Fig. 1: The targeting of C3G in platelets decreases Rap1 activation, resulting in impaired platelet activation and aggregation.
From: C3G contributes to platelet activation and aggregation by regulating major signaling pathways
a Tail-bleeding assays were performed in C3G-KO (KO) mice and their wild-type (WT) controls at weaning. The scatter plots represent the bleeding times of 13–15 animals of each genotype. The mean values are shown. The Mann–Whitney U test was performed. b Washed blood from C3G-KO and C3G-wt mice was stimulated with 0.5 U/ml or 1 U/ml thrombin and incubated with anti-CD62P-FITC to determine the percentage of platelets presenting P-selectin on their surface or with JON/A-PE antibody to determine the percentage of platelets in which the integrin αIIbβ3 was activated. The histograms represent the mean ± SD of the percentage of labeled platelets. c Histograms represent the mean ± SD of the number of platelet aggregates formed upon 1 U/ml thrombin stimulation for the indicated time periods. d Washed blood from C3G-KO and C3G-wt mice was stimulated with PMA at the indicated concentrations and incubated with anti-CD62P-FITC. The histograms represent the mean ± SD of the percentage of labeled platelets. e Washed blood from C3G-KO and C3G-wt mice was stimulated with 10 μM ADP in combination with Alexa 488-fibrinogen, and labeled platelets were detected by flow cytometry. The histograms represent the mean ± SD of the percentage of labeled platelets. Platelets from C3G-KO and C3G-wt mice were stimulated with (f) 0.2 or 2 μM PMA for 5 min, (g) 0.2 or 1 U thrombin for 5 min (upper) or 1 U thrombin for 1 or 5 min (lower). The levels of Rap1-GTP were determined by pulldown assay and immunoblotting. Values are relative to the control value (f) or to the value for thrombin in wild-type platelets (g) and were normalized against total Rap1. The Rap1-GTP/Total Rap1 ratio is indicated beneath the blots. h Kaplan–Meier survival plots for mice after the induction of acute pulmonary thromboembolism (i.v. injection of a lethal dose of collagen/norepinephrine). Survival was significantly increased in C3G-KO mice (p = 0.0409, log-rank test). Two of six C3G-KO mice survived the lethal treatment. i The histograms show the percentage of reduction in blood platelets induced by the i.v. injection of a sublethal dose of collagen/norepinephrine. Blood was withdrawn before the injection and 4 min after injection. The Mann–Whitney U test was performed. j Representative H&E staining of lung sections from mice treated with a lethal dose of collagen/norepinephrine. Thrombi (arrowheads) appeared as eosinophilic material with borders, with partial or total affectation of the lumen of the small and medium caliber blood vessels located mainly on the periphery of the pulmonary lobes. Bar: 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001. wt: wild-type; T: thrombin.
