Anti-inflammatory and antithrombotic activity of Desmolaris. (A) Thrombosis. A paper filter imbibed with 7.5% FeCl₃ was applied to the carotid artery, and blood flow was monitored with a perivascular flow probe for 60 minutes or until stable occlusion took place. Fifteen minutes before injury, Desmolaris was injected into the caudal veins of the mice. Each symbol represents one individual. *P < .05 (analysis of variance [ANOVA] with Dunnett posttest). (B) Bleeding time. Bleeding was caused by a tail transection after IV infection of Desmolaris as the indicated concentrations. Absorbance at 540 nm (hemoglobin concentration) was used to estimate blood loss. *P < .05 (ANOVA with Dunnett posttest). (C) aPTT and PT ex vivo. The aPTT or PT reagents were added to plasma collected from mice injected with Desmolaris (250 µg/kg) or PBS (control). Clot was determined using a coagulometer (n = 6; *P < .05; t test; NS, nonsignificant). The ratio of clotting times is shown. Absolute values: PT; 11.67 ± 0.33 seconds; aPTT, 49.99± 4 seconds. (D) Vascular permeability (Miles assay). A total of 100 μL of a mixture containing Evan’s blue and Desmolaris (500 μg/kg) was injected IV into the tails of mice. Five minutes later, 40 μL of polyphosphate (polyP; 0.2 mg/mL) was injected intradermally. After 20 minutes, animals were sacrificed and skin removed to allow sites of injection to be photographed. (E) Evans blue extravasation was estimated after extraction with formamide and reading at 620 nm. Results are the average of experiments obtained with 4 animals for each condition. *P < .05 (ANOVA). (F) Desmolaris blocks the inflammatory effects of FXa. Posterior paw edema was induced by intradermal injection of 30 μL of FXa (10 μg, 7.3 μM) in the presence of PBS (squares), or FXa previously incubated with 10 μM of Desmolaris (triangles). Edema caused by 30 μL of PBS only is shown by circles. Edema formation (increase in paw thickness in millimeters) was estimated with a caliper before injection of FXa or after 15, 30, 45, and 60 minutes. Four posterior paws were used for each data point. *P < .05 (ANOVA). (G) Kaplan-Meier survival curves. Mortality associated with thromboembolism triggered by an IV injection of collagen (0.8 mg/kg) and epinephrine (60 μg/kg) after administration of either PBS (control) or Desmolaris. Animals that were alive 30 minutes after the challenge were considered to be survivors. Each symbol represents one individual. *P < .05 (log-rank test). (H) Histology of the lung. Hematoxylin and eosin-stained lung sections of control (PBS) and Desmolaris-treated (1000 μg/kg) mice. Animals were euthanized 5 minutes after collagen and epinephrine injection. Representative images from 3 animals for each condition. Bars represent 100 μm.