A recombinant soluble form of the integrin alpha IIb beta 3 (GPIIb-IIIa) assumes an active, ligand-binding conformation and is recognized by GPIIb-IIIa-specific monoclonal, allo-, auto-, and drug-dependent platelet antibodies.
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 microg/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.
Peterson
J A
JA
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, USA.
Visentin
G P
GP
Newman
P J
PJ
Aster
R H
RH
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001323:Autoantibodies / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D007518:Isoantibodies / Q000378:metabolism; D008024:Ligands; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D011802:Quinidine / Q000494:pharmacology; D011803:Quinine / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D012995:Solubility; D013420:Sulfamethoxazole / Q000494:pharmacology; D013921:Thrombocytopenia / Q000139:chemically induced / Q000276:immunology
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D007518:Isoantibodies; D008024:Ligands; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins; D011803:Quinine; D011802:Quinidine; D013420:Sulfamethoxazole