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Blood, Vol. 92 No. 6 (September 15), 1998:
pp. 2053-2063
A Recombinant Soluble Form of the Integrin
 IIb 3 (GPIIb-IIIa) Assumes an
Active, Ligand-Binding Conformation and Is Recognized by
GPIIb-IIIa-Specific Monoclonal, Allo-, Auto-, and
Drug-Dependent Platelet Antibodies
Julie A. Peterson,
Gian P. Visentin,
Peter J. Newman, and
Richard
H. Aster
From The Blood Research Institute of The Blood Center of Southeastern
Wisconsin and Departments of Medicine, Pathology, Cellular Biology, and
Pharmacology, Medical College of Wisconsin, Milwaukee, WI.
The IIb-IIIa glycoprotein complex is a favored target for allo-,
auto-, and drug-dependent antibodies associated with immune thrombocytopenia. A soluble, recombinant form of the GPIIb-IIIa heterodimer that could be produced in large quantities and maintained in solution without detergent could provide a useful experimental tool
for the study of platelet-reactive antibodies, but previous attempts to
produce such a construct have yielded only small quantities of the end
product. Using a baculovirus expression system and the dual-promoter
transfer vector P2Bac, we were able to express soluble GPIIb-IIIa
complex (srGPIIb-IIIa) lacking cytoplasmic and transmembrane domains in
quantities of about 1,000 µg/L, about 40 times greater than reported
previously. The high yield achieved may be related to inclusion of the
entire extracellular region of the GPIIb light chain in the construct.
srGPIIb-IIIa reacts spontaneously with fibrinogen, and this interaction
is totally inhibited by the peptide RGDS. Reactions of 24 GPIIb-IIIa-specific antibodies evaluated (12 monoclonal, 3 allo-specific, 3 auto-specific, and 6 drug-dependent) with srGPIIb-IIIa
were indistinguishable from reactions with platelet GPIIb-IIIa. Thus,
srGPIIb-IIIa spontaneously assumes an active, ligand-binding
conformation and contains epitopes for all monoclonal and human
antibodies tested to date. srGPIIb-IIIa can be produced in large
quantities, can readily be modified by site-directed mutagenesis, and
should facilitate identification of epitopes recognized by
GPIIb-IIIa-specific antibodies, study of the mechanism(s) by which
certain drugs promote antibody binding to GPIIb-IIIa in drug-induced
thrombocytopenia and structure-function relationships of GPIIb-IIIa.
© 1998 by The American Society of Hematology.
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