RHOA and RAC1 inhibition prevent the cold-induced mislocalization of glycosylation enzymes. (A) Representative example of confocal microscopy images of platelets stained with anti-GalNACT1-3 and a secondary antibody conjugated to fluorescein isothiocyanate (FITC). Magnification bar = 1 μm. (B) Quantification of intracytoplasmic expression of GalNac T1-3. A minimum of 20 platelets from each group was analyzed and averaged from 2 independent experiments. (C) Measurement of UDP-galactosyltransferase and (D) syalyl transferase activities in lipid raft/endocytic microdomains, which were not present in either RHOA or RAC1 inhibited cold-stored platelets. Three independent experiments with triplicates in each group were performed. (E) Cold-stored platelet phagocytosis by phorbol 12-myristate 13-acetate–activated THP-1 cells is prevented by S-G04 in 7-day stored human platelets. Three independent experiments with triplicates in each group were performed. (F-G) NSG mice were pretreated with clodronate to deplete the macrophages, sublethally irradiated to induce moderate thrombocytopenia, and 3 × 109 platelets were stored for 7 (F) or 10 (G) days and infused at 24 hours after aspirin administration. Tail bleeding analysis was done at 6 hours after the platelet infusion. In vitro experiments were run in triplicate in 3 independent experiments. In vivo experiments included a minimum of 5 mice per group in 2 independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.
