drugs are u nder development that block Fb binding to GPIIb-IIIa and inhibit plate let aggregation.A thorough understanding of the relationship between c irculating drug levels and the extent of GPIIb-IIIa receptor occupancy in humans is crucial for safe and efficacious use of these agents. De scribed here is the development of a new technique for measurement of GPIIb-IIIa receptor occupancy. in this assay, activated human platelet s are incubated with biotinylated fibrinogen (Fb-biotin) followed by a ntibiotin-FITC. The extent of Fb binding is determined using flow cyto metric analysis. Our results indicate that Fb-biotin binds rapidly to activated platelets and its detection is dependent on incubation tempe rature. Platelets that were pre-incubated with the GPIIb-IIIa antagoni st echistatin were inhibited from binding Fb-biotin in a concentration -dependent manner. The fluorescence of processed samples was stable fo r two weeks in the cold. The assay described here is simple, cost effe ctive, and can be adapted for use in clinical evaluations of these new drugs. (C) 1998 Wiley-Liss, Inc.