Karpatkin S, Xia J, Patel J, Thorbecke G J
Department of Medicine, New York University Medical School, NY 10016.
Blood. 1992 Dec 15;80(12):3164-72.
Serum platelet-reactive and glycoprotein (GP) IIb-GPIIIa-reactive IgG and F(ab')2 was examined in 39 patients with classic autoimmune thrombocytopenic purpura (ATP), two patients with anti-PLA1 antibody and 25 control subjects in an enzyme-linked immunosorbent assay. IgG was purified by diethyl aminoethyl chromatography and centrifuged at 100,000g before testing of the supernatant. Significant IgG binding (threefold to fourfold control IgG binding) was noted with 8 of 17 ATP patients' IgG, 2 anti-PLA1 IgGs, and 2 ATP patients with multiple platelet transfusions. However, F(ab')2 fragments of nine of nine positive ATP IgGs were nonreactive; F(ab')2 from the two anti-PLA1 and two multiply transfused ATP IgGs were as reactive as their intact IgG. Antiplatelet or anti-GPIIb-GPIIIa reactivity of ATP IgG could be adsorbed to fixed platelets or solid-phase GPIIb-GPIIIa and eluted with 0.1 mol/L glycine, pH 2.5. However, binding of IgG to GPIIb-GPIIIa could not be inhibited with F(ab')2 of ATP IgG or Fc fragments of control subjects. When platelet- or GPIIb-GPIIIa-reactive ATP IgG was applied to a Sephacryl 300 gel filtration column, no reactivity was noted in the 7S region, whereas anti-PLA1 localized to this region. Antiplatelet or anti-GPIIb-GPIIIa reactivity was noted in the void volume and accompanied by a high molecular weight protein region. An immunoblot of the void volume fraction with goat antihuman IgG (gamma chain) antibody showed high molecular weight bands greater than 250 Kd, which after reduction converted to a 55-Kd heavy-chain band. Fresh samples of ATP and control IgG processed within 1 to 2 days of blood withdrawal had no reactivity for GPIIb-GPIIIa. After storage at -20 degrees C for greater than 3 months, 5 of 19 ATP IgG became reactive, whereas 16 of 16 controls were nonreactive. Thus, platelet-reactive IgG of ATP sera appears to be caused by the development of IgG aggregates held together by disulfide bonds that develop on storage, and is not F(ab')2 mediated.
采用酶联免疫吸附试验检测了39例经典自身免疫性血小板减少性紫癜（ATP）患者、2例抗PLA1抗体患者及25名对照者血清中血小板反应性及糖蛋白（GP）IIb-GPIIIa反应性IgG和F(ab')2。IgG通过二乙氨基乙基层析法纯化，在检测上清液前以100,000g离心。17例ATP患者中的8例IgG、2例抗PLA1 IgG以及2例多次接受血小板输注的ATP患者出现显著的IgG结合（为对照IgG结合的三到四倍）。然而，9例阳性ATP IgG中的9例F(ab')2片段无反应性；来自2例抗PLA1和2例多次输血的ATP IgG的F(ab')2与完整IgG一样具有反应性。ATP IgG的抗血小板或抗GPIIb-GPIIIa反应性可吸附至固定化血小板或固相GPIIb-GPIIIa上，并用0.1 mol/L甘氨酸（pH 2.5）洗脱，但ATP IgG的F(ab')2或对照者的Fc片段均不能抑制IgG与GPIIb-GPIIIa的结合。当将血小板反应性或GPIIb-GPIIIa反应性ATP IgG应用于Sephacryl S-300凝胶过滤柱时，在7S区域未观察到反应性，但抗PLA1定位于该区域。在空体积及高分子量蛋白区域观察到抗血小板或抗GPIIb-GPIIIa反应性。用山羊抗人IgG（γ链）抗体对空体积部分进行免疫印迹显示分子量大于250 Kd 的高分子量条带，还原后转变为55-Kd重链条带。在采血后1至2天内处理的ATP和对照IgG新鲜样本对GPIIb-GPIIIa无反应性；在-20℃储存超过3个月后，19例ATP IgG中的5例变得具有反应性，而16例对照均无反应性。因此，ATP血清中的血小板反应性IgG似乎是由储存时形成的二硫键连接的IgG聚集体的形成所导致，而非F(ab')2介导。
