Threonine-145/Methionine-145 variants of baculovirus produced recombinant ligand binding domain of GPIbα express HPA-2 epitopes and show equal binding of von Willebrand factor

Abstract

Glycoprotein (GP) Ib is the functionally dominant subunit of the platelet GPIb-IX-V receptor complex, with the von Willebrand factor (vWF) binding site residing on the amino-terminus. A threonine for methionine-145 replacement of GPIb is associated with the human platelet antigen (HPA)-2 system. To study the structural and functional consequences of this mutation, both forms of GPIb were expressed as calmodulin fusion proteins in insect cells. Both recombinant proteins were recognized by their respective alloantibodies, independent of glycosylation or intactness of disulfide bonds, and gave similar results to platelet-derived GPIb in antibody detection assays. Resonant mirror studies showed that vWF binding was not affected by the HPA-2 mutation; however, vWF binding was partially inhibited by IgG HPA-2 antibodies. Our data are compatible with an involvement of the leucine-rich repeat domain of GPIb in vWF binding and indicate that recombinant GPIb may be used to detect HPA-2 antibodies. (Blood. 2000;95:205-211)