Extracellular epitopes of platelet glycoprotein Ib alpha reactive with
serum antibodies from patients with chronic idiopathic thrombocytopenic
purpura
R He, DM Reid, CE Jones and NR Shulman
Clinical Hematology Branch, National Institute of Diabetes, Digestive, and
Kidney Diseases, National Institutes of Health, Bethesda, MD 20892- 1370,
USA.
Glycoproteins (GPs) IIb/IIIa and Ib/IX are principal targets of
autoantibodies (autoAbs) in idiopathic thrombocytopenic purpura (ITP).
Platelet-associated Abs against GPIIb/IIIa primarily recognize
discontinuous or nonlinear epitopes (Fujisawa et al, Blood 81:1284, 1993).
This study focused on whether Abs against the extracellular domain of
GPIb/IX might react with short linear amino acid (aa) sequences of GPIb
alpha. Complementary DNAs (cDNAs) coding for two overlapping fragments of
GPIb alpha were amplified, cloned into pFLAG.2 plasmids, and expressed in
Escherichia coli DH5 alpha competent cells as FLAG fusion proteins, which
were purified by anti-FLAG immunoaffinity chromatography. Of 16 selected
ITP sera containing anti- GPIb/IX, 6 reacted in microtiter
radioimmunoassays (RIAs) with recombinant protein fragment 2 (aas 240 to
485); 1 also with fragment 1 (aas 1 to 247). When synthetic peptides
corresponding to 4 segments of fragment 2 with high antigenic indices (P1
to P4) were used as targets in RIAs, all 6 sera reacted with P2 (aas 326 to
346); 1 also reacted with P4 (aas 389 to 412). P2 was shown to be present
on the surface of intact platelets by adsorption studies, and anti-P2 was
detected in direct eluates of platelets from ITP patients. Glycocalicin in
solution effectively competed with immobilized P2 for anti-P2; P2 in
solution was a less effective competitor. Epitope scanning with a panel of
synthetic 15-mer peptides localized the P2 epitope to the sequence,
TKEQTTFPP. Epitope definition may offer insight into the pathophysiology of
and more specific treatments for ITP.
Volume 86,
Issue 10,
pp. 3789-3796,
11/15/1995
Copyright © 1995 by The American Society of Hematology
