Anticoagulation increases alveolar hemorrhage in mice infected with influenza A

Figure 1. Anticoagulating mice with warfarin or dabigatran etexilate. Wild‐type (WT) mice were untreated or treated with 2 or 4 μg/mL of warfarin via drinking water for 3 (A) or 7 (B) days and the level of anticoagulation was assessed by measuring the prothrombin time (PT). (C) WT mice were untreated or treated with dabigatran etexilate (DE) containing chow (10 mg/g of chow) for 3 days and the level of anticoagulation was assessed by measuring the activated partial thromboplastin time (aPTT). (D) Bleeding times of untreated WT mice or mice‐receiving warfarin (4 μg/mL, drinking water) or DE (10 mg/g of chow) for 3 days were determined using the saphenous vein injury model. Data are represented as an average hemostasis time after clot disruptions. WT mice were untreated or treated with warfarin via drinking water (2 or 4 μg/mL) or DE via chow (10 mg/g of chow) for 3 days before being infected with influenza A virus (IAV) intranasally. (E) Levels of thrombin–antithrombin complexes (TATc) in bronchoalveolar lavage fluid (BALF) of control (C; white), and 2 μg/mL (W2; light gray) or 4 μg/mL (W4; dark gray) of warfarin‐treated mice before and 7 days after IAV infection. (F) Levels of TATc in BALF…

Figure 2. Anticoagulation with warfarin or dabigatran etexilate increases alveolar hemorrhage in mice after influenza A infection. Wild‐type mice were untreated or treated with warfarin via the drinking water (2 or 4 μg/mL) or dabigatran etexilate (DE) via chow (10 mg/g of chow) for 3 days and were infected with influenza A virus (IAV) intranasally. (A) (Left) Gross appearance of bronchoalveolar lavage fluid (BALF) from three representative mice receiving with warfarin before and 7 days after IAV infection. (Right) Levels of hemoglobin (Hb) in BALF of control mice (C; white), and 2 μg/mL (W2; light gray) or 4 μg/mL (W4; dark gray) of warfarin‐treated mice before and 7 days after IAV infection. (B) (Left) Gross appearance of BALF from three representative mice with or without DE chow 7 days after IAV infection. (Right) Levels of Hb in BALF of control (white) or DE‐fed (black) mice at 7 days after IAV infection. Data were analyzed by two‐way ANOVA for A, and by Student's t‐test for B. Data are represented as mean ± SEM, and statistical significances are indicated as * (P < 0.05) between groups, or ## ( P < 0.01) versus uninfected control of the respective group. The numbers of mice used are…

Figure 3. Effect of anticoagulation on morbidity and mortality in mice after influenza A infection. Wild‐type mice were untreated or treated with low‐ (2 μg/mL; W2) (A and B) and high‐ (4 μg/mL; W4) (C and D) dose warfarin via drinking water, or dabigatran etexilate (DE) via chow (10 mg/g of chow; DE) (E and F) for 3 days and were infected with influenza A virus (IAV) intranasally. The different groups are designated with different colors: blue indicates mice receiving anticoagulants alone; black indicates IAV alone; and red indicates mice that received anticoagulation and were infected. Body weights before infection were set to 100%. Survival (mean ± SEM; n = 7–27 per group) and body weight data were analyzed by log‐rank test (A, C, and E), or by two‐way ANOVA (B, D, and F). Statistical significance is indicated as * (P < 0.05) between IAV‐infected mice with and without anticoagulants.

Figure 4. Effect of anticoagulation on vascular permeability after influenza A infection. Wild‐type mice were untreated or treated with high‐ (4 μg/mL; W4) dose warfarin via drinking water, or dabigatran etexilate (DE) via chow (10 mg/g of chow) for 3 days and were infected with influenza A virus (IAV) intranasally for 3 days. Mice treated with warfarin (W4) or DE without IAV infection were used as controls. Vascular permeability was determined by measuring levels of Evans blue in the lungs. Data were analyzed by one‐way ANOVA. Statistical significance is indicated as * (P < 0.05) between IAV‐infected mice with and without anticoagulants.

Figure 5. Effect of anticoagulation on IAV genomes and protein in the bronchoalveolar lavage fluid. Wild‐type mice were untreated or treated with low (2 μg/mL; W2) and high (4 μg/mL; W4) doses of warfarin or dabigatran etexilate (DE) for 3 days before influenza A infection. (A) Levels of influenza A genomes in the lungs were measured in control and anticoagulated mice 7 days after infection. Levels of total protein (B) and albumin (C) in the bronchoalveolar lavage fluid in control and anticoagulated mice 7 days after infection.

Figure 6. Effect of anticoagulation on white blood cell counts and inflammatory mediators in the bronchoalveolar lavage fluid. Wild‐type mice were untreated or treated with low (2 μg/mL; W2) and high (4 μg/mL; W4) doses of warfarin or dabigatran etexilate (DE) for 3 days before influenza A infection. Levels of white blood cells (WBC) and various cytokines/chemokines in the bronchoalveolar lavage fluid (BALF) were measured 7 days after influenza A infection. (Panel A) shows levels of WBC in the different groups. (Panels B and C) show the effect of warfarin and DE on levels of cytokines/chemokines in mice treated with high‐dose warfarin or DE, respectively. Data (mean ± SEM; n = 4–17 per group) were analyzed by Student's t‐test.