Platelet Factor 4 and heparins in NETosis and Sepsis

摘要 脓毒症是一种对感染的多器官、功能障碍反应。项目4的重点是发布的角色 中性粒细胞胞外陷阱(Net)，其捕获微生物的能力，以及网络降解产物(NDP，例如， 无细胞DNA和组蛋白)在器官损伤中的作用以及阳离子趋化因子和血小板因子的相互作用 4(PF4)和带有Net的聚阴离子肝素。在表现上，许多败血症患者有压倒性的 通过阻断氨基精氨酸脱亚胺酶4来防止净释放(净释放)的网络释放量 否则，加速净溶解在预防发病率和死亡率方面将是无效的。我们建议在脓毒症中 净稳定、加强微生物捕获和/或NDP封存将是保护性的。我们的研究 定义了可以实现其中一个或多个目标的三个相关战略。我们建议更好地 了解这些策略的潜在机制(S)、止血/血栓形成的含义以及它们 潜在的治疗效果。具体目标(SA)#1：了解小的、积极的蛋白质对 篮网。PF4和其他小的、阳性的蛋白质(如硫酸鱼精蛋白(ProSO4))紧凑网络，减少 它们被DNA酶裂解。PF4而不是ProSO4可以阻止NDP的释放，这是两者之间的一个重要区别 阳离子蛋白将被探索，以了解紧凑网络在脓毒症中的影响。PF4增强 微生物捕获和保护血管内皮细胞免受净损伤。ProSO4是否对网络产生类似影响 鉴于其释放NDPs的生物学特性，将在微流体系统和小鼠内毒素/脓毒症中进行研究 模特们。这两种阳离子蛋白都将与其他网络导向治疗药物进行比较或结合 选择。这些阳离子的副作用，特别是在止血方面，将被探索。犬只和狗的试点试验 人(H)PF4将被注入患有自发性腹膜炎的狗身上，长期目标是在 这个大型动物败血症环境。SA#2：加强PF4对NETS影响的研究。我们发现了 单抗KKO可增强PF4与Net的结合，进一步增加对DNase的抵抗力。一个 FC修饰的KKO变体在体外和小鼠败血症模型中对净有害影响具有保护作用。这个 KKO和其他抗PF4抗体如何影响网络生物学的潜在机制(S)将被定义。KKO 将在表达HPF4的小鼠身上进行输液研究，以观察疗效和血栓并发症 在败血症犬体内联合输注kko和hpf4的初步研究中。SA#3：了解ODSH对英语教师的影响 败血症。我们之前已经证明了脱硫肝素ODSH，它具有明显较低的抗凝血剂 在组蛋白输注小鼠模型中具有保护作用，并将探讨其机制基础。 在体外和在SA#1的小鼠脓毒症模型中。我们将继续剂量递增研究 自发性腹膜炎犬的ODSH以确定最终临床试验的最大耐受量。 项目4中的这些研究应该会促进我们对PF4和肝素与 Net在败血症中的作用，并为这种毁灭性的临床状态带来了新的治疗方法。

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.