CLEC-2 contributes to thrombus stability in vitro and in vivo independently of hemITAM signaling. (A) The surface area of aggregates formed on collagen under flow conditions was significantly reduced in Y7A knockin (KI) chimeric samples only after preincubation with 5 µg/mL INU1 Fab′ fragments. Shear rate: 1000 s−1; data presented as means with error bars of standard deviations, n = 8. Representative images shown from the 5 fields of view analyzed per mouse. Scale bar represents 50 µm. (B) Y7A KI chimeric mice had normal tail bleeding times in comparison with wild-type mice, however, after injection of 2 µg/g of INU1 Fab′ fragments, Y7A KI mice had significantly destabilized hemostasis, with significantly more mice bleeding until the end of the observation period. The horizontal line denotes the mean time to cessation of bleeding, and each circle represents 1 animal. (C) Time to vessel occlusion in the mechanical injury of the aorta thrombosis model. Chimeric Y7A KI mice were able to form occlusive thrombi after injury, however, after injection of 2 µg/g of INU1 Fab′ fragments, Y7A KI mice had significantly destabilized thrombosis, reflecting the results of Pf4-cre CLEC-2 KO mice analyzed in parallel. The horizontal line denotes the mean time to vessel occlusion, and each circle represents 1 animal (D) There were no significant differences between any of the indicated mouse genotypes in the time to the first appearance of thrombi in the ferric chloride injury model of mesenteric arterioles. The horizontal line denotes the mean time to thrombi appearance, and each circle represents 1 animal. (Ei) Time to vessel occlusion in the ferric chloride injury model of mesenteric arterioles. (Eii) Representative images of injured vessels from the different mouse genotypes at the indicated time points after injury. There were no significant differences between vessel occlusion times in chimeric wild-type and Y7A KI mice, however, there was a significant reduction in vessels occluded within the observation period in chimeric Y7A KI mice treated with 2 µg/g of INU1 Fab′ fragments. This destabilization in thrombosis reflected the results of Pf4-cre CLEC-2 KO mice analyzed in parallel. The horizontal line denotes the mean time to vessel occlusion, each circle represents 1 arteriole, and 2 vessels were injured per animal. Platelets were visualized for microscopy with an anti-GPIX DyLight488-conjugated antibody derivative administered to the mice at the start of the experiment. Scale bars represent 20 µm. ***P < .001; ns, not significant.
