Abstract
Background and aimsPlatelets play a fundamental role in the increased atherothrombotic risk related to central obesity since they show hyperactivation and lower sensitivity to antiplatelet therapy in obese patients. The main goal of this study was to identify platelet biomarkers related to the risk of atherothrombosis in obese patients, confirm platelet activation levels in these patients, and identify altered activation pathways. MethodsPlatelets were obtained from cohorts of obese patients and age- and sex-matched lean controls. Biochemical and proteome analyses were done by two-dimensional differential in-gel electrophoresis (2D-DIGE), mass spectrometry, and immunoblotting. Functional and mechanistic studies were conducted with aggregation assays and flow cytometry. ResultsWe confirmed an up-regulation of αIIb and fibrinogen isoforms in platelets from obese patients. A complementary platelet aggregation approach showed platelets from obese patients are hyper-reactive in response to collagen and collagen-related peptide (CRP), revealing the collagen receptor Glycoprotein VI (GPVI) signalling as one of the altered pathways. We also found the active form of Src (pTyr418) is up-regulated in platelets from obese individuals, which links proteomics to aggregation data. Moreover, we showed that CRP-activated platelets present higher levels of tyrosine phosphorylated PLCγ2 in obese patients, confirming alterations in GPVI signalling. In line with the above, flow cytometry studies show higher surface expression levels of total GPVI and GPVI-dimer in obese platelets, both correlating with BMI. ConclusionsOur results suggest a higher activation state of SFKs-mediated signalling pathways in platelets from obese patients, with a primary involvement of GPVI signalling.
Highlights
Thirty-four patients admitted to the hospital with a diagnosis of severe “healthy” obesity (BMI ≥ 40) and 34 age- and gender-matched lean healthy controls were included in the study (Supplementary Table 2)
By using a specific anti-Src antibody, we demonstrated that the active form of Src is up-regulated in platelets from a cohort of 29 “healthy” obese patients with BMI ≥ 40 compared to matched lean healthy controls (Fig. 3)
After immunoprecipitation with the 4G10 antiphosphotyrosine antibody, we saw an increase of PLCγ2 tyr phosphorylation in the obese group compared to lean matched-controls, which suggests a hyperactivation of Glycoprotein VI (GPVI) signalling in line with the results shown above (Fig. 4)
Summary
Central obesity corresponds to an excess of intra-abdominal adipose tissue [2] This accumulation leads to the intrinsic dysfunction of adipose tissue, together with insulin resistance, endothelial dysfunction, systemic inflammatory state and oxidative stress. The main goal of this study was to identify platelet biomarkers related to the risk of atherothrombosis in obese patients, confirm platelet activation levels in these patients, and identify altered activation pathways. A complementary platelet aggregation approach showed platelets from obese patients are hyper-reactive in response to collagen and collagen-related peptide (CRP), revealing the collagen receptor Glycoprotein VI (GPVI) signalling as one of the altered pathways. We showed that CRPactivated platelets present higher levels of tyrosine phosphorylated PLCγ2 in obese patients, confirming alterations in GPVI signalling. In line with the above, flow cytometry studies show higher surface expression levels of total GPVI and GPVI-dimer in obese platelets, both correlating with BMI
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