Organization of the glycoprotein (GP) IIb/IIIa heterodimer on resting human platelets studied by flow cytometric energy transfer.
Glycoprotein IIb/IIIa is a heterodimer of glycoproteins IIb and IIIa which serves as the inducible receptor for fibrinogen and other adhesive proteins at the surface of platelets. Although a model of the quaternary structure of the GPIIb/IIIa molecule has been constructed in solution by Calvete et al. [Biochem. J. 282 (1992) 523], a corresponding model at the surface of intact platelets is still missing. In the present work conformation and lateral distribution of the GPIIb/IIIa heterodimer were studied at a nanometer resolution on the surface of resting human platelets under physiological conditions. The experiments were based on dual wavelength flow cytometric detection of fluorescence resonance energy transfer and application of a panel of monoclonal antibodies raised against well described binding sites. Monodisperse distribution of the GPIIb/IIIa heterodimer has been observed and a detailed three-dimensional proximity map of antibody binding sites was constructed on the platelet membrane, under physiological conditions, for the first time. Our data support the view that the GPIIb subunit is in a bent conformation. A detailed analysis of the K(d)-values and the number of binding sites for a set of monoclonal antibodies was also carried out giving supplementary data for the topology of the binding sites. Our results provide a refinement of the membrane-topology of the GPIIb/IIIa heterodimer.
Mátyus
L
L
Department of Biophysics and Cell Biology, University of Debrecen, Nagyerdei krt 98, H-4012 Debrecen, Hungary. lmatyus@jaguar.dote.hu
Bene
L
L
Hársfalvi
J
J
Alvarez
M V
MV
González-Rodríguez
J
J
Jenei
A
A
Muszbek
L
L
Damjanovich
S
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism*; D019281:Dimerization; D004735:Energy Transfer; D005434:Flow Cytometry; D006801:Humans; D051379:Mice; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D019039:Platelet Glycoprotein GPIIb-IIIa Complex