Platelet Factor 4 and heparins in NETosis and Sepsis

血小板因子 4 和肝素在 NETosis 和脓毒症中的作用

• 批准号: 10656307
• 负责人: Mortimer Poncz
• 金额: $ 58.89万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-10 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10656307
• 关键词: Acceleration; Affect; Animals; Antibiotics; Antibodies; Anticoagulants; Arginine deiminase; Bacteria; Binding; Biology; Blood; Blood Platelets; Canis familiaris; Cations; Cause of Death; Charge; Clinical; Clinical Trials; Complex; Complication; Cytolysis; DNA; Deoxyribonuclease I; Deoxyribonucleases; Disease; Dose; Endothelium; Endotoxemia; Generations; Goals; Grant; Health; Hemostatic Agents; Hemostatic function; Heparin; Histones; Human; In Vitro; Infection; Infusion procedures; Injury; Intervention; Leukocytes; Life; Lipopolysaccharides; Maximum Tolerated Dose; Microbe; Microfluidics; Modeling; Monoclonal Antibodies; Morbidity - disease rate; Mus; Organ; Outcome; PF4 Gene; Pathologic; Patient Care; Patients; Peritonitis; Physiological; Pilot Projects; Predisposition; Process; Protamine Sulfate; Protein-arginine deiminase; Proteins; Resistance; Role; Sepsis; System; Testing; Therapeutic; Toxic effect; Translating; Treatment Efficacy; United States; Variant; cell free DNA; chemokine; efficacy study; extracellular; in vitro testing; in vivo; microbial; microbicide; mortality; mouse model; neutrophil; novel therapeutics; pilot trial; polyanion; pre-clinical; prevent; response; septic; septic patients; side effect; therapy development; thrombotic; thrombotic complications

ABSTRACT Sepsis is a multi-organ, dysfunctional response to infection. PROJECT 4 focuses on the role of released neutrophil extracellular traps (NETs), its ability to entrap microbes, and NET-degradation products (NDPs, e.g., cell-free DNA and histones) in organ damage, and on the interactions of the cationic chemokine, platelet factor 4 (PF4) and the polyanion heparin with NETs. On presentation, many septic patients have an overwhelming amount of released NETs so that preventing NET release (NETosis) by blocking peptidylarginine deiminase 4 or accelerating NET lysis would be ineffective in preventing morbidity and mortality. We propose that in sepsis NET stabilization, enhanced microbe entrapment, and/or NDP sequestration would be protective. Our studies have defined three related strategies that can achieve one or more of these goals. We propose to better understand the underlying mechanism(s) of these strategies, hemostatic/thrombotic implications, and their potential therapeutic efficacy. Specific Aim (SA) #1: Understanding small, positive protein effects on NETs. PF4 and other small, positive proteins (e.g., protamine sulfate (ProSO4)) compact NETs, decreasing their lysis by DNases. PF4, but not ProSO4, prevents NDP release, an important difference between these cationic proteins that will be explored to understand the impact of compacting NETs in sepsis. PF4 enhances microbe entrapment and protects the endothelium from NET injury. Whether ProSO4 similarly effects NET biology given that it releases NDPs will be pursued in a microfluidic system and in murine endotoxic/sepsis models. Both cationic proteins will be compared to or in conjunction with other NET-directed therapeutic options. The side-effects of these cations, especially on hemostasis, will be explored. Pilot trials of canine and human (h) PF4s will be infused in dogs with spontaneous peritonitis with the long-term goal of a clinical trial in this large animal sepsis setting. SA#2: Studies on enhancing the effect of PF4 on NETs. We have found that a monoclonal antibody KKO that enhances PF4 binding to NETs further increases DNase resistance. An Fc-modified KKO variant protects against NET deleterious effects in vitro and in mice models of sepsis. The underlying mechanism(s) of how KKO, and other anti-PF4 antibodies effect NET biology will be defined. KKO infusion studies will be pursued in mice expressing hPF4 looking at efficacy and thrombotic complications and in pilot studies of co-infused KKO and hPF4 in septic dogs. SA#3: Understanding ODSH effects on NETs in sepsis. We have previously shown that the desulfated heparin ODSH, which has markedly lower anticoagulant effects than heparin, is protective in a histone infusion murine model and will explore its mechanistic basis in vitro and in murine sepsis models as in SA#1. We will continue a dose escalation study on the efficacy of ODSH in dogs with spontaneous peritonitis to define maximal tolerated dose for an eventual clinical trial. These studies in PROJECT 4 should advance our understanding of the interactions of PF4 and heparins with NETs in sepsis and lead to novel therapies for this devastating clinical state.

抽象的 败血症是对感染的多器官功能失调的反应。项目4专注于发布的作用 中性粒细胞外陷阱（净），其捕获微生物的能力和净降解产物（例如NDPS，例如， 无细胞的DNA和组蛋白）在器官损伤中，以及阳离子趋化因子，血小板因子的相互作用 4（PF4）和带网的聚苯蛋白肝素。在介绍中，许多化粪池患者有压倒性的 释放的网量，从而通过阻断肽基金蛋白脱节酶4来防止净释放（Netosis） 或加速净裂解将无效预防发病率和死亡率。我们在败血症中提出 净稳定，增强的微生物夹带和/或NDP隔离将具有保护性。我们的研究 已经定义了三种相关策略，可以实现这些目标中的一个或多个。我们建议更好 了解这些策略的潜在机制，止血/血栓形成含义及其 潜在的治疗功效。特定目的（SA）＃1：了解小蛋白质对 网。 PF4和其他小的阳性蛋白质（例如硫酸质（Proso4））紧凑型网，减少 他们裂解了DNass。 PF4，但不是ProSO4可防止NDP发布，这是其中一个重要的区别 阳离子蛋白将被探索以了解败血症中压实网的影响。 PF4增强 微生物夹带并保护内皮免受净损伤。 ProSO4是否同样效果网络 鉴于生物学将在微流体系统和鼠内毒素/败血症中释放NDP 型号。两种阳离子蛋白将与其他净定向治疗方法进行比较或结合 选项。将探索这些阳离子的副作用，尤其是在止血上。犬的试点试验 人（H）PF4将被注入自发性腹膜炎的狗，其长期目标是进行临床试验 这个大型动物败血症的环境。 SA＃2：有关增强PF4对网的影响的研究。我们找到了 增强PF4结合网的单克隆抗体KKO进一步增加了DNase耐药性。一个 FC修饰的KKO变体可预防体外和败血症小鼠模型的净有害作用。这 将定义KKO和其他抗PF4抗体效应净生物学的基本机制。 Kko 在表达HPF4的小鼠中，将进行输注研究，以研究疗效和血栓形成并发症以及 在化粪池中共同注入的KKO和HPF4的试点研究中。 SA＃3：了解ODSH对网的影响 败血症。我们以前已经表明，脱硫ODSH显着降低了抗凝剂 效果比肝素是在组蛋白输注鼠模型中具有保护性的作用，并将探索其机械基础 体外和鼠类败血症模型中的SA＃1。我们将继续一项剂量升级研究 自发性腹膜炎的狗的ODSH定义最终临床试验的最大耐受剂量。 项目4中的这些研究应提高我们对PF4和肝素与肝素与 败血症的网中的网，并为这种毁灭性的临床状态提供新的疗法。