CD148 positively regulates platelet aggregate formation on collagen under flow and thrombus formation in vivo. (Ai) Anticoagulated blood from wild-type (WT) and CD148 transmembrane-knockout (KO) mice was flowed through collagen-coated capillary tubes at 1000 s−1. Platelets were fluorescently labeled with DiOC6 before being flowed. Representative images were taken in real time by fluorescence microscopy (10 μm scale bar). (Aii) Differential interference contrast (DIC) images of fixed platelets on collagen fibrils after being flowed through collagen-coated capillary tubes at 1000 s−1 for 4 minutes (10 μm scale bar). (Aiii) High magnification DIC images of adherent platelets from panel (Aii) (5 μm scale bar). Results are representative of 3 WT and 3 KO mice. See also Figure S7 Videos S5,S6. (B-D) The functional role of CD148 in thrombosis and hemostasis was investigated using 3 different in vivo assays: (B) the tail bleeding assay, (C) the ferric chloride injury model, and (D) the laser injury model. (B) CD148 transmembrane-knockout (CD148 TM-KO) mice exhibited prolonged bleeding compared with litter-matched WT mice, after excision of a small portion of the tail tip. Symbols represent individual mice. Horizontal lines intersecting datasets represent the mean. (Ci) FcR γ-chain–deficient (γ-chain−/−) and (Cii) CD148 TM-KO mice exhibited delayed vascular occlusion after ferric chloride–induced injury of mesenteric arterioles compared with litter-matched WT mice. (Di) Platelets from WT and CD148 TM-KO mice were fluorescently labeled ex vivo with rat anti–mouse αIIb primary antibody and Alex488-conjugated secondary antibody before being reintroduced into recipient mice. Arterioles in cremaster muscles of recipients were subsequently injured by laser, and the accumulation of platelets (green) into the thrombi was assessed. Representative images from 5 WT and 5 CD148 TM-KO mice are shown. (Dii) Each curve represents the median integrated thrombus fluorescence intensity in arbitrary units (a.u.) for 25 thrombi induced in 5 mice of each genotype. See also Figure S7 Videos S7,S8.
