ResearchActivated protein C ameliorates coagulopathy but does not influence outcome in lethal H 1 N 1 influenza : a controlled laboratory study

Introduction: Influenza accounts for 5 to 10% of community-acquired pneumonias and is a major cause of mortality. Sterile and bacterial lung injuries are associated with procoagulant and inflammatory derangements in the lungs. Activated protein C (APC) is an anticoagulant with anti-inflammatory properties that exert beneficial effects in models of lung injury. We determined the impact of lethal influenza A (H1N1) infection on systemic and pulmonary coagulation and inflammation, and the effect of recombinant mouse (rm-) APC hereon. Methods: Male C57BL/6 mice were intranasally infected with a lethal dose of a mouse adapted influenza A (H1N1) strain. Treatment with rm-APC (125 μg intraperitoneally every eight hours for a maximum of three days) or vehicle was initiated 24 hours after infection. Mice were euthanized 48 or 96 hours after infection, or observed for up to nine days. Results: Lethal H1N1 influenza resulted in systemic and pulmonary activation of coagulation, as reflected by elevated plasma and lung levels of thrombin-antithrombin complexes and fibrin degradation products. These procoagulant changes were accompanied by inhibition of the fibrinolytic response due to enhanced release of plasminogen activator inhibitor type-1. Rm-APC strongly inhibited coagulation activation in both plasma and lungs, and partially reversed the inhibition of fibrinolysis. Rm-APC temporarily reduced pulmonary viral loads, but did not impact on lung inflammation or survival. Conclusions: Lethal influenza induces procoagulant and antifibrinolytic changes in the lung which can be partially prevented by rm-APC treatment. Introduction Influenza A infection is a major cause of morbidity and mortality: Seasonal influenza A infection causes over 200,000 hospitalizations and approximately 41,000 deaths in the United States annually, being the seventh leading cause of mortality [1]. Besides its regular seasonal character, influenza A, due to the introduction and adaptation of novel hemagglutinin subtypes from other mammals or birds resulting in antigenic shifts, has the potential to cause pandemics, as the pandemics in 1918, 1957 and 1968 have shown [2]. Currently, a novel influenza A (H1N1) strain from swine origin has evolved to a pandemic, now worldwide causing major concern for the near future [3]. Although the greatest proportion of mortality caused by influenza A infection is due to secondary bacterial pneumonia and cardiovascular complications, influenza itself is also an important cause of communityacquired pneumonia (CAP), causing 5 to 10% of CAPcases [4-7]. As such, influenza is a major concern for pulmonologists and intensive care physicians [8]. Severe infection and inflammation have been closely linked to activation of coagulation and downregulation of anticoagulant mechanisms and fibrinolysis [9]. In bacterial pneumonia, pulmonary activation of coagulation as well as downregulation of the anticoagulant protein C (PC) pathway and fibrinolysis have been demonstrated [10-12]. Beside anticoagulant properties, activated (A)PC has been shown to have profibrinolytic, anti-inflamma* Correspondence: m.schouten@amc.uva.nl 1 Center for Experimental and Molecular Medicine (CEMM), Academic Medical Center, University of Amsterdam, Meibergdreef 9, Room G2-130, 1105 AZ, Amsterdam, The Netherlands Full list of author information is available at the end of the article BioMed Central © 2010 Schouten et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Schouten et al. Critical Care 2010, 14:R65 http://ccforum.com/content/14/2/R65 Page 2 of 10 tory, anti-apoptotic and other cytoprotective properties [13]. Downregulation of the PC pathway has been correlated to disease severity and mortality in severe bacterial pneumonia and sepsis [14,15] and continuous intravenous administration of recombinant human (rh-) APC for four days (Human Activated Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) trial) has been shown not only to downregulate activation of coagulation, but also to reduce inflammation and improve survival in patients with severe sepsis [16]. The benefical effect of rh-APC in this trial seemed especially prominent in patients with severe sepsis due to pneumonia [17]. While much research has been done on coagulation activation during severe bacterial infection, data on coagulation activation in viral infection like influenza are sparse. Evidence that influenza can be associated with coagulation activation comes from a clinical study in pediatric patients hospitalized for severe influenza [18] and from a recent study showing elevated plasma levels of thrombinantithrombin complexes (TATc) in mice infected with a non-lethal dose of influenza A [19]. Interestingly, and as mentioned above, many elderly patients with