Low concentrations of thrombin are generated after vascular injury and are important for thrombosis in vivo. (A) Western blot detection of 0.2 mg/mL purified human and mouse α−thrombin protein using an antithrombin monoclonal antibody. (B) In vivo imaging of thrombin generation (green) during thrombosis captured using intravital microscopy after laser-induced cremaster arteriolar wall injury in wild-type mice injected with 2 mg/kg Alexa Fluor 488–conjugated antithrombin monoclonal antibody or an Alexa Fluor 488–conjugated control IgG. Arrows indicate the direction of blood flow. Also provided are movies of thrombin generation and platelet thrombus formation (supplemental Movies 1 and 2) and representative images of time-dependent changes in thrombin concentration and platelet thrombus formation (supplemental Figure 2). (C) Median integrated fluorescence intensity over time for Alexa Fluor 488–conjugated (AF 488) antithrombin antibodies and control IgGs. Based on the standard curve (D), 4 nM denotes the approximate concentration of thrombin generated after 260 seconds of laser injury. (D) A standard curve was computed using imaging of fluorescence intensities of known concentrations of AF 488–conjugated thrombin monoclonal antibody under equivalent intravital instrument settings as in A. (E) Quantification of integrated fluorescence intensity at 260 seconds (Mann Whitney U test,*P < .05; 30 thrombi in 4 mice per group). (F) Median integrated fluorescence intensity over time for wild-type mice infused with 0.3 mg/kg BW of dabigatran or vehicle control. The kinetics of platelet accumulation were plotted as the median fluorescence intensity as a function of time in 30 thrombi in 3 to 4 mice per group. For images, also see supplemental Figure 3.