Localization of human platelet autoantigens to the cysteine-rich region of glycoprotein IIIa.
The object of this study was to further localize autoantigenic structures on IIb-IIIa and, if possible, to precisely identify the epitopes recognized by human autoantibodies. In this paper, we identify a 50-kD chymotryptic fragment of IIIa that is recognized by a high percentage of human autoantibodies, typified by the prototype IgG autoantibody RA, which binds to IIIa on intact platelets as well as in an immunoblot assay under nonreduced conditions. Using an immunoblot assay, a carboxy-terminal region of this fragment (33 kD) that contains the cysteine-rich domains of IIIa was found to carry the epitope(s) recognized by the prototype autoantibody RA. The amino-terminal amino acid sequence of the reduced 33-kD fragment, the smallest fragment that retains the RA epitope, is XPSQQDEXSP, and that of the reduced 50-kD fragment is IVQVTFD. This indicates that the 33-kD fragment consists of approximately 175 amino acids beginning at residue 479 and extending at least through residues 636-654, while the 50-kD fragment spans the same region but begins at residue 427. It is apparent that the 33-kD fragment is generated from the 50-kD fragment by additional chymotryptic hydrolysis but remains associated because of the multiple disulfide bonds that are characteristic of this cysteine-rich domain. Sera from 48% of patients with chronic ITP and 2 of 8 patients with acute ITP contain antibodies that bind to the 50-kD fragment in an ELISA. Antibodies of the same specificity are also found in one-third of patients with either secondary immune thrombocytopenia or apparent non-immune thrombocytopenia. We conclude that the 50-kD cysteine-rich region of IIIa is a frequent target of autoantibodies in ITP, but that such antibodies may also be present in cases of thrombocytopenia that cannot be linked to an apparent autoimmune process.
Kekomaki
R
R
Blood Center of Southeastern Wisconsin, Milwaukee 53233.
Dawson
B
B
McFarland
J
J
Kunicki
T J
TJ
D001323:Autoantibodies / Q000276:immunology; D001324:Autoantigens / Q000032:analysis*; D001792:Blood Platelets / Q000276:immunology*; D003545:Cysteine / Q000032:analysis; D000939:Epitopes / Q000032:analysis; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis; D010980:Platelet Membrane Glycoproteins / Q000032:analysis / Q000276:immunology*; D011487:Protein Conformation; D011696:Purpura, Thrombocytopenic / Q000276:immunology
D001323:Autoantibodies; D001324:Autoantigens; D000939:Epitopes; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D003545:Cysteine