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The aggregation of cells bearing recombinant integrin albB3 (platelet GPIIb-IIIa) has been analyzed by two-color flow cytometry. As in normal platelets, aggregation requires functional af3, "activation" of af3, and fibrinogen (fg) binding to

Shear rate can affect protein adsorption and platelet aggregation by regulating both the collision frequency and the capture efficiency (alpha). These effects were evaluated in well defined shear field in a micro-couette for shear rate G = 10 - 1000 s-1. The rate of protein binding was independent of G, shown for adsorption of albumin to latex beads and PAC1 to activated platelets. The initial aggregation rate for ADP-activated platelets in citrated platelet-rich plasma followed second order kinetics at the initial platelet concentrations between 20,000 and 60,000/microliters. alpha values, which dropped nearly fivefold for a 10-fold increase in G, were approximately proportional to G-1, contrary to a minor drop predicted by the theory that includes protein cross-bridging. Varying ADP concentration did not change alpha of maximally activated platelet subpopulations, suggesting that aggregation between unactivated and activated platelets is negligible. Directly blocking the unoccupied but activated GPIIb-IIIa receptors without affecting pre-bound Fg on "RGD"-activated, fixed platelets (AFP) by GRGDSP or Ro 43-5054 eliminated aggregation, suggesting that cross-bridging of GPIIb-IIIa on adjacent platelets by fibrinogen mediates aggregation. Alpha for AFP remained maximal (approximately 0.24) over 25-75% Fg occupancy, otherwise decreasing rapidly, with a half-maximum occurring at around 2% occupancy, suggesting that very few bound Fg were required to cause significant aggregation.

Dynamic and quantitative studies of the binding of fibrinogen (Fg) to its receptor, GPIIb-IIIa, on activated platelets, leading to platelet aggregation, are best studied with fluorescently-labelled Fg by flow cytometry. Due to conflicting reports on the functionality of FITC-labelled Fg, we have developed a reproducible and 'mild' labelling of fibrinogen with FITC-celite at pH 7.4-8.5 for direct and dynamic studies of specific Fg binding to activated platelets evaluated for native platelet-rich plasma, for washed platelets, and for activated, fixed platelets. We have demonstrated the equivalence of FITC-labelled and unlabelled Fg for binding to activated GPIIb-IIIa receptors, and in the rate and extent of mediating platelet aggregation. We found that FITC-Fg labelled at pH > or = 9 had reduced to absent specific binding to activated platelets, whether using soluble FITC or FITC-celite. The FITC-labelled Fg must be diluted 3-fold with unlabelled Fg when evaluating maximal Fg binding to activated platelets in order to prevent autoquenching of the FITC-Fg which leads to underestimation of Fg levels. The dissociation constant (KD) of Fg on stable preparations of activated, fixed platelets, determined with FITC-Fg binding to platelets by flow cytometry, was in the range reported for 125I-labelled Fg, 70-255 nm with Bmax = 10000-25000 Fg per platelet (n = 20). The FITC-Fg was used to monitor Fg binding to activated platelets directly by plasma, as well as to evaluate platelet subpopulations which maximally bind Fg according to the concentration of ADP used as activator. It is expected that this 'mildly' labelled FITC-Fg will stimulate further studies of platelet activation directly in native anticoagulated blood/plasma, for both basic and clinical research.

Thrombospondin-1 (TSP) may, after secretion from platelet ␣ granules, participate in platelet aggregation, but its mode of action is poorly understood. We evaluated the capacity of TSP to form inter-platelet crossbridges through its interaction with fibrinogen (Fg), using either Fg-coated beads or Fg bound to the activated GPIIbIIIa integrin (GPIIbIIIa*) immobilized on beads or on activated fixed platelets (AFP), i.e. in a system free of platelet signaling and secretion mechanisms. Aggregation at physiological shear rates (100 -2000 s ؊1 ) was studied in a microcouette device and monitored by flow cytometry. Soluble TSP bound to and induced aggregation of Fg-coated beads dose-dependently, which could be blocked by the amino-terminal heparin-binding domain of TSP, TSP18. Soluble TSP did not bind to GPIIbIIIa*-coated beads or AFP, unless they were preincubated with Fg. The interaction of soluble TSP with Fg-GPIIbIIIa*-coated beads or Fg-AFP resulted in the formation of aggregates via Fg-TSP-Fg cross-bridges, as demonstrated in a system where direct cross-bridges mediated by GPIIbIIIa*-Fg on one particle and free GPIIbIIIa* on a second particle were blocked by the RGD mimetic Ro 44 -9883. Soluble TSP increased the efficiency of Fg-mediated aggregation of AFP by 30 -110% over all shear rates and GPIIbIIIa* occupancies evaluated. Surprisingly, TSP binding to Fg already bound to its GPIIbIIIa* receptor appears to block the ability of this occupied Fg to recognize another GPIIbIIIa* receptor, but this TSP can indeed cross-bridge to another Fg molecule on a second platelet. Finally, TSP-coated beads could directly coaggregate at shear rates from 100 to 2000 s ؊1 . Our studies provide a model for the contribution of secreted TSP in reinforcing inter-platelet interactions in flowing blood, through direct Fg-TSP-Fg and TSP-TSP cross-bridges.

The kinetics of adhesion of platelets to fibrinogen (Fg) immobilized on polystyrene latex beads (Fg-beads) was determined in suspensions undergoing Couette flow at well-defined homogeneous shear rates. The efficiency of platelet adhesion to Fg-beads was compared for ADP-activated versus "resting" platelets. The effects of the shear rate (100-2000 s(-1)), Fg density on the beads (24-2882 Fg/microm(2)), the concentration of ADP used to activate the platelets, and the presence of soluble fibrinogen were assessed. "Resting" platelets did not specifically adhere to Fg-beads at levels detectable with our methodology. The apparent efficiency of platelet adhesion to Fg-beads readily correlated with the proportion of platelets "quantally" activated by doses of ADP, i.e., only ADP-activated platelets appeared to adhere to Fg-beads, with a maximal adhesion efficiency of 6-10% at shear rates of 100-300 s(-1), decreasing with increasing shear rates up to 2000 s(-1). The adhesion efficiency was found to decrease by only threefold when decreasing the density of Fg at the surface of the beads by 100-fold, with only moderate decreases in the presence of physiologic concentrations of soluble Fg. These adhesive interactions were also compared using activated GPIIbIIIa-coated beads. Our studies provide novel model particles for studying platelet adhesion relevant to hemostasis and thrombosis, and show how the state of activation of the platelet and the local flow conditions regulate Fg-dependent adhesion.

Platelet aggregation, which occurs within seconds of activation, is generally considered to be mediated by fibrinogen binding to glycoprotein IIb-IIIa which becomes expressed as a fibrinogen receptor (FbR) on the activated platelet surface. This receptor expression has, however, only been measured to date at relatively long activation times (greater than 15 min). We have therefore developed a theoretical and experimental approach for determining FbR expression within seconds of platelet activation using flow cytometry. The fluorescently labeled IgM monoclonal antibody FITC-PAC1, was used to report on the GPIIb-IIIa receptor for Fb (FbR). Human citrated platelet-rich plasma (PRP; diluted 1:10) was incubated with adenosine diphosphate (ADP) or phorbol myristate acetate (PMA) for varying times (tau = 0-10 s, out to 60 min), followed by incubation with fluorescein isothiocyanate (FITC)-PAC1 antibody at saturating concentrations. The time course of FITC-PAC1 binding was then measured for these variously preactivated samples (different tau) from the mean platelet-bound fluorescence (Fl), determined for greater than or equal to 5 s of PAC1 addition by dilution quenching and determination of fluorescence intensity histograms with the FACSTAR or FACSCAN (Becton-Dickinson Canada, Mississauga, Ontario) flow cytometers. Both rapid, initial rate of increase in Fl (nu) (related to PAC1 on-rates) and maximal extent of increase (Flmax) were thus determined for different tau values. These measurements yield the rate of formation of FbR (k1), and both the rate (k2) and efficiency (alpha) of binding of PAC1 to FbR as a function of activator type and time of action. We have found that ADP appears to cause rapid, maximal expression of FbR within 1-3 s (k1 greater than 20 min-1), whereas PMA expresses FbR in a slow, biphasic manner (k1 - 0.01 and 0.2 min-1). However, k2 and alpha for maximal PMA activation are about two and three times greater, respectively, than for maximal ADP-activation. Moreover, k2 decreases with post ADP activation time. These differences are discussed in terms of altered FbR organization and accessibility. This kinetic approach can be widely used to analyze the dynamics and organization of molecules on cell surfaces by flow cytometry, including studies of size-dependent subpopulations (see Part II, Frojmovic, M., and T. Wong. 1991. Biophys. J. 59:828-837).

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