Platelet Factor 4 and heparins in NETosis and Sepsis

摘要 脓毒症是一种多器官、功能失调的感染反应。项目4侧重于释放的作用 中性粒细胞胞外陷阱（NET），其捕获微生物的能力，以及NET降解产物（NDP，例如， 细胞游离DNA和组蛋白）在器官损伤中的作用，以及阳离子趋化因子、血小板因子 4（PF 4）和聚阴离子肝素与NET。在介绍，许多脓毒症患者有压倒性的 释放NET的量，从而通过阻断肽基精氨酸脱亚胺酶4来防止NET释放（NETosis） 或加速NET溶解在预防发病率和死亡率方面是无效的。我们认为在败血症中 NET稳定化、增强的微生物截留和/或NDP螯合将是保护性的。我们的研究 已经确定了三个相关的战略，可以实现这些目标中的一个或多个。我们建议更好地 了解这些策略的潜在机制、止血/血栓形成影响及其 潜在的治疗效果。具体目标（SA）#1：了解小的，积极的蛋白质影响， NET。PF 4和其他小的阳性蛋白（例如，硫酸鱼精蛋白（ProSO 4））紧凑NET，减少 它们被DNA酶裂解。PF 4，而不是ProSO 4，阻止NDP释放，这是它们之间的一个重要区别 阳离子蛋白，将探讨了解脓毒症的影响压实NET。PF 4增强 微生物截留和保护内皮免受NET损伤。ProSO 4是否同样影响NET 考虑到其释放NDP，将在微流体系统和鼠内毒素/脓毒症中进行生物学研究 模型这两种阳离子蛋白质将与其他NET导向的治疗药物进行比较或与其他NET导向的治疗药物联合使用。 选项.将探讨这些阳离子的副作用，特别是对止血的副作用。犬科动物和 人（h）PF 4将输注到患有自发性腹膜炎的犬中，长期目标是进行以下临床试验： 这种大型动物败血症的情况SA #2：关于增强PF 4对NET的作用的研究。我们发现 增强PF 4与NET结合的单克隆抗体KKO进一步增加了DNA酶抗性。一个 Fc修饰的KKO变体在体外和脓毒症小鼠模型中保护免受NET有害作用。的 将定义KKO和其他抗PF 4抗体如何影响NET生物学的潜在机制。KKO 将在表达hPF 4的小鼠中进行输注研究，观察疗效和血栓形成并发症， 在败血症犬中共输注KKO和hPF 4的初步研究中。SA#3：了解ODSH对NET的影响， 败血症我们先前已经证明，具有显著较低抗凝活性的肝素ODSH 在组蛋白输注小鼠模型中具有保护作用，并将探讨其在 如SA#1中的体外和鼠脓毒症模型。我们将继续进行剂量递增研究， 自发性腹膜炎犬中的ODSH，以确定最终临床试验的最大耐受剂量。 项目4中的这些研究应该促进我们对PF 4和肝素与 NET在脓毒症中的作用，并为这种毁灭性的临床状态带来新的治疗方法。

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.