Measurement Conditions and Effects of Antiplatelet Agents on Fibrinogen Binding to Activated Platelets by Flow Cytometry

Abstract

Activated platelets express fibrinogen receptors on their surface, bind fibrinogen, and then aggregate each other. The platelet-bound fibrinogen was measured by flow cytometry using a fluorescein isothiocyanate (FITC)-conjugated polyclonal anti-fibrinogen antibody. Platelets were activated with ADP and incubated with FITC-antifibrinogen antibody, then the percentage of platelets showing positive anti-fibrinogen antibody binding and the relative fluorescence intensity of those platelets were measured. The percentage increased with increasing the ADP concentration, showing 90% at 5μM ADP, although about 10% of platelets failed to bind fibrinogen even at 50μM ADP. The relative fluorescence intensity increased as the ADP concentration increased from 0.1 to 50μM, indicating that the average number of bound fibrinogen to each platelet increased. The percentage of fibrinogen binding to unstimulated platelets increased with time lag between obtaining venous blood and starting incubation of the samples. The ADP-induced fibrinogen binding was not significantly affected by the time lag from 30 to 120 minutes. The ADP-induced fibrinogen binding was inhibited by prostaglandin I2 and its analogue, a fibrinogen receptor antagonist, and a ADP receptor antagonist, but not by aspirin. ADP-concentration-dependent increases were more prominent in the fibrinogen binding to platelets than in the expression of p-selectin on platelet surface. These studies indicate that platelet-bound fibrinogen can be quantitated by means of flow cytometry, and that this method may be useful for the detection of sensitive or activated platelets.