Soluble TLT-1 partially rescues bleeding and tissue damage in ALI model. (A) LPS-treated treml1^−/− mice were injected IV with smTLT-1, and platelets were evaluated before injection (time 0), 1, 3, and 6 hours postinjection. Platelets were stained with voldemort and anti-rabbit-fitc secondary, and anti GPIIα IIIα-PE. (B) Quantification of the percentage of TLT-1 positive cells relative to WT (gray column). (C) Immunofluorescent staining of lungs from WT, treml1^−/−, or treml1^−/− mice treated with sTLT-1, 24 hours after LPS inhalation. CD41, red; TLT-1, yellow; 4′,6-diamidino-2-phenylindole, blue. (D-H) Treml1^−/− mice were pretreated with either sTLT-1 or vehicle and then subjected to intranasal LPS challenge and evaluated at 24 hours. (D) Representative images of gross BALF and whole lungs, illustrating reduced bleeding in treml1^−/− mice pretreated with sTLT-1. Pretreating treml1^−/− mice with sTLT-1 reduced RBC counts (E), neutrophil influx into BALF (F), and PI staining of mouse lungs (G and H). Scale bar represents 100 μM (n = 5-6 mice per group; *P ≤ .05, **P ≤ .01, ***P ≤ .001, ****P ≤ .0001, 1-way ANOVA). Confocal images were processed using Nikon Confocal Microscope and analyzed using NIS element viewer 4.20, magnification ×20. All cytometric quantifications were done using BD Accuri C6 software (BD Biosciences). MFI, mean fluorescent intensity.