Localization of human platelet autoantigens to the cysteine-rich region of glycoprotein IIIa.

Abstract

The object of this study was to further localize autoantigenic structures on Ilb-Ila and, if possible, to precisely identify the epitopes recognized by human autoantibodies. In this paper, we identify a 50-kD chymotryptic fragment of i11a that is recognized by a high percentage of human autoantibodies, typified by the prototype IgG autoantibody RA, which binds to I11a 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 i11a 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 fiagment is IVQVTFD. This indicates that the 33-kD fragment consists of -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 of8 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 nonimmune thrombocytopenia. We conclude that the 50-kD cysteine-rich region of lIla 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. (J. Clin. Invest. 1991. 88:847-854.)

Mapping early conformational changes in αIIb and β3 during biogenesis reveals a potential mechanism for αIIbβ3 adopting its bent conformation

“…Antibodies AP5 and LIBS2 have previously been shown to recognize epitopes on the ␤3 PSI and ␤TD domains, respectively ( Figure 3A). 22,32,33,44 They bind to only a small fraction of mature ␣IIb␤3 complexes on unactivated platelets, and their binding is markedly enhanced by ligand binding to the receptor or by EDTA treatment. After 2 to 4 hours of chase, both of these antibodies precipitated nearly as much ␤3 as the non-LIBS mAb 7H2 ( Figure 3B).…”

Immunodominant epitopes on glycoprotein IIb-IIIa recognized by autoreactive T cells in patients with immune thrombocytopenic purpura

“…[25][26][27][28][29][30][31][32] Previously reported antigenic epitopes include a 33-kd chymotryptic core fragment of GPIIIa, encoding cysteine-rich domains, 25 a carboxyl-terminal cytoplasmic portion of GPIIIa, 26 a 65-kd chymotryptic carboxyl-terminal fragment of IIb␣, 27 and amino acid residues 231-238 of IIb␣, 28 which are included in IIb␣18-259. However, several recent studies showed that anti-GPIIb-IIIa antibodies, especially plateletassociated antibodies, recognize cation-dependent conformational epitopes expressed exclusively on the GPIIb-IIIa complex.…”

Platelet-associated anti–GPIIb-IIIa autoantibodies in chronic immune thrombocytopenic purpura recognizing epitopes close to the ligand-binding site of glycoprotein (GP) IIb

“…On the other hand, epitopes for serum anti-GPIIb-IIIa autoantibodies have not been localized in the N-terminal region of GPIIb-IIIa (the cytoplasmic domain of GPIIIa, a 50-kd chymotryptic fragment of GPIIIa containing the cysteine-rich repeat, and a C-terminal 65-kd chymotryptic fragment of GPIIb heavy chain). [9][10][11] Previous studies suggested that PA anti-GPIIb-IIIa autoantibodies may differ in specificity from serum antibodies even in the same patient. 16,41 We clearly demonstrated that affinity-purified serum antibodies from patients whose PA antibodies showed the impaired binding to KO GPIIb-IIIa equally reacted with KO and WT GPIIb-IIIa.…”

“…4,5 It has been demonstrated that platelet-associated (PA) autoantibodies, rather than serum antibodies, play a key role in platelet destruction. 6,7 Although a few studies demonstrate the localization of autoantigens on GPIIb or GPIIIa for serum antibodies, [8][9][10][11] characterization of the antigenic epitope(s) for PA autoantibodies remains elusive. [12][13][14] Dissociation of the GPIIb-IIIa complex into free GPIIb and GPIIIa by EDTA treatment markedly impaired the reactivity of PA anti-GPIIb-IIIa autoantibodies, suggesting that most PA autoantibodies recognize cation-dependent conformation(s) of GPIIbIIIa.…”