influenza infections suffer from cardiovascular complications. At present it is unknown whether APC can influence the procoagulant and inflammatory response to lethal influenza A infection. Therefore, in the present study we sought to establish the effect of recombinant mouse (rm)APC treatment on local and systemic activation of coagulation and fibrinolysis during lethal H1N1 influenza A in mice and moreover determined the effect of rm-APC on lung inflammation, pulmonary viral loads and survival. We here show, that lethal H1N1 influenza A infection is associated with both pulmonary and systemic activation of coagulation and inhibition of fibrinolysis. Moreover, we show that rm-APC treatment, started 24 hours after the onset of infection, partially prevents these hemostatic derangements, but does not impact on lung inflammation or survival. Materials and methods Animals Male C57BL/6 mice were purchased from Charles River (Maastricht, the Netherlands) and maintained in the animal facility of the Academic Medical Center (University of Amsterdam) according to national guidelines with free access to food and water. Ten-week-old mice were used in experiments. All experiments were approved by the Institutional Animal Care and Use Committee of the Academic Medical Center. Experimental infection and treatment Influenza infection was induced by intranasal instillation of a lethal dose (28,000 copies) of influenza A/PR/8/34 (H1N1, ATCC no. VR-95; Rockville, MD, USA), as described [20,21]. This infectious dose was chosen based on a previous study from our laboratory showing that it caused lethality in C57BL/6 mice which could be delayed by eliminating signalling via the proinflammatory receptor for advanced glycation end products (RAGE) [20]. Recombinant murine (rm-) APC and buffer control were generated by Eli Lilly & Co (Indianapolis, IN, USA) as described [22]. Rm-APC (2 mg/ml) was diluted in sterile pyrogen-free saline to a concentration of 625 μg/ml. The buffer control was diluted likewise. Uninfected mice were euthanized before or at one, four or eight hours (n = 4 per time point) after a single intraperitoneal injection of 125 μg of rm-APC (200 μl) to determine plasma APC-levels. In infection experiments, from 24 hours after infection on, mice were treated every eight hours for a maximum of three days with 125 μg of rm-APC or buffer. Sample harvesting and processing, and determination of viral copies were done as described (n = 8 per group at each time point) [20,21]. Assays M-APC levels were measured by an enzyme capture assay [23]. TATc (Behringwerke AG, Marburg, Germany), fibrin degradation products (FDP) [24], plasminogen activator inhibitor type-1 antigen (PAI-1) [25], myeloperoxidase (MPO; HyCult Biotechnology, Uden, the Netherlands), interleukin (IL)-1β, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2 (all R&D Systems, Minneapolis, MN, USA) were measured by ELISA. Plasminogen activator activity (PAA) was determined by an amidolytic assay [26]. Tumor necrosis factor (TNF)-α, IL-6, IL-12p70, IL-10 and interferon (IFN)-γ were measured by cytometric bead array (CBA) multiplex assay (BD Biosciences, San Jose, CA, USA). Histology and immunohistochemistry Paraffin lung sections were stained with haematoxylin and eosin or fluorescein isothiocyanate-labeled antimouse Ly-6G mAb (Pharmingen, San Diego, CA, USA) as described [27]. To score lung inflammation, the lung surface was analyzed with respect to the following parameters: bronchitis, interstitial inflammation, oedema, endothelialitis, pleuritis and thrombus formation. Each parameter was graded on a scale of 0 to 4 (0: absent, 1: mild, 2: moderate, 3: severe, 4: very severe). The total histopathological score was expressed as the sum of the scores for the different parameters, the maximum being 24. Ly-6G stained slides were photographed with a microscope equipped with a digital camera (Leica CTR500, Leica Microsystems, Wetzlar, Germany). Stained areas were analysed with Image Pro Plus (Media Cybernetics, Bethesda, MD, USA) and expressed as percentage of the Schouten et al. Critical Care 2010, 14:R65 http://ccforum.com/content/14/2/R65 Page 3 of 10 surface area. The average of 10 pictures was used for analysis. Statistical analysis Data are expressed as box-and-whisker diagrams (depicting the smallest observation, lower quartile, median, upper quartile and largest observation), medians with interquartile ranges or as survival curves. Differences between groups were determined with Kruskal-Wallis, Mann-Whitney U test or log rank test. Analyses were performed using GraphPad Prism version 4.0 (GraphPad Software, San Diego, CA, USA). P-values less than 0.05 were considered statistically significant. Results Plasma m-APC levels after single dose administration of rm-APC To determine plasma levels of m-APC after single dose administration of rm-APC, uninfected mice were injected intraperitoneally