Affinity purification of heparin-dependent antibodies to platelet factor 4 developed in heparin-induced thrombocytopenia: biological characteristics and effects on platelet activation
Affinity purification of heparin-dependent antibodies to platelet factor 4 developed in heparin-induced thrombocytopenia: biological characteristics and effects on platelet activation
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DOI：
10.1046/j.1365-2141.2000.02034.x
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发表时间：
2000-05-01
影响因子：
6.5
通讯作者：
Gruel, Y
中科院分区：
文献类型：
--
作者：
Amiral, J;Pouplard, C;Gruel, Y
用亲和层析法从3例肝素诱导的血小板减少症（HIT）患者血浆中纯化肝素血小板因子4（H-PF 4）复合物抗体。从每个血浆中，用pH 7.5的2 M NaCl洗脱最大量的抗体（峰1），并且使用pH 2.5的0.1M甘氨酸/0.5M NaCl获得剩余的抗体（峰2）。在酶联免疫吸附试验（ELISA）中，我们发现每个患者都产生了具有不同特征的PF 4抗体。在患者1中，峰1 IgG几乎仅与H-PF 4复合物反应，而峰2 IgG与PF 4具有类似的反应性，无论是否存在肝素，患者2表达伊加、IgM和IgG的混合物，并且两种级分均单独与PF 4结合或与H-PF 4复合物结合。终于来了患者3中的IgG仅与H-PF 4结合，并且与单独的PF 4不反应。使用[C-14]-5-羟色胺释放测定，在三名患者中产生的抗体，并表现出最强的能力，激活血小板与肝素是那些具有最高的亲和力H-PF 4。这些结果有力地支持了这样的假设，即针对PF 4的HIT抗体在它们对靶抗原的亲和力和特异性方面是异质的，这可能极大地影响它们激活血小板的能力和它们的致病性。
Antibodies to heparin platelet factor 4 (H-PF4) complexes were purified from the plasma of three patients with heparin-induced thrombocytopenia (HIT) using affinity chromatography. From each plasma, the largest amount of antibodies was eluted with 2 M NaCl at pH 7.5 (peak 1) and the remainder was obtained using 0.1 M glycine/0.5 M NaCl at pH 2.5 (peak 2). In an enzyme-linked immunosorbent assay (ELISA), we then showed that each patient had developed antibodies to PF4 displaying different characteristics. In patient 1, peak 1 IgG reacted almost exclusively with H-PF4 complexes, whereas peak 2 IgG had similar reactivity with PF4 whether or not heparin was present, Patient 2 expressed a mixture of IgA, IgM and IgG and both fractions bound to PF4 alone or to H-PF4 complexes. Finally. IgG in patient 3 only bound to H-PF4 and was unreactive with PF4 alone. Using [C-14]-serotonin release assays, the antibodies developed in the three patients and exhibiting the strongest ability to activate platelets with heparin were those having the highest affinity to H-PF4. These results strongly support the hypothesis that HIT antibodies to PF4 are heterogeneous regarding their affinity and specificity for target antigens and this may greatly influence their ability to activate platelets and their pathogenicity.