Structure-based Design of Rational PF4 Inhibitors in HIT

HIT 中合理 PF4 抑制剂的基于结构的设计

• 批准号: 9900853
• 负责人: Douglas Brock Cines
• 金额: $ 57.22万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9900853
• 关键词: Antibodies; Antibody Avidity; Anticoagulant therapy; Anticoagulants; Anticoagulation; Antidotes; Antigen-Antibody Complex; Antigens; Antithrombin III; Autoimmune Process; Binding; Biochemical; Biological Assay; Biophysics; Bypass; CRISPR/Cas technology; Cardiac; Cardiovascular Surgical Procedures; Cells; Clinical Research; Complement; Complex; Crystallization; Detection; Diagnosis; Diagnostic; Disease; Enzyme-Linked Immunosorbent Assay; Exposure to; Generations; Hemorrhage; Hemostatic Agents; Heparin; Heparin Binding; Human; In Vitro; Interruption; Intervention; Knock-in Mouse; Lead; Mediating; Monoclonal Antibodies; Mus; Operative Surgical Procedures; PF4 Gene; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Pharmaceutical Preparations; Physiological; Plasma; Platelet Activation; Predictive Value of Tests; Prevalence; Property; Recurrence; Risk; Role; Safety; Sampling; Structure; Symptoms; Testing; Therapeutic; Thrombocytopenia; Thromboplastin; Thrombosis; Transgenic Organisms; Variant; base; biophysical analysis; biophysical techniques; clinical Diagnosis; clinically relevant; cofactor; design; experimental study; heparin cofactor; heparin-induced thrombocytopenia; high risk; in vivo; inhibiting antibody; inhibitor/antagonist; monocyte; monomer; mouse model; mutant; novel; novel diagnostics; polyanion; preservation; prevent; receptor; tool

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.

摘要 肝素诱导的血小板减少症（HIT）是由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。对于那些有血栓的人 并发症，替代抗凝剂不能减轻症状，仅提供部分保护， 复发性血栓形成，并有出血的风险，没有解毒剂。过度诊断是因为 目前的测定不能区分致病性和非致病性抗PF 4抗体（Ab）。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，非 抗凝剂管理我们最近描述了PF 4与肝素复合物的晶体结构- 基于HIT的五聚体和HIT样鼠（m）Ab（KKO）。我们发现，抗PF 4 mAb（RTO）可以阻止 PF 4形成四聚体和更高级的免疫复合物，防止血小板被HIT抗体激活， 体外实验和体内实验表明，HIT Abs可引起血小板减少和血栓形成。基于这些结构，我们 开发了I类PF 4单体突变体，其不能形成四聚体，从而抑制繁殖 寡聚PF 4/UFH复合物结合KKO和人HIT Ab，激活FcRγIIA受体，抑制 组织因子的表达，并导致UFH/抗凝血酶III辅因子活性的抑制比天然PF 4少。 我们还开发了形成抗原阴性四聚体的II类PF 4突变体作为替代方法。在 在这个建议中，我们将分析这两类PF 4变异体，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。具体目标（SA）#1。有效性、安全性， I类和II类PF 4突变体的作用机制和结构。这些研究将研究PF 4， 肝素和HIT Ab寡聚化形成更高级抗原复合物。我们将决定 这些PF 4变体在体外和HIT小鼠模型中阻断免疫复合物的形成，同时产生 在这种血栓性疾病中具有良好的止血特性。SA #2。I类的生成和表征 人和小鼠PF 4小鼠。体内研究将使用CRISPR/Cas9技术进行扩展， 表达I类单体PF 4变体的小鼠，并检查它们在HIT鼠模型中的作用。SA #3。 I类单体PF 4变体区分致病性与非致病性HIT Ab的效用。 I类PF 4单体柱将用于分离结合四聚体PF 4/聚阴离子的致病性HIT Ab 来自非致病性Ab的复合物，其结合单体PF 4并阻断致病性Ab的结合。我们将 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。一起 拟议的研究结合了联合收割机生物物理学研究、新型小鼠模型和涉及人类的新假设 样本，这将提供更好地了解免疫复合物的形成在HIT，发展一种疾病- 特异性ELISA，并可能为合理的非抗凝方法提供基础，以补充 当代抗血栓治疗以减轻这种严重的自身免疫性血栓形成病症。