GPVI surface expression and signalling pathway activation are increased in platelets from obese patients: Elucidating potential anti-atherothrombotic targets in obesity

Figures

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  Fig. 1. 2D-DIGE-based differential proteome analysis of platelets from obese and lean individuals: up-regulation of fibrinogen features in obese patients. (A-i) Representative image of the analysis in grey scale. The figure shows the location on the 2D gels of those spots that are differentially regulated when comparing obese patients and lean matched-controls. Protein identifications are shown by the identification numbers in Supplementary Tables 3 and 4. Spots corresponding to fibrinogen are indicated with a black arrow. (A-ii) Enlarged images of representative fibrinogen spots found to be up-regulated in obese patients by proteomics. (B) Representative 2D-Western blot images of fibrinogen on pools of samples from obese-patients’ platelets and lean matched-controls (n = 22 per group). GAPDH was used as loading control (1D-western blot). Western blot experiments were run in triplicate. OB: severe obese patients; C: lean matched-controls; IB: immunoblot.

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  Fig. 2. Platelet aggregation in response to GPVI activation is increased in obese patients compared to lean matched-controls. PRP and washed platelets were stimulated with different GPVI agonists. (A) PRP was stimulated with CRP-XL (0.1, 0.15 or 0.2 μg/mL) or Horm collagen (0.5, 0.75 or 1 μg/mL) to trigger aggregation. (B) Washed platelets were stimulated with CRP-XL (0.4, 0.5 or 1 μg/mL) and Horm collagen (1, 2 or 3 μg/mL). Results are presented as the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001. p-values and cohort data are shown in Supplementary Table 5. OB: severe obese patients; C: lean-matched controls.

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  Fig. 3. P-Src (pTyr 418) levels are augmented in platelets from obese patients. (A) 1D-Western blot analysis of Src-pTyr418, Src pan and GAPDH protein expression levels in platelets from individual samples (29 obese patients and 29 lean matched-controls). Images are representative of the results obtained and show samples distributed in two gels. (B) Densitometry graphs showing the mean values ± SD of Src (Tyr 418) of 30 obese patients and their lean matched-controls corrected by Src and the loading control (GAPDH), p-values are 0.037 and 0.024 respectively. Moreover, densitometry graph showing the mean values ± SD of Src corrected by the loading control (GAPDH) indicates no differences between obese and control groups (p-value = 0.97). OB: severe obese patients; C: lean matched-controls; IB: immunoblot; *p < 0.05.

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  Fig. 4. PLCγ2 phosphorylation levels are increased in platelets from obese patients following CRP-XL stimulation. Immunoblot analysis of PLCɣ2 following immunoprecipitation with the anti-phosphorylation 4G10 mAB. Severe obese and lean matched controls are compared using 4 × 108 platelets per immunoprecipitation. All samples were stimulated with CRP (1 μg/mL; 90 s), as indicated in the Methods section. (A) Representative images corresponding to individual patients are shown. Samples were distributed in two gels; a vertical line indicates a failed well. (B) Densitometry graph showing the mean values ± SD for all patients (severe obese: 12; lean matched controls: 10). Significant differences are indicated: ***p < 0.0001. IP: immunoprecipitation; IB: immunoblot.

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  Fig. 5. Higher expression levels of GPVI dimer and total GPVI in the surface of platelets from obese patients, with a positive correlation with BMI. The study was performed with a subgroup of patients (12 severe obese patients and their 12 lean matched-controls) and measured in triplicate. (A) GPVI dimer and total levels of GPVI are increased in the obese group. Fluorescence values are shown as mean ± SD. Significant differences are indicated as follow: *p < 0.05, **p < 0.01. (B) Positive correlation between BMI and total GPVI and GPVI dimer was found using Spearman's test.