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

尽管除了重症监护病房的支持外，还试图进行积极的源头控制，但最近英国爆发的静脉吸毒者中炭疽芽孢杆菌(B.anthracis)软组织感染的死亡率非常高(在40多名患者中超过40%)。其中许多患者的一个显著发现是明显的凝血障碍和血小板减少症。这些情况极大地增加了患者清创的难度。虽然在以前的炭疽暴发中，凝血障碍和血小板减少的实验室证据并不一致，但胸腔积液和脑膜炎经常被描述为出血性。因此，凝血功能紊乱、过度纤溶和血小板消耗或破坏可能在炭疽病的发病机制中起重要作用。了解这些过程的基础将对未来炭疽病的靶向治疗非常重要。 炭疽病与几种毒力因素有关，这些因素可能导致凝血障碍、纤溶和血小板减少或血小板功能障碍。一方面，炭疽病产生致命性毒素和水肿性毒素(分别为LeTx和ETX)。LeTx抑制是一种锌依赖的蛋白水解酶，它可以破坏MAPK通路，该通路在天然免疫、细胞周期和复制以及其他重要的宿主功能中起重要作用。水肿性毒素具有钙调素依赖的腺苷环化酶活性，并使细胞内cAMP水平升高到非常高的水平。这两种毒素都有可能改变凝血、纤溶和血小板功能。然而，作为一种革兰氏阳性细菌，炭疽菌有一个肽聚糖细胞壁，它也可以通过刺激炎症途径来破坏这些功能。虽然这种与LeTx或ETX相关的异常可能最好是用毒素抑制剂来治疗，但细胞壁引起的异常可能需要其他形式的治疗，如抗炎治疗。 本方案的目的是在先前建立的大鼠模型中直接比较LeTx、ETX和炭疽细胞壁肽聚糖对凝血、纤溶和血小板的影响。在现已完成的实验中，动物们被要求使用先前实验中开发的方法，24小时注入这三种成分中的一种。在输液过程中，以及从24到48小时，动物进行了一系列的凝血、纤溶和血小板研究。我们以前开发了测量这种啮齿动物的凝血酶原(PT)和部分凝血活酶(PTT)时间、纤维蛋白原水平和凝血酶抗凝血酶(TAT)水平的技术。其他措施包括组织因子、蛋白C、抗凝血酶III和纤溶酶原激活物抑制物。在这项研究中，炭疽细胞壁肽聚糖具有显著的凝血和炎症作用，而同样致命剂量的LeTx和ETX则没有。这项工作之前已经发表过。 [邱普，李y，施洛奇J，崔旭，孙杰，Trin h L，Kubler-Kielb J，Vinogradov E，Mani H，Al-Hamad M，Fitz Y，Eichacker PQ(2013)炭疽芽孢杆菌细胞壁肽聚糖但非致死性或水肿性毒素产生的变化与弥漫性血管内凝血一致。J传染病208：978-89) 在这项工作的基础上，已经完成了其他研究，以检验抗炎剂在炭疽细胞壁肽聚糖攻击环境中的保护作用。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。对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药对炭疽菌PGN攻击没有保护作用。 一份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.