Sequestration of anti-platelet GPIIIa antibody in rheumatoid factor immune complexes of human immunodeficiency virus 1 thrombocytopenic patients.

Abstract

Human immunodeficiency virus 1-related idiopathic thrombocytopenic purpura (HIV-1-ITP) patients have a 4-fold increased percentage of CD5+ B cells and a 4.8-fold increased percentage of serum immune complexes precipitated by polyethylene glycol (PEG-ICs) compared to control subjects, as reported previously. Since CD5+ B cells produce predominantly IgM rheumatoid factor (RF) vs. Fc of IgG and PEG-ICs contain high levels of IgM, we looked for the presence of RF in the immune complexes of HIV-1-ITP patients. PEG-ICs were adsorbed to protein A and dissociated with acid, and IgM and IgG were purified by gel filtration and affinity chromatography. Solid-phase ELISA was used to measure antibody specificity vs. platelets, Fc, and HIV-1 gp120, p24, and CD4. Dissociated IgG antibody reacted with platelets, HIV-1 gp120, p24, and CD4, but not with Fc. Serum IgG did not react with platelets or Fc but did react with HIV-1 gp120, p24, and CD4. Both PEG-IC IgM and serum IgM reacted with Fc as well as the other four antigens. Control IgM and IgG were unreactive. Isolated IgM from PEG-ICs relocated approximately 50% of the IgG preincubated with IgM to the Vo region of a G200 gel-filtration column. Anti-platelet IgG but not IgM could be affinity-purified from fixed platelets. Both F(ab')2 fragments of anti-platelet IgG and the total PEG-IC bound to platelets in a saturation-dependent manner. F(ab')2 of anti-platelet IgG inhibited 50% binding of PEG-IC to platelets at an F(ab')2/complex ratio of 3:1 (wt/wt). Scatchard analysis revealed two classes of binding sites: high-affinity Kd values of 0.8-1.8 nM and lower-affinity Kd values of 6.6-12.3 nM with respective numbers of binding sites of 44,000-57,000 and 122,000-256,000 (n = 4). Anti-platelet IgG of 6/6 patients precipitated GPIIIa from platelet lysates of surface 125I-labeled platelets. Platelet count correlated inversely with anti-platelet IgG (r = -0.73; P < 0.01; n = 27). Thus, PEG-ICs of HIV-1-ITP patients contain IgM RF, which sequesters serum anti-platelet IgG containing anti-GPIIIa. Anti-platelet IgG contributes to binding of immune complexes to platelets and correlates with thrombocytopenia.

Implications for Induction of Autoimmunity via Activation of B-1 Cells byHelicobacter pyloriUrease

“…In addition to such gastroduodenal disorders, H. pylori infection is associated with various autoimmune diseases such as rheumatoid arthritis (22), Sjögren's syndrome (12), and idiopathic thrombocytopenic purpura (ITP) (17). In the case of ITP, the binding ability of anti-platelet-specific immunoglobulin G (IgG) is enhanced by rheumatoid factors (RFs) that may sequester IgG (26). The marked improvement in platelet counts after H. pylori eradication (14) indicates a direct correlation between the pathogenicity of ITP and H. pylori infection.…”

Platelet particle formation by anti–GPIIIa49-66 Ab, Ca2+ ionophore A23187, and phorbol myristate acetate is induced by reactive oxygen species and inhibited by dexamethasone blockade of platelet phospholipase A2, 12-lipoxygenase, and NADPH oxidase

“…36 The antiplatelet Ab was eluted with 0.1 M glycine buffer (pH 2.5) and further purified on a GPIIIa49-66 peptide-affinity column, as described. 36 …”

Complement-independent Ab-induced peroxide lysis of platelets requires 12-lipoxygenase and a platelet NADPH oxidase pathway

“…Affinity purification of antiplatelet GPIIIa49-66. IgG was isolated by polyethylene glycol-precipitable immunocomplexes (8), followed by protein A affinity chromatography, G-200 gel filtration, affinity chromatography and affinity purification on 2% paraformaldehyde-fixed platelets as described (7). The antiplatelet Ab was eluted with 0.1 M glycine buffer, pH 2.9, and further purified on a GPIIIa49-66 peptide-affinity column, as described (7).…”