Dynamic measurements of the platelet membrane glycoprotein IIb-IIIa receptor for fibrinogen by flow cytometry. II. Platelet size-dependent subpopulations

Abstract

Platelet aggregation has previously been shown to occur within 1 s of activation with 100 microM adenosine diphosphate (ADP) for both large (L) and small (S) platelet subpopulations, but L platelets were about twofold more sensitive and more rapidly recruited into microaggregates than were S platelets after correcting for differences in platelet surface area. Because platelet aggregation normally requires fibrinogen binding to glycoprotein IIb-IIIa receptors (FbR) expressed on the activated platelet surface, we wished to compare the kinetics and nature of FbR expression induced by ADP for L versus S platelets, and to measure size-dependent differences in FbR expression for platelets maximally activated with phorbol myristate acetate (PMA). We presented the theory and methodology in Part I (Frojmovic, M., T. Wong, and T. van de Ven. 1991. Biophys. J. 59:815-827) for measuring the rate of FbR expression (k1) and both the rate (k2) and efficiency (alpha) of binding of PAC1 to FbR as a function of activation conditions from the initial on-rate of FITC-PAC1 to FbR (V) and the maximal number of FbR expressed: these are measured, respectively, from the initial rate of increase in platelet-bound fluorescence (v) and the maximal increase in mean fluorescence (Flmax). We extended these analyses to L and S platelets, selected by electronic gating of forward scatter profiles (FSC), with corresponding fluorescence (Fl) histograms retrieved analytically. Platelet size (V) and surface area (SA), determined directly for cells separated with a cell sorter, were highly correlated with FSC, allowing v and Flmax values to be expressed per unit area of membrane for L:S comparisons. Surprisingly, ADP activation appeared to express all FbR within 1-3 s of ADP activation for both L and S platelets, whereas k1 was similar for PMA activation. In addition, L platelets maximally expressed two and three times more FbR per unit area than did S platelets when maximally stimulated, respectively, with ADP or PMA. Whereas k2 was independent of platelet size for a given activator, the efficiency of PAC1 binding (alpha), per unit area of membrane, was two times greater for L than for S platelets, for either ADP or PMA activation. Our data suggest that the FbR structure, its microenvironment, or its surface organization may vary with platelet size or activator type. Major reorganization of FbR and/or its environment appears to occur after approximately 5 min of ADP activation equally for both L and S platelets. A model is presented to account for size-dependent differences in FbR expression with implications for regulation of platelet aggregation.