Complete loss of both PIP5KIγ isoforms disrupts the integrin-dependent integrity of the membrane cytoskeleton and the stabilization of platelet adhesion. (A) Differential interference contrast micrograph that shows that PIP5KIγ-null platelets form membrane tethers by pulling on a fibrinogen-coated bead that was touched to the surface of the cell and then moved apart. (B) PIP5KIγ−/− platelets that were analyzed for their ability to form membrane tethers were quantified by an optical trap. The graph illustrates that pulling on the membrane-attached fibrinogen-coated bead will only stretch the membrane when membrane-bound integrins are not anchored to the underlying cytoskeleton. Comparable results are obtained with platelets lacking PIP5KIγ (PIP5KIγ−/− MLC-2v Cre+) or wild-type platelets exposure to a pharmacologic G-actin sequestering agent, Latrunculin A. Tethers were rarely seen in wild-type platelets. (C-D) Wild-type platelets or platelets lacking PIP5KIγ were perfused over immobilized collagen at a wall shear stress of 1 dyn/cm2 for 10 minutes. After changing to platelet-free buffer, the wall shear stress was increased every 20 seconds up to 40 dyn/cm2, and the platelet covered area was measured. (C) Representative micrograph of platelet-covered areas remaining at a wall shear stress of 40 dyn/cm2. (D) Data for all wall shear stresses. (E) Wild-type platelets or platelets lacking PIP5KIγ were perfused over immobilized von Willebrand factor at the indicated wall shear stress, and mean rolling velocities were measured. The data in panels D and E represent the mean ± standard error of the mean of 5 experiments. *P < .05; **P < .01 relative to wild-type platelets at the same wall shear stress.
