Threonine-145/methionine-145 variants of baculovirus produced recombinant ligand binding domain of GPIbalpha express HPA-2 epitopes and show equal binding of von Willebrand factor.
Glycoprotein (GP) Ibalpha 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 GPIbalpha is associated with the human platelet antigen (HPA)-2 system. To study the structural and functional consequences of this mutation, both forms of GPIbalpha 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 GPIbalpha 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 GPIbalpha in vWF binding and indicate that recombinant GPIbalpha may be used to detect HPA-2 antibodies. (Blood. 2000;95:205-211)
Li
C Q
CQ
Department of Hematology, University of Cambridge, National Blood Service East Anglia, Cambridge, UK.
Garner
S F
SF
Davies
J
J
Smethurst
P A
PA
Wardell
M R
MR
Ouwehand
W H
WH
D019943:Amino Acid Substitution; D000818:Animals; D000906:Antibodies; D016824:Antigens, Human Platelet / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae; D015153:Blotting, Western; D002460:Cell Line; D014644:Genetic Variation; D006801:Humans; D007313:Insecta; D007700:Kinetics; D008715:Methionine; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013912:Threonine; D014162:Transfection; D014841:von Willebrand Factor / Q000378:metabolism*
D000906:Antibodies; D016824:Antigens, Human Platelet; D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D013912:Threonine; D008715:Methionine