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.

抽象的 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，发生于 对于血栓患者，普通肝素 (UFH) 仍然是标准抗凝剂。 并发症，替代抗凝剂不能减轻症状，只能提供部分保护 复发性血栓并存在出血风险，但没有解毒剂，因此 HIT 被过度诊断。 目前的检测无法区分致病性和非致病性抗 PF4 抗体 (Abs)。 更好地了解 HIT 的发病机制，以开发更好的诊断方法和合理的、针对疾病的、非 我们最近描述了 PF4 与肝素复合物的晶体结构。 基于五聚体和 HIT 样鼠 (m) 抗体 (KKO) 我们证明了一种抗 PF4 单克隆抗体 (RTO) 可以预防。 PF4 形成四聚体和高阶免疫复合物，可防止 HIT Abs 激活血小板 基于这些结构，我们研究了 HIT 抗体在体外引起的血小板减少和血栓形成。 开发了 I 类 PF4 单体突变体，无法形成四聚体，从而抑制繁殖 寡聚 PF4/UFH 复合物结合 KKO 和人 HIT Abs，激活 FcRγIIA 受体，抑制 组织因子的表达，并且与天然 PF4 相比，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。 在这个提案中，我们将分析这两类 PF4 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（SA）#1。 I 类和 II 类 PF4 突变体的作用机制和结构这些研究将探讨 PF4 如何作用。 肝素和 HIT Ab 寡聚形成更高级的抗原复合物。 这些 PF4 变体在体外和 HIT 小鼠模型中阻断免疫复合物形成，同时产生 这种血栓性疾病的良好止血特性。 I 类药物的产生和表征。 将使用 CRISPR/Cas9 技术扩展人类和小鼠 PF4 小鼠的体内研究。 小鼠表达 I 类单体 PF4 变体，并检查它们在 HIT 小鼠模型中的作用。 I 类单体 PF4 变体用于区分致病性和非致病性 HIT 抗体的用途。 I 类 PF4 单体柱将用于分离结合四聚体 PF4/聚阴离子的致病性 HIT 抗体 来自非致病性抗体的复合物结合单体 PF4 并阻断致病性抗体的结合。 测试一个新的假设，即 HIT 在缺乏这种阻断抗体的患者中更为普遍。 拟议的研究结合了生物物理学研究、新颖的小鼠模型和涉及人类的新假设 样本将有助于更好地了解 HIT 中免疫复合物的形成，开发一种疾病- 特异性 ELISA，并可能为合理的非抗凝方法补充提供基础 现代抗血栓治疗可减轻这种严重的自身免疫性血栓性疾病。