Arterioscler Thromb Vasc Biol In Vivo Imaging of Thrombin Activity in Experimental Thrombi With Thrombin-Sensitive Near-Infrared Molecular Probe by Farouc A. Jaffer, Ching-Hsuan Tung, Robert E. Gerszten, and Ralph Weissleder Arterioscler Thromb Vasc Biol Volume 22(11):1929-1935 November 1, 2002 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. NIRF imaging of endogenous thrombin activity in human blood clots. Figure 1. NIRF imaging of endogenous thrombin activity in human blood clots. A, NIRF signal evolution over time. The thrombin probe (TP) NIRF signal was amplified 18-fold over 24 hours (P=0.008). At 24 hours, the NIRF signal was highest in the TP group (P=0.008 versus TP+hirudin [H], P=0.007 versus control probe [CP], P=0.008 versus CP+H, and P=0.006 versus blood). Hirudin, a direct thrombin inhibitor, suppressed TP activation by 82.4%. B, Representative light and NIRF images at 15 minutes and 24 hours, from 1 experiment. The NIRF signal was initially detected at the margin of the semisolid blood clot and became homogeneous over time, which was likely due to diffusion. NIRF images at both time points have been windowed equally. AU indicates arbitrary units. Farouc A. Jaffer et al. Arterioscler Thromb Vasc Biol. 2002;22:1929-1935 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. In vivo fluorescence reflectance imaging of thrombin activity within a trauma-induced hematoma. Figure 2. In vivo fluorescence reflectance imaging of thrombin activity within a trauma-induced hematoma. The thrombin probe was rapidly activated in vivo after tail hematoma formation, resulting in a large detectable NIRF signal from the hematoma. On average, the NIRF signal from the hematoma was 94±108% greater than the adjacent tissue background (P=0.039). In this example, light and NIRF images were acquired 12 minutes after injury. Farouc A. Jaffer et al. Arterioscler Thromb Vasc Biol. 2002;22:1929-1935 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. FeCl3 model of murine venous thrombosis. Figure 3. FeCl3 model of murine venous thrombosis. A, FeCl3-soaked filter paper was applied directly on top of the vessel for 3 minutes. B, Occlusive (solid arrows) and nonocclusive thrombus (dashed arrow) formation was evident within 20 minutes and persisted for hours. C, Pathology demonstrated a distended and densely red blood cell–packed vein, consistent with an occlusive venous thrombosis (T). The adjacent artery was patent. V indicates vein; A, artery; and N, nerve. Farouc A. Jaffer et al. Arterioscler Thromb Vasc Biol. 2002;22:1929-1935 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. In vivo optical imaging of thrombin activity in thrombosis. Figure 4. In vivo optical imaging of thrombin activity in thrombosis. A, In the acute thrombus model (thrombin probe injected 1 hour after thrombus formation), the light image demonstrates darker clotted segments within the femoral vein after application of FeCl3 (arrows). B, NIRF image demonstrates focal signal in areas of thrombosis, particularly within side branches (arrows and dashed box). C, Fusion NIR image shows focal areas of high fluorescence signal within microthrombi. D, Another example of thrombin activity in acute thrombi is shown. Focal NIRF signal enhancement is visible along the margins of the side branch as well as areas of nonocclusive thrombi in the main femoral vein (darker regions in the dashed boxes). Note that the NIRF signal tended to occur just at the margins of nonocclusive thrombi, presumably in areas of lower flow, such as the vessel wall. E, NIRF image of thrombin activity in subacute occlusive thrombi is shown. Thrombin probe was injected 12 hours after thrombus formation. NIRF signal enhancement was again seen in several venous side branches. F, NIRF image of thrombi with use of control NIR fluorochrome does not reveal focal signal enhancement within thrombi. Images were acquired 60 to 90 minutes after probe injection and have been windowed individually. V indicates vein; A, artery. Farouc A. Jaffer et al. Arterioscler Thromb Vasc Biol. 2002;22:1929-1935 Copyright © American Heart Association, Inc. All rights reserved.

Figure 5. Fluorescence microscopy of thrombin probe activation within an occlusive venous thrombus. Figure 5. Fluorescence microscopy of thrombin probe activation within an occlusive venous thrombus. Tissue sections were obtained from a mouse injected with the thrombin probe 1 hour after thrombus formation (at the anatomic section denoted by the dashed arrow in Figure 4b). A, Segment of thrombosed vein showing dense cluster of erythrocytes (dashed box). Original magnification ×20 (hematoxylin and eosin [H&E] stain). B High-magnification view of the occlusive thrombus. Original magnification ×100 (H&E stain, oil immersion). C, Control fluorescence image of adjacent frozen section (fluorescein channel). Original magnification ×40. Unlike nonspecific background vessel wall fluorescence, the thrombus did not significantly fluoresce in the control channel. Asterisk denotes fracture of thrombus during histological processing. D, NIRF fluorescence image of adjacent frozen tissue section showing bright signal from center of occlusive thrombus, consistent with thrombin probe activation (NIRF channel). Original magnification ×40. Fluorescence images have been windowed equally. Farouc A. Jaffer et al. Arterioscler Thromb Vasc Biol. 2002;22:1929-1935 Copyright © American Heart Association, Inc. All rights reserved.