Flow cytometric analysis of platelet activation and fibrinogen binding

Abstract

Human blood platelets can be activated by a variety of physiological activators such as adenosine diphosphate (ADP), thrombin or collagen, leading to activation of GPIIb-IIIa into a high-affinity receptor for Fg (FgR), binding of fibrinogen (Fg), and subsequent platelet aggregation required for normal hemostasis. Although enormous progress has been made in the biochemistry of platelet activation, of the platelet membrane GPIIb-IIIa, and of solution Fg, much less is known of the dynamics of expression of FgR, of its occupancy by Fg, and of their relation to the dynamics of platelet aggregation. Since platelet activation and aggregation occur within ∼1 s of stirring with activators such as ADP, a methodology was required for determining the rapid dynamics of expression of FgR and binding of Fg, and their correlation with platelet aggregation kinetics. We therefore developed the theoretical and experimental base for determining these dynamic changes under non-equilibrium conditions, using fluorescently-labelled probes and flow cytometry. This approach has yielded a novel general technique for assessing the rapid dynamics of any cell surface molecule, as well as unexpected new insights into the kinetic expression and nature of FgR formed on platelet surfaces activated with ADP and PMA. The same approach has been extended to an analysis of the size-dependent (subpopulation) behaviour of platelets in expressing FgR, obtainable by analytically selecting platelets of different size from forward scatter profiles obtained in studies of the whole population. Parallel measurements of kinetics of platelet recruitment into microaggregates and expression and Fg occupancy of FgR as a function of ADP concentration, led to an unexpected new model for platelet activation and recruitment based once again on the selective recruitment of platelet subpopulation and an 'all or none, quantal' response of any single platelet in expressing all of its FgR and becoming recruitable for aggregation, but at a critical ADP concentration dependent on its own subpopulation characteristics. This approach has also led to novel insights into problems associated with platelet 'activation' arising with different isolation procedures. Dynamic binding studies of Fg to FgR on activated platelets has become possible using appropriately FITC-labelled Fg and flow cytometry. This has also led to studies of the relation between shear-dependent capture efficiency of platelets into doublet formation and the fraction of Fg-occupied receptors. In addition, we have successfully used FITC-labelled human and bovine Fg to demonstrate a delayed expression of FgR and Fg binding to ADP-activated platelets from bleeding Simmental cattle, although the final expression of numbers and accessibility of FgR, measured at equilibrium, were normal. Some future directions for dynamic flow cytometric studies of platelet activation and function are discussed.