Two-step binding mechanism of fibrinogen to alpha IIb beta 3 integrin reconstituted into planar lipid bilayers.
The platelet integrin alpha IIb beta 3 binds to fibrinogen and thus mediates platelet aggregation after stimulation. This integrin was isolated from human platelets and reconstituted into lipid vesicles. As judged by electron microscopy the integrin incorporated adequately only into 1,2-dimyristoylglycero-3-phosphocholine/1,2-dimyristoylphosphatidy lglycerol vesicles after removal of the detergent by adsorption to Bio-Beads. These vesicles were then used to generate planar lipid bilayers. The binding of fluorochrome labeled fibrinogen or the peptide ligand Gly-Arg-Gly-Asp-Ser-Pro-Cys (GRGDSPC) was monitored by total internal reflection fluorescence microscopy and a solid phase binding assay. Analysis of the kinetics revealed fast reversible formation of a fibrinogen/integrin precomplex (KD = 50 nM) followed by formation of a stable irreversible complex. This transition was monitored by measuring the fraction of precomplex which could be dissociated by addition of excess Gly-Arg-Gly-Asp-Ser (GRGDS). For the peptide, the KD was 1200 nM, and the rates of association and dissociation were faster than the time resolution of the method. Similar KD values were found by inhibition of fibrinogen binding to alpha IIb beta 3 in the immobilized receptor assay. Since the binding of fibrinogen was irreversible, KD values were dependent on the time period between fibrinogen incubation and peptide addition. These and results by other authors point to the biological importance of the biphasic binding process of fibrinogen to its receptor on platelets.
Müller
B
B
Department of Biophysical Chemistry, University of Basel, Switzerland.
Zerwes
H G
HG
Tangemann
K
K
Peter
J
J
Engel
J
J
D000595:Amino Acid Sequence; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data
D016023:Integrins; D008051:Lipid Bilayers; D005340:Fibrinogen