Structure-based Design of Rational PF4 Inhibitors in HIT

摘要 肝素诱导的血小板减少症(HIT)是由在体内形成的免疫复合物引起的血栓性疾病 普通肝素(UFH)仍为标准抗凝剂的设置。对于那些患有血栓的人 并发症，替代抗凝剂不能缓解症状，只能提供部分保护 反复血栓形成，有出血的风险，目前还没有解药。HIT被过度诊断是因为 目前的检测方法不能区分致病性和非致病性抗PF4抗体。有必要 更好地了解HIT的发病机制，以开发更好的诊断和合理的、疾病特异性的、非 抗凝剂管理。我们最近描述了PF4与肝素的络合物的晶体结构。 基于五聚体和一种类似小鼠(M)的抗体(Kko)。我们证明了一种抗PF4单抗(RTO)可以防止 PF4不形成四聚体和更高序度的免疫复合体，防止HIT抗体激活血小板 体外和体内HIT抗体引起的血小板减少和血栓形成。基于这些结构，我们 开发出不能形成四聚体从而抑制繁殖的I类PF4单体突变体 结合KKO和人HIT抗体的寡聚体Pf4/UFH复合体，激活FCRγIIA受体，抑制 与天然的PF4相比，组织因子的表达对UFH/抗凝血酶III辅因子活性的抑制较小。 我们还开发了形成抗原阴性四聚体的II类PF4突变体作为替代方法。在……里面 在这项建议中，我们将分析这两类PF4变体，以更好地了解HIT的发病机制 并开发新的诊断和潜在的治疗工具。特定目标(SA)#1.有效性、安全性、 I类和II类pf4突变体的作用机制和结构。这些研究将考察PF4、 肝素和Hit Abs寡聚形成更高有序的抗原复合体。我们将确定是否 这些PF4变异体在体外和在HIT小鼠模型中阻止免疫复合物的形成，同时产生 在这种血栓性疾病中有良好的止血效果。SA#2.第I类的生成和表征 人和小鼠PF4小鼠。将使用CRISPR/Cas9技术扩展体内研究以开发 表达I类单体PF4变异体的小鼠，并在HIT小鼠模型中检查它们的效果。SA#3. I类单体PF4变异体用于区分致病性和非致病性HIT抗体。 I类PF4单体柱将用于分离结合四聚体PF4/聚阴离子的致病HIT抗体 来自非致病抗体的结合单体PF4并阻断致病抗体结合的复合体。我们会 测试一个新的假设，即HIT在缺乏这种阻断抗体的患者中更普遍。团结在一起 拟议的研究结合了生物物理学研究、新的小鼠模型和涉及人类的新假说 将提供更好地了解HIT中免疫复合体形成的样本，会发展成一种疾病- 特异性的酶联免疫吸附试验，并可能为合理的非抗凝剂补体方法提供基础 现代抗血栓治疗，以减轻这种严重的自身免疫性血栓形成障碍。

ABSTRACT Heparin-induced thrombocytopenia (HIT) is thrombotic disorder caused by immune complexes that develops in settings where unfractionated heparin (UFH) remains the standard anticoagulant. For those with thrombotic complications, alternative anticoagulants do not mitigate symptoms, provide only partial protection against recurrent thrombosis and carry a risk of bleeding for which there is no antidote. HIT is over-diagnosed because current assays do not distinguish pathogenic from non-pathogenic anti-PF4 antibodies (Abs). There is need for better understanding the pathogenesis of HIT to develop better diagnostics and rational, disease-specific, non- anticoagulant management. We recently described the crystal structure of PF4 in complex with a heparin- based pentamer and a HIT-like murine (m) Ab (KKO). We showed that an anti-PF4 mAb (RTO) that prevents PF4 from forming tetramers and higher-ordered immune complexes, prevents platelet activation by HIT Abs in vitro, and thrombocytopenia and thrombosis caused by HIT Abs in vivo. Based on these structures, we developed Class I PF4 monomeric mutants that are unable to form tetramers, and thereby inhibit propagation of oligomeric PF4/UFH complexes that bind KKO and human HIT Abs, activate FcRγIIA receptors, inhibit expression of tissue factor and cause less inhibition of UFH/antithrombin III cofactor activity than native PF4. We also developed Class II PF4 mutants that form antigen-negative tetramers as an alternative approach. In this proposal, we will analyze these two classes of PF4 variants to better understand the pathogenesis of HIT and to develop novel diagnostic and potential therapeutic tools. Specific Aims (SA)#1. Efficacy, safety, mechanism of action and structure of Class I and II PF4 mutants. These studies will examine how PF4, heparin and HIT Abs oligomerize to form higher-ordered antigenic complexes. We will determine whether these PF4 variants block immune complex formation in vitro and in a murine model of HIT while generating a favorable hemostatic profile in this thrombotic disease. SA#2. Generation and characterization of Class I human and mouse PF4 mice. In vivo studies will be extended using CRISPR/Cas9 technology to develop mice expressing Class I monomeric PF4 variants and to examine their effects in a murine model of HIT. SA#3. Utility of Class I monomeric PF4 variants to distinguish pathogenic from non-pathogenic HIT Abs. Class I PF4 monomer columns will be used to separate pathogenic HIT Abs that bind tetrameric PF4/polyanion complexes from non-pathogenic Abs that bind monomeric PF4 and block binding of pathogenic Abs. We will test a novel hypothesis that HIT is more prevalent in patients lacking such blocking Abs. Together the proposed studies combine biophysical studies, novel murine models and a new hypothesis involving human samples that will provide a better understanding of immune complex formation in HIT, develop a disease- specific ELISA, and potentially offer a basis for a rational non-anticoagulant approach to complement contemporary anti-thrombotic therapy to mitigate this serious autoimmune thrombotic disorder.