The Effects of Anthrax Toxins and Cell Wall on Coagulation and Thrombosis

尽管在重症监护室的支持下，在积极的源头控制的尝试，死亡率在最近的英国爆发的炭疽杆菌（B。炭疽菌）软组织感染的比例非常高（在40多名患者中超过40%）。在这些患者中，一个值得注意的发现是明显的凝血功能障碍和血小板减少症。这些情况使患者的清创工作变得非常复杂。虽然凝血功能障碍和血小板减少症的实验室证据在以前的炭疽爆发中没有一致的报道，但胸腔积液和脑膜炎经常被描述为出血性的。因此，凝血功能的破坏，过度的纤维蛋白溶解和血小板的消耗或破坏可能在炭疽的发病机制中起重要作用。了解这些过程的基础对于将来有针对性地治疗炭疽病将是重要的。 炭疽与几种毒力因子有关，这些毒力因子可能导致凝血病、纤维蛋白溶解和血小板减少症或血小板功能障碍。一方面，炭疽产生致死毒素和水肿毒素（分别为LeTx和ETx）。LeTx抑制是一种锌依赖性蛋白酶，其破坏在先天免疫、细胞周期和复制以及其他必需宿主功能中重要的MAPK途径。水肿毒素具有钙调蛋白依赖性腺苷酸环化酶活性，并将细胞内cAMP增加到非常高的水平。这两种毒素都有可能改变凝血、纤维蛋白溶解和血小板功能。然而，作为革兰氏阳性细菌，炭疽具有肽聚糖细胞壁，其也可以通过刺激炎症途径来破坏这些功能。虽然与LeTx或ETx相关的这种异常可能最好通过毒素抑制剂治疗，但细胞壁诱导的异常可能需要替代形式的治疗，例如抗炎治疗。 本方案的目的是在先前开发的大鼠模型中直接比较LeTx、ETx和炭疽细胞壁肽聚糖对凝血、纤维蛋白溶解和血小板的影响。在现已完成的实验中，使用先前实验中开发的方法，用这三种组分之一的24小时输注来激发动物。在输注期间以及24 - 48小时，对动物进行了一系列凝血、纤维蛋白溶解和血小板研究。我们以前开发的技术，以测量凝血酶原（PT）和部分凝血活酶（PTT）的时间，纤维蛋白原水平，凝血酶抗凝血酶（达特）水平在这种啮齿动物物种。 其他指标包括组织因子、蛋白C、抗凝血酶III和纤溶酶原激活物抑制剂。 在这项研究中，炭疽细胞壁肽聚糖具有明显的凝血和炎症作用，而类似的致死剂量的LeTx和ETx没有。 这项工作以前发表过。 (Qiu p，Li y，Shiloach J，Cui X，Sun J，Trinh L，Kubler-Kielb J，Vinogradov E，Mani H，Al-Hamad M，Fitz Y，Eichacker PQ（2013）Bacillus anthracis Cell Wall Peptidoglycan but Not Lethal or Edema Toxins Produces Changes Consistent With Disseminated Intravascular Coagulation in a Rat Model. J Infection Diseases 208：978-89） 基于这项工作，已经完成了额外的研究，检查抗炎剂在炭疽细胞壁肽聚糖挑战的设置中的保护作用。在这些研究中，高剂量和中剂量的皮质类固醇对炭疽PGN攻击具有高度保护作用。肿瘤坏死因子可溶性受体的研究表明，这种高度选择性的抗炎剂，与炭疽PGN挑战没有保护性。 描述这些研究的手稿正在审查中。

Despite attempts at aggressive source control in addition to intensive care unit support, mortality in the recent UK outbreak of Bacillus anthracis (B. anthracis) soft tissue infection among intravenous drug abusers was very high (greater than 40% in more than 40 patients). A noticeable finding among many of these patients was a marked coagulopathy and thrombocytopenia. These conditions greatly complicated efforts at debridement in patients. While laboratory evidence of coagulopathy and thrombocytopenia has not been consistently reported on in prior anthrax outbreaks, pleural fluid collections and meningitis have frequently been described as hemorrhagic. Thus disruption of coagulation, excessive fibrinolysis and platelet consumption or destruction may play an important role in the pathogenesis of anthrax. Understanding the basis for these processes will be important for targeted treatment of anthrax in the future. Anthrax is associated with several virulence factors, which could potentially contribute to coagulopathy, fibirnolysis and thrombocytopenia or platelet dysfunction. On the one hand, anthrax produces lethal and edema toxins (LeTx and ETx respectively). LeTx inhibits is a zinc dependent protease which disrupts MAPK pathways important in innate immunity, cell cycling and replication and other essential host functions. Edema toxin has calmodulin dependent adenyl cyclase activity and increases intracellular cAMP to very high levels. Both toxins have the potential to alter both coagulation, fibrinolysis and platelet function. However, as a gram-positive bacteria, anthrax has a peptidoglycan cell wall which could also disrupt these functions via stimulation of inflammatory pathways. While such abnormalities related to LeTx or ETx might be best treated by toxin inhibitors, cell wall induced abnormalities might require alternate forms of therapy such as anti-inflammatory ones. The purpose of the present protocol has been to directly compare the effects of LeTx, ETx and anthrax cell wall peptidoglycan on coagulation, fibrinolysis and platelets in a previously developed rat model. In experiments now completed, animals were challenged with 24-hour infusions of one of these three components using methods developed in prior experiments. During infusion, as well as from 24 to 48 hours, animals had serial coagulation, fibrinolysis and platelet studies performed. We previously developed techniques to measure prothrombin (PT) and partial thromboplastin (PTT) times, fibrinogen levels, and thrombin anti-thrombin (TAT) levels in this rodent species. Other measures included tissue factor, protein C, anti-thrombin III, and plasminogen activator inhibitor. In this study anthrax cell wall peptidoglycan had marked coagulopathic and inflammatory actions, while similarly lethal doses of LeTx and ETx did not. This work was previously published. (Qiu p, Li y, Shiloach J, Cui X, Sun J, Trinh L, Kubler-Kielb J, Vinogradov E, Mani H, Al-Hamad M, Fitz Y, Eichacker PQ (2013) Bacillus anthracis Cell Wall Peptidoglycan but Not Lethal or Edema Toxins Produces Changes Consistent With Disseminated Intravascular Coagulation in a Rat Model. J Infection Diseases 208:978-89) Based on this work, additional studies have been completed examining the protective effects of anti-inflammatory agents in the setting of anthrax cell wall peptidoglycan challenge. In these studies a high and medium dose of corticosteroids were highly protective with anthrax PGN challenge. Studies with tumor necrosis factor soluble receptor showed that this highly selective anti-inflammatory agent, was not protective with anthrax PGN challenge. A manuscript describing these studies is under review.