Rational design and characterization of platelet factor 4 antagonists for the study of heparin-induced thrombocytopenia.
Rational design and characterization of platelet factor 4 antagonists for the study of heparin-induced thrombocytopenia.
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DOI：
10.1182/blood-2012-01-406801
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发表时间：
2012-06
期刊：
影响因子：
20.3
通讯作者：
B. Sachais;A. Rux;D. Cines;S. Yarovoi;Lee Garner;S. Watson;Jillian L. Hinds;J. Rux
中科院分区：
文献类型：
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作者：
B. Sachais;A. Rux;D. Cines;S. Yarovoi;Lee Garner;S. Watson;Jillian L. Hinds;J. Rux
肝素诱导的血小板减少症（HIT）患者尽管在管理方面有所改善，但仍存在复发性血栓栓塞并发症的风险。HIT由优先识别肝素和血小板因子4（PF 4）四聚体的超大复合物（ULC）的抗体引起。我们先前证明了变体PF 4（K50 E）形成二聚体，但不四聚化或形成ULC。在这里，我们确定了预测结合PF 4的二聚体-二聚体界面附近的小分子，干扰PF 4四聚体。通过计算机模拟筛选该位点结合的小分子库，我们鉴定出4种在微摩尔浓度下抑制四聚化的化合物，命名为PF 4拮抗剂（PF 4As）。PF 4As还抑制致病性ULC的形成，并且这些PF 4As中的3种促进预先形成的ULC的分解。为了表征PF 4As抑制细胞活化的能力，我们开发了一种耐用且可重现的试验，该试验使用DT 40细胞通过FcγRIIA测量HIT抗体的细胞活化。PF 4A抑制HIT抗体对DT 40细胞的Fcγ RIIA依赖性活化以及血小板活化，如通过5-羟色胺释放测量的。PF 4A为探讨HIT的病理生理学提供了新的工具。他们还可以提供洞察到新的，疾病特异性治疗的血栓栓塞并发症的治疗HIT的发展。
Patients with heparin-induced thrombocytopenia (HIT) remain at risk for recurrent thromboembolic complications despite improvements in management. HIT is caused by antibodies that preferentially recognize ultralarge complexes (ULCs) of heparin and platelet factor 4 (PF4) tetramers. We demonstrated previously that a variant PF4(K50E) forms dimers but does not tetramerize or form ULCs. Here, we identified small molecules predicted to bind PF4 near the dimer-dimer interface and that interfere with PF4 tetramerization. Screening a library of small molecules in silico for binding at this site, we identified 4 compounds that inhibited tetramerization at micromolar concentrations, designated PF4 antagonists (PF4As). PF4As also inhibited formation of pathogenic ULCs, and 3 of these PF4As promoted the breakdown of preformed ULCs. To characterize the ability of PF4As to inhibit cellular activation, we developed a robust and reproducible assay that measures cellular activation by HIT antibodies via FcγRIIA using DT40 cells. PF4As inhibit FcγRIIA-dependent activation of DT40 cells by HIT antibodies as well as platelet activation, as measured by serotonin release. PF4As provide new tools to probe the pathophysiology of HIT. They also may provide insight into the development of novel, disease-specific therapeutics for the treatment of thromboembolic complications in HIT.