Cold storage of platelets induces abnormal membrane distribution of GPIbα and lipid raft formation, which can be prevented by RHOA inhibition. (A) Human GPIbα analysis by confocal microscopy (magnification bar = 1 μm). The green color shows GP1B expression. Arrows represent areas of abnormally distributed membrane GPIbα. (B) Representative example of immunoblot analysis of cold-stored platelets from WT and RhoA^Δ/Δ mice treated with either RHOA inhibitor, RAC inhibitor, or both for Cav1 and Flot1, showing induction of lipid rafts upon cold storage. This induction can be prevented by the RHOA and RAC inhibitors. (C) Representative example of immunoblot analysis in purified lipid rafts (Triton X-100/sucrose gradient) of 3-day cold-stored human platelets analyzed for the expression of FLOT1 and CAV1. (D) Representative example of immunoblot analysis in purified lipid rafts (Triton X-100/sucrose gradient) of 7-day cold-stored human platelets analyzed for the expression of FLOT1 and CAV1. Density quantification was performed using ImageJ software, and ratios were calculated after normalization of the RT-vehicle control. Data were reproduced in at least 2 independent experiments.