Javier Modrego

Different expression of proteins in platelets from aspirin-resistant and aspirin-sensitive patients

The aim of the present study was to analyse differences in the protein expression profile between platelets from aspirin (ASA)-resistant patients and ASA-sensitive patients. We analysed platelets from 51 clinically stable coronary ischaemic patients taking ASA (100 mg/day) divided into ASA-resistant (n=25) and ASA-sensitive (n=26) based on a platelet functionality test (PFA-100). Proteins associated with cytoskeleton, energetic metabolism, oxidative stress, inflammation and cell survival were analysed by two-dimensional electrophoresis and mass spectrometry. The expression of two gelsolin precursor isotypes and one F-acting capping protein isotype was decreased in ASA-resistant platelets (p<0.05). The expression of glyceraldehyde 3-phosphate dehydrogenase was increased in the ASA-resistant platelets (1751.1 + or - 220.6 vs. 4273.3 + or - 971.7, 95% confidence interval [CI] 1815.11 to 4061.2, p=0.001). It was accompanied by a reduced expression and activity of 1,6-bisphosphate aldolase in platelets without changes in the content of pyruvate. A reduced expression of gluthathione-S-transferase and the protein disulfide isomerase isotype 1 was found in ASA-resistant platelets. The protein expression of the chloride intracellular channel isotype 1 was increased in ASA-resistant platelets (21.3 + or - 3.8 vs. 48.8 + or - 6.0, CI 29.5 to 45.95, p=0.03) while the expression of two HSP60 and two HSP71 isotypes was decreased. No changes were observed in proteins associated with inflammation. In conclusion, ASA-resistant and ASA-sensitive platelets are different in terms of the level of expression of proteins associated with mechanisms such as energetic metabolism, cytoskeleton, oxidative stress and cell survival which may be associated with their different ability to respond to ASA.

Proteomic changes related to "bewildered" circulating platelets in the acute coronary syndrome.

Acute coronary syndromes (ACS) are associated with platelet activation. The aim of the present study was to study the protein expression level associated with glycolysis, oxidative stress, cytoskeleton and cell survival in platelets obtained during an ACS. Platelets from 42 coronary ischemic patients, divided into patients admitted within 24 h after the onset of chest pain (ACS group; n=16) and patients with stable coronary ischemic disease (CAD, n=26), were analyzed using proteomics. The expression levels of proteins involved in cellular cytoskeleton (F‐actin capping, β‐tubulin, α‐tubulin isotypes 1 and 2, vinculin, vimentin and two Ras‐related protein Rab‐7b isotypes), glycolysis pathway (glyceraldehyde‐3‐phosphate dehydrogenase, lactate dehydrogenase and two pyruvate kinase isotypes) and cellular‐related antioxidant system (manganese superoxide dismutase) and even the expression and activity of glutathione‐S‐transferase were significantly reduced in platelets from ACS patients compared to CAD patients. Moreover, reduction in the expression of proteins associated with cell survival such as proteasome subunit β type 1 was also observed in ACS platelets compared with CAD platelets. Principal component and logistic regression analysis suggested the existence of factors (proteins) expressed in the platelets inversely associated with acute coronary ischemia. In summary, these results suggest the existence of circulating antioxidant, cytoskeleton and glycolytic‐“bewildered” platelets during the acute phase of a coronary event.

Comparative Expression of Proteins in Left and Right Atrial Appendages From Patients With Mitral Valve Disease at Sinus Rhythm and Atrial Fibrillation

Proteomics and Atrial Appendages. Introduction: The objective was to compare by proteomics the expression of proteins associated with the cytoskeleton, energetic metabolism, and cardiac cytoprotection between left atrial appendages (LAA) and right atrial appendages (RAA) obtained from patients with mitral valve disease both in sinus rhythm (SR, n = 6) and in permanent atrial fibrillation (AF, n = 11).

Expression of cytoskeleton and energetic metabolism-related proteins at human abdominal aortic aneurysm sites

OBJECTIVE The purpose of this study was to evaluate the expression of proteins related to cytoskeleton and energetic metabolism at abdominal aortic aneurysm (AAA) sites using proteomics. Several remodeling-related mechanisms have been associated with AAA formation but less is known about the expression of proteins associated with cytoskeleton and energetic metabolism in AAAs. METHODS AAA samples (6.73 ± 0.40 cm size) were obtained from 13 patients during elective aneurysm repair. Control abdominal aortic samples were obtained from 12 organ donors. Proteins were analyzed using two-dimensional electrophoresis and mass spectrometry. RESULTS The expression of filamin was increased in the AAA site compared to control abdominal aortic samples while microfibril-associated glycoprotein-4 isotype 1, annexin A5 isotype 1, and annexin A2 were reduced compared with control abdominal aortic samples. Reduction in expression level of energetic metabolism-associated proteins such as triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, and cytosolic aldehyde dehydrogenase was also observed in AAAs compared to controls. Reduction of triosephosphate isomerase expression was also observed by Western blot, which was accompanied by diminished triosephosphate isomerase activity. At the AAA site, pyruvate dehydrogenase expression was reduced and the content of both lactate and pyruvate was increased with respect to controls without changes in lactate dehydrogenase activity. CONCLUSIONS The present results suggest that an anaerobic metabolic state may be favored further to reduce the expression of cytoskeleton-related proteins. The better knowledge of molecular mechanism involved in AAAs may favor development of new clinical strategies.

Plasma desmoplakin I biomarker of vascular recurrence after ischemic stroke

J. Neurochem. (2012) 121, 314–325.

New circulating biomarkers for predicting cardiovascular death in healthy population.

There is interest to analyse newer biomarkers to identify healthy individuals at risk to develop cardiovascular disease (CVD) incidents and death. To determine in healthy individuals new circulating protein biomarkers, whose systemic levels may be associated with the risk of future development of CVD incidents and death. The study was performed in 82 individuals from the Malmö Diet and Cancer study cohort, free from CVD of whom 41 developed CVD and 41 did not. Plasma proteins related to inflammation and thrombo‐coagulating processes were analysed. α1‐antitrypsin isotype 3 plasma levels were significantly higher while apolipoprotein J plasma levels were lower in participants that developed CVD incidents than those that did not develop acute cardiovascular episode. Of 82 participants, 17 died by CVD causes. There were proteins whose expression in plasma was significantly higher in participants suffering CVD death as compared with those that did not die by CVD. These proteins included: fibrinogen β‐chain isotypes 1 and 3, fibrinogen‐γ‐chain isotype 2, vitamin D‐binding protein isotypes 1, 2 and 3, α1‐antitrypsin isotