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.

The biology of extracellular vesicles with focus on platelet microparticles and their role in cancer development and progression

“…It has been shown that following PS binding with annexin V, the fusion of PMPs with target cell is blocked that prevents exchange of their cargo [15]. PMPs may also be eliminated by serum phospholipase A2 activity [3, 92]. It is still unclear which of the abovementioned mechanisms is the most significant mean of PMP elimination.…”

Relationships of glycoproteins IIb-IIIa and Ib content with mean platelet volume and their genetic polymorphisms

“…Leytin et al [22] calculated that large platelets contained about twice more GPIIb-IIIa than small ones, whereas the density of GPIIb-IIIa per surface area unit was the same in both fractions. Comparison of platelet subpopulations has also indicated that, upon activation, large platelets express higher amounts of activated GPIIb-IIIa (PAC-1 antibody binding) and P-selectin [23,24]. Interindividual variations of adhesion molecules number could be influenced not only by the size of platelets which is routinely assessed by measuring mean platelet volume (MPV) but also by their genetic polymorphisms.…”

Quantitative detection of platelet GPIIb-IIIa receptor antagonist activity using a flow cytometric method

“…The platelet glycoprotein (GP) IIb-IIIa is the most abundant platelet integrin with 50,000-70,000 sites per platelet (1). When platelets are activated, membrane-surface GPIIb-IIIa binds soluble fibrinogen (Fb) in a key, early step of platelet aggregation and thrombus formation (2).…”