Karpatkin S, Nardi M A, Hymes K B
Department of Medicine, New York University Medical School, NY 10016.
Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2263-7. doi: 10.1073/pnas.92.6.2263.
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.
如先前报道，与对照受试者相比，人类免疫缺陷病毒1相关的特发性血小板减少性紫癜（HIV-1-ITP）患者的CD5⁺ B细胞百分比增加了4倍，聚乙二醇沉淀的血清免疫复合物（PEG-ICs）百分比增加了4.8倍。由于CD5⁺ B细胞主要产生IgM类风湿因子（RF）而非IgG的Fc，且PEG-ICs含有高水平的IgM，我们研究了HIV-1-ITP患者免疫复合物中RF的存在情况。PEG-ICs吸附于蛋白A并经酸解离，IgM和IgG通过凝胶过滤和亲和层析纯化。采用固相ELISA检测抗体对血小板、Fc以及HIV-1 gp120、p24和CD4的特异性。解离的IgG抗体与血小板、HIV-1 gp120、p24和CD4反应，但不与Fc反应。血清IgG不与血小板或Fc反应，但与HIV-1 gp120、p24和CD4反应。PEG-IC IgM和血清IgM均与Fc以及其他四种抗原反应。对照IgM和IgG无反应。从PEG-ICs中分离的IgM将与IgM预孵育的约50%的IgG重新定位到G200凝胶过滤柱的Vo区域。抗血小板IgG而非IgM可从固定血小板中亲和纯化。抗血小板IgG的F(ab')₂片段和总PEG-IC均以饱和依赖方式与血小板结合。抗血小板IgG的F(ab')₂在F(ab')₂/复合物比例为3:1（重量/重量）时抑制50%的PEG-IC与血小板结合。Scatchard分析显示有两类结合位点：高亲和力Kd值为0.8 - 1.8 nM，低亲和力Kd值为6.6 - 12.3 nM，各自的结合位点数为44,000 - 57,000和122,000 - 256,000（n = 4）。6/6例患者的抗血小板IgG从表面¹²⁵I标记血小板的血小板裂解物中沉淀出GPIIIa。血小板计数与抗血小板IgG呈负相关（r = -0.73；P < 0.01；n = 27）。因此，HIV-1-ITP患者的PEG-ICs含有IgM RF，其隔离了含有抗GPIIIa的血清抗血小板IgG。抗血小板IgG有助于免疫复合物与血小板的结合，并与血小板减少症相关。
