Extracellular epitopes of platelet glycoprotein Ib alpha reactive with serum antibodies from patients with chronic idiopathic thrombocytopenic purpura.
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.
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Clinical Hematology Branch, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-1370, USA.
Reid
D M
DM
Jones
C E
CE
Shulman
N R
NR
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D016824:Antigens, Human Platelet / Q000737:chemistry / Q000276:immunology*; D001323:Autoantibodies / Q000097:blood / Q000276:immunology*; D001327:Autoimmune Diseases / Q000097:blood / Q000276:immunology*; D002908:Chronic Disease; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D005260:Female; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000276:immunology*; D016553:Purpura, Thrombocytopenic, Idiopathic / Q000097:blood / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology
D016824:Antigens, Human Platelet; D001323:Autoantibodies; D018076:DNA, Complementary; D000939:Epitopes; D010446:Peptide Fragments; D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins