Thrombi are larger and more stable in ceacam1−/− mice both in vitro and in vivo. (A) Images of thrombus formation in response to ferric chloride induced vascular injury was visualized in arterioles of wild-type versus ceacam1−/− mice over time. The different lengths of time after ferric chloride application are indicated. Note that ceacam1−/− arterioles formed larger thrombi over time than did wild-type control arterioles (n = 15). (B-D) Quantitative analysis of arterial thrombogenesis of wild-type (●), ceacam1+/− (■), and ceacam1−/− (▴) arterioles. Compared with wild-type and ceacam1+/−, arterioles, ceacam1−/− arterioles exhibited a significantly larger thrombus area at 2 minutes (3294 ± 223.4 vs 2859 ± 294 vs 6212 ± 268.7 μm2, respectively; ***P < .001; n = 15), greater stability in thrombi formed (2.40 ± 0.21 vs 1.05 ± 0.12 vs 4.07 ± 0.37, respectively; ***P < .001; n = 15), and greater thrombus volume (49 430 ± 4602 vs 63 680 ± 5478 vs 122 400 ± 6794 μm3, respectively; ***P < .001; n = 15). The primary stability of the thrombi was scored from 1 to 10, with 1 being 0% to 10% occupancy and 10 being 91% to 100% occupancy (ie, complete vessel occlusion) monitored over time. (E,F) Platelet thrombus formation after inhibition of GPVI using monoclonal antibody JAQ1 administration to ceacam1+/+ and ceacam1−/− mice compared with control IgG treated ceacam1+/+ and ceacam1−/− or untreated ceacam1+/+ and ceacam1−/− mice. Mice received either 100 μg control IgG or JAQ1 antibody and were left for 5 days before ferric chloride injury and intravital microscopy. Compared with untreated ceacam1−/− or control IgG treated ceacam1−/− arterioles, JAQ1 treated ceacam1−/− arterioles exhibited a 3-fold smaller thrombus volume at 2 minutes (135 500 ± 2137 vs 134 000 ± 1837 vs 40 400 ± 1127 μm3, respectively; ***P < .001; n = 10 arterioles from 3 mice/group) and a 2-fold lower stability score at 2 minutes (6.150 ± 0.212 vs 5.700 ± 0.300 vs 3.350 ± 0.212, respectively; ***P < .001; n = 10 arterioles from 3 mice/group). In contrast, compared with untreated ceacam1+/+ or control IgG treated ceacam1+/+ arterioles, JAQ1 treated ceacam1+/+ arterioles exhibited a 3-fold smaller thrombus volume at 2 minutes (107 800 ± 3526 vs 102 100 ± 2607 vs 32 050 ± 599 μm3, respectively; ***P < .001; n = 10 arterioles from 3 mice/group) and a moderately lower stability score at 2 minutes (2.750 ± 0.261 vs 3.000 ± 0.236 vs 2.150 ± 0.076, respectively; ***P < .001; n = 10 arterioles from 3 mice/group). (G) DiOC6-labeled whole blood of wild-type, ceacam1+/−, and ceacam1−/− mice was perfused over 100 μg/mL type I fibrillar collagen-coated microslides at a wall shear rate of 1800 seconds−1. Thrombi (1-μm sections) were imaged at 4 minutes with a Zeiss Axiovert 135 inverted microscope (Carl Zeiss) using a 60×/0.4 NA objective at 37°C and captured with an Axiocam MRm camera (Carl Zeiss), and thrombus volume was quantified using Slidebook software (Intelligent Imaging Innovations, Denver, CO). Each data point showed on the graph represents the thrombus volume for each individual mouse performed independently.
