Mutation of the 3 C-terminal tyrosines of LAT affect platelet thrombi formation on collagen under flow. DiOC6-labeled platelets in whole blood were perfused through a collagen-coated microcapillary at a shear rate of 1500 s−1 for 2 minutes. (A) Thrombus formation was visualized with a 40×/1.3 NA; long working distance objective in real time and then imaged using transmitted light microscopy. (B) After a washing step with phosphate-buffered saline for 2 minutes at the same shear rate to remove nonadherent cells, slides were visualized using differential interference contrast microscopy. Representative images of platelet adhesion from WT and Lat3YF mice are shown. A representative time course for both WT (top panel) and Lat3YF (bottom panel) platelet accumulation on collagen is shown (see “In vitro flow-based adhesion studies” for complete image acquisition information). (C) Area covered by platelet thrombi and (D) thrombus volume were measured at 2 surface locations in each of 3 different experiments (mean ± SEM). **P < .005 versus WT, according to Student t test. Scale bar equals 20 μm.
