Measurement of fibrinogen binding to platelets in whole blood by flow cytometry: a micromethod for the detection of platelet activation

Abstract

Platelet fibrinogen binding in whole blood has been measured in vitro by flow cytometry using a commercially available, fluorescein isothiocyanate (FITC)-conjugated polyclonal antifibrinogen antibody. Fibrinogen-antifibrinogen immune complexes were formed in experimental conditions approaching antigen-antibody equivalence, but optimal reaction conditions in which their formation was prevented or minimized could be achieved. Immune complex formation was associated with fibrinogen binding to unstimulated platelets but did not significantly affect ADP-induced fibrinogen binding. Half-maximal fibrinogen binding occurred at about 0.4 microM ADP, and ADP-induced fibrinogen binding continued progressively during 20 min incubation with 10 microM ADP. Fibrinogen binding correlated closely with platelet glycoprotein IIb-IIIa expression in members of a family with Glanzmann's thrombasthenia, and, in double labelling experiments, with the binding of PAC1, a monoclonal antibody that binds to GP IIb-IIIa only after the exposure of fibrinogen receptors. These studies show that platelet fibrinogen binding can be reliably measured in whole blood by means of a polyclonal antifibrinogen antibody which does not discriminate between plasma and platelet-bound fibrinogen, despite the presence of an approximately 100-fold excess of the former.