AT inhibits HRPII-mediated inflammation and coagulation. Mice were injected IP with saline or HRPII (10 µg/g) and AT (20 µg/g) followed by euthanizing mice after 3.5 hours, perfusing the lungs with PBS, dissecting the lower right lobe of the lung, and dissolving it in the tissue lysis buffer. (A) Lung tissue lysates were immunoblotted for VCAM1 and ICAM1 and β-actin. Densitometric analysis of expression of these proteins is presented. (B) The barrier-protective effect of AT in mice injected with both AT and HRPII was analyzed after 3 hours by IV injection of 1% Evans blue dye. After 30 minutes, animals were euthanized and perfused with PBS, lung tissue samples were collected, and vascular permeability was measured from the amount of Evans blue dye leaked into the lung as described in "Materials and methods." (C) Plasma level of VWF was measured by ELISA using a commercial kit. (D) Blood platelet counts were determined using a veterinary hematology analyzer. (E) Lung cryosections were fixed, permeabilized, and incubated with anti-CD41 (rat) and anti-CD31 (rabbit) antibodies followed by Alexa Fluor 555-conjugated anti-rat and Alexa Fluor 488-conjugated anti-rabbit antibodies. DAPI was used to stain the nucleus. The arrows indicate platelet-rich thrombus. Relative intensity of CD41 is presented.