Fig. 7: C3G participates in the activities of the TXA2 pathway in platelets.
From: C3G contributes to platelet activation and aggregation by regulating major signaling pathways
a TgC3G expression negatively regulates the production of TXB2 induced by thrombin. Platelets from mice of the different genotypes under study were stimulated with thrombin (1 U/ml) for 3.5 min at 37 °C while stirring, and secreted TXA2 was determined by detecting its breakdown product, TXB2, by LC/MS/MS. The histograms represent the mean ± SD of the amount of TXB2 (pmol) per 105 platelets. b C3G inhibits cPLA2 phosphorylation. Representative immunofluorescence confocal microscopy images of tgC3G and C3G-KO platelets and their corresponding controls treated with 0.2 U or 1 U/ml thrombin, as indicated, and stained with anti-phospho-cPLA2_Alexa Fluor 568 (red) and phalloidin (green). The images of wtC3G/tgC3G and C3G-wt/C3G-KO platelets correspond to two different experiments, but all images from each experiment were taken at the same exposure time. Bar: 2 μm. c The histograms represent the mean ± SD (n > 10) of the fluorescence intensity (arbitrary units) of phospho-cPLA2 relative to the levels of total cPLA2 (Fig. S7a), as quantified by ImageJ. d tgC3G and tgC3GΔCat platelets showed different sensitivities to the activity of aspirin. Transgenic platelets and their controls were pretreated with 2 mM aspirin for 5 min and then stimulated for 15 min with 1 U/ml thrombin or 10 μM ADP. The histograms represent the mean ± SD of the percentage of inhibition of P-selectin expression on the surface or activation of integrin αIIbβ3 in platelets treated with thrombin + aspirin or ADP + aspirin, compared to agonist-treated platelets. e Left: Representative immunofluorescence confocal microscopy images of platelets of each genotype under each treatment condition (resting, thrombin treatment, thrombin + aspirin treatment) stained with anti-pTyr504-C3G_Cy5 (red) and phalloidin (green) were taken at the same exposure time. Bar: 2 μm. Right: The histograms represent the mean ± SD of the fluorescence intensity (arbitrary units) of pTyr504-C3G (p-C3G) relative to the phalloidin signal, as quantified by ImageJ. f Platelets were pretreated with 2 mM aspirin for 5 min and then stimulated with 0.2 U/ml thrombin for an additional 5 min. Rap1-GTP was isolated by pulldown with GST-RalGDS-RBD and detected by immunoblotting with anti-Rap1 antibodies. Values are relative to the thrombin value in control platelets and were normalized against total Rap1. The Rap1-GTP/Total Rap1 ratio is indicated beneath the blots. *p < 0.05, **p < 0.01, ***p < 0.001. tg: transgenic; wt: wild-type; T: thrombin, Asp: aspirin.
