Reversibility of G04-mediated inhibition of RHOA allows clotting activity of platelets in vivo and prevents cold storage–associated deficiencies in adhesion, formation of microparticles, and myosin light chain (MLC) activation. (A) Tail bleeding time analysis in C57BL/6 mice. C57BL/6 mice were sublethally irradiated (2.5 Gy), administered aspirin 1 week later, treated with aspirin, and 24 hours later transfused with either RT or 7-day cold-stored congenic platelets stored in plasma with or without R-G04 (75 μM). Data are derived from 2 independent experiments with 5 mice per group. (B) Representative example of human platelet spreading on fibrinogen, and confocal microscopy of phalloidin-stained specimens. Cold-stored platelets were stored in either vehicle or G04 for 7 days. Arrows depict platelet aggregates. The white outlined square highlights a representative area of platelet singlets. (C) Quantification of measurement of platelet spreading in panel B. A minimum of 20 platelets per group were analyzed and averaged from 2 independent experiments. (D) Representative example of electron micrograph sections (1500× original magnification) of platelets stored at RT with vehicle or in cold conditions with or without R-G04. Arrows depict microparticles of variable sizes, <1 μm in diameter. Magnification bar = 5 μm. (E) Quantification of microparticle content of platelets stored at RT in vehicle or after cold storage with or without R-G04. A minimum of 10 fields similar to the ones presented in panel D were analyzed and averaged from 2 biological replicates. (F) Transmission electron microscopy analysis of 7-day RT-stored platelets in vehicle control or stored in cold conditions with or without R-G04 (10 000× larger images and 1500× original magnification insets). Arrows depict dilated cisterns in platelets stored in cold conditions in the absence of R-G04, which were not evident in cold-stored platelets in the presence of R-G04 or in platelets stored at RT. Magnification bar = 0.1 μm. (G) Representative example of immunoblot analysis of 7-day cold-stored human platelets blotted for phosphorylated MLC (Ser19; p-MLC) showing that cold storage induces p-MLC, which can be prevented by RHOA and RAC inhibition. 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. ∗∗∗P < .001.
Figure 3.

Reversibility of G04-mediated inhibition of RHOA allows clotting activity of platelets in vivo and prevents cold storage–associated deficiencies in adhesion, formation of microparticles, and myosin light chain (MLC) activation. (A) Tail bleeding time analysis in C57BL/6 mice. C57BL/6 mice were sublethally irradiated (2.5 Gy), administered aspirin 1 week later, treated with aspirin, and 24 hours later transfused with either RT or 7-day cold-stored congenic platelets stored in plasma with or without R-G04 (75 μM). Data are derived from 2 independent experiments with 5 mice per group. (B) Representative example of human platelet spreading on fibrinogen, and confocal microscopy of phalloidin-stained specimens. Cold-stored platelets were stored in either vehicle or G04 for 7 days. Arrows depict platelet aggregates. The white outlined square highlights a representative area of platelet singlets. (C) Quantification of measurement of platelet spreading in panel B. A minimum of 20 platelets per group were analyzed and averaged from 2 independent experiments. (D) Representative example of electron micrograph sections (1500× original magnification) of platelets stored at RT with vehicle or in cold conditions with or without R-G04. Arrows depict microparticles of variable sizes, <1 μm in diameter. Magnification bar = 5 μm. (E) Quantification of microparticle content of platelets stored at RT in vehicle or after cold storage with or without R-G04. A minimum of 10 fields similar to the ones presented in panel D were analyzed and averaged from 2 biological replicates. (F) Transmission electron microscopy analysis of 7-day RT-stored platelets in vehicle control or stored in cold conditions with or without R-G04 (10 000× larger images and 1500× original magnification insets). Arrows depict dilated cisterns in platelets stored in cold conditions in the absence of R-G04, which were not evident in cold-stored platelets in the presence of R-G04 or in platelets stored at RT. Magnification bar = 0.1 μm. (G) Representative example of immunoblot analysis of 7-day cold-stored human platelets blotted for phosphorylated MLC (Ser19; p-MLC) showing that cold storage induces p-MLC, which can be prevented by RHOA and RAC inhibition. 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. ∗∗∗P < .001.

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