Dynamic measurements of the platelet membrane glycoprotein IIb-IIIa receptor for fibrinogen by flow cytometry. I. Methodology, theory and results for two distinct activators.

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