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

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Abstract

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 are now underway examining the protective effects of anti-inflammatory agents in the setting of anthrax cell wall peptidoglycan challenge. Preliminary experiments from an initial study examining the effects of corticosteroid treatment in PGN challenged animals have been completed and were presented at the 2016 International Conference of the ATS. In those studies a very high dose of corticosteroids were highly protective with anthrax PGN challenge. Additional studies have now been conducted showing that lower doses of corticosteroid than originally investigated, continued to be highly protective. This dose de-escalation is ongoing and after these studies are completed, a new set of studies will examine whether tumor necrosis factor soluble receptor, a highly selective anti-inflammatory agent, will also be protective in the setting of anthrax PGN challenge.