Figure 1.
Murine cold-stored platelets exhibit induced RHOA activation upon cold storage and reduced survival upon congenic infusion, whereas RHOA inhibitors reduce RHOA activity in cold-stored human platelets. (A) Schematic experimental design for pulldown assay with murine platelets (top); representative example of effector binding pulldown assays on murine platelets stored in different conditions (bottom). Murine platelets were isolated and stored at 4°C for indicated time points. Pulldown assays were performed using glutathione S-transferase (GST)–rhotekin domain bound beads to pull down RHOA-guanosine triphosphate (GTP) and GST-PAK beads to pull down RAC-GTP and CDC42-GTP. Pooled platelets from 3 mice were used per group. Two independent experiments were performed. (B) Schematic experimental design for congenic transfusion of murine platelet for in vivo survival analysis. (C) Blood platelet survival in unconditioned C57BL/6 mice transfused with platelets stored for 3.5 hours at RT or cold temperature; 3 × 108 congenic platelets were transfused per mouse. RT + vehicle stored platelets, cold + vehicle, and cold + R-G04/NSC/CASIN treatment groups are depicted. A minimum of 10 mice per group in 2 independent experiments were analyzed. Representative example of 2 independent experiments with similar results. (D) Area under the curve (AUC) of data depicted in panel C. (E) Representative example of pulldown assays using either rhotekin beads or GST-PAK beads with lysates prepared from 1 × 108 platelets per group from human RT-stored or cold-stored platelets for 7 days. Quantification was done using image densitometry. (F) Representative example of 2 independent experiments for pulldown assays using either GST-rhotekin beads or GST-PAK beads with lysates prepared from 1 × 108 platelets per group from human RT-stored or cold-stored platelets for 7 days in PAS-3M/plasma with or without G04 (75 μM) and NSC (30 μM) treatment. Density quantification was performed using ImageJ software and ratios were calculated after normalization of the RT-vehicle control. (G) Recovery of 7-day stored human platelets transfused to clodronate-treated, sublethally irradiated thrombocytopenic NSG immunodeficient mice. Data depict results from a minimum of 3 mice per group, in each of the 3 independent experiments. (H) AUC of data depicted in panel G. (I) Cold-stored platelet phagocytosis by phorbol 12-myristate 13-acetate–activated THP-1 cells is prevented by G04 in 7-day stored human platelets. Data were reproduced in at least 3 independent experiments performed in triplicate. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.

Murine cold-stored platelets exhibit induced RHOA activation upon cold storage and reduced survival upon congenic infusion, whereas RHOA inhibitors reduce RHOA activity in cold-stored human platelets. (A) Schematic experimental design for pulldown assay with murine platelets (top); representative example of effector binding pulldown assays on murine platelets stored in different conditions (bottom). Murine platelets were isolated and stored at 4°C for indicated time points. Pulldown assays were performed using glutathione S-transferase (GST)–rhotekin domain bound beads to pull down RHOA-guanosine triphosphate (GTP) and GST-PAK beads to pull down RAC-GTP and CDC42-GTP. Pooled platelets from 3 mice were used per group. Two independent experiments were performed. (B) Schematic experimental design for congenic transfusion of murine platelet for in vivo survival analysis. (C) Blood platelet survival in unconditioned C57BL/6 mice transfused with platelets stored for 3.5 hours at RT or cold temperature; 3 × 108 congenic platelets were transfused per mouse. RT + vehicle stored platelets, cold + vehicle, and cold + R-G04/NSC/CASIN treatment groups are depicted. A minimum of 10 mice per group in 2 independent experiments were analyzed. Representative example of 2 independent experiments with similar results. (D) Area under the curve (AUC) of data depicted in panel C. (E) Representative example of pulldown assays using either rhotekin beads or GST-PAK beads with lysates prepared from 1 × 108 platelets per group from human RT-stored or cold-stored platelets for 7 days. Quantification was done using image densitometry. (F) Representative example of 2 independent experiments for pulldown assays using either GST-rhotekin beads or GST-PAK beads with lysates prepared from 1 × 108 platelets per group from human RT-stored or cold-stored platelets for 7 days in PAS-3M/plasma with or without G04 (75 μM) and NSC (30 μM) treatment. Density quantification was performed using ImageJ software and ratios were calculated after normalization of the RT-vehicle control. (G) Recovery of 7-day stored human platelets transfused to clodronate-treated, sublethally irradiated thrombocytopenic NSG immunodeficient mice. Data depict results from a minimum of 3 mice per group, in each of the 3 independent experiments. (H) AUC of data depicted in panel G. (I) Cold-stored platelet phagocytosis by phorbol 12-myristate 13-acetate–activated THP-1 cells is prevented by G04 in 7-day stored human platelets. Data were reproduced in at least 3 independent experiments performed in triplicate. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal