Characterization of New Monoclonal PF4-Specific Antibodies as Useful Tools for Studies on Typical and Autoimmune Heparin-Induced Thrombocytopenia
Characterization of New Monoclonal PF4-Specific Antibodies as Useful Tools for Studies on Typical and Autoimmune Heparin-Induced Thrombocytopenia
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DOI：
10.1055/s-0040-1717078
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发表时间：
2020-10-21
影响因子：
6.7
通讯作者：
Greinacher, Andreas
中科院分区：
文献类型：
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作者：
Vayne, Caroline;Nguyen, Thi-Huong;Greinacher, Andreas
肝素诱导的血小板减少症（HIT）通常是由血小板活化免疫球蛋白G（IgG）抗体（Ab）与肝素（H）复合引起的。不太常见的“自身免疫性”HIT与典型HIT的区别在于没有肝素的血小板活化以及抗PF 4/H和抗PF 4 IgG的存在。我们开发了三种具有人Fc部分的鼠单克隆抗PF 4 Abs，1 E12、1C 12和2 E1，类似于自身免疫性HIT Abs。目的与肝素依赖性单克隆抗PF 4/H Abs 5 B 9和KKO相比，表征1 E12、1C 12和2 E1。方法采用酶联免疫吸附试验（ELISA）检测抗人PF 4和PF 4/H抗体的相互作用，单分子力谱、等温滴定量热法和动态光散射。采用血清素释放试验和肝素诱导的血小板活化试验检测血小板活化情况。结果1C 12、1 E12和2 E1对PF 4/H复合物的亲和力高于5 B 9和KKO，与人源3型抗体的亲和力相当。只有1C 12、1 E12、2 E1和第3组Ab与天然PF 4形成大的复合物，并且在没有肝素的情况下活化血小板。PF 4上1C 12、1 E12和2 E1的预测结合位点与KKO和5 B 9的不同，但彼此接近。结论1C 12、1 E12和2 E1可作为研究自身免疫性HIT病理生理的工具。2 E1为HIT Ab的新结合机制提供了证据。
Background Heparin-induced thrombocytopenia (HIT) is typically caused by platelet-activating immunoglobulin G (IgG) antibodies (Abs) against platelet factor 4 (PF4) complexed with heparin (H). Much less frequent "autoimmune" HIT is distinguished from typical HIT by platelet activation without heparin and the presence of both anti-PF4/H and anti-PF4 IgG. We developed three murine monoclonal anti-PF4 Abs with a human Fc-part, 1E12, 1C12, and 2E1, resembling autoimmune HIT Abs.Objectives To characterize 1E12, 1C12, and 2E1 in comparison to the heparin-dependent monoclonal anti-PF4/H Abs 5B9 and KKO, and polyclonal Abs from patients with typical HIT (group-2) and autoimmune HIT (group-3).Methods Interactions of Abs with PF4 and PF4/H were studied by enzyme-linked-immunosorbent assay, single-molecule force spectroscopy, isothermal titration calorimetry, and dynamic light scattering. Serotonin release assay and heparin-induced platelet activation assay were used to assess platelet activation. The binding sites of monoclonal Abs on PF4 were predicted in silico (MAbTope method).Results 1C12, 1E12, and 2E1 displayed higher affinity for PF4/H complexes than 5B9 and KKO, comparable to human group-3 Abs. Only 1C12, 1E12, 2E1, and group-3 Abs formed large complexes with native PF4, and activated platelets without heparin. The predicted binding sites of 1C12, 1E12, and 2E1 on PF4 differed from those of KKO and 5B9, but were close to each other. 2E1 exhibited unique bivalent binding, involving its antigen recognition site to PF4 and charge-dependent interactions with heparin.Conclusion 1C12, 1E12, and 2E1 are tools for studying the pathophysiology of autoimmune HIT. 2E1 provides evidence for a new binding mechanism of HIT Abs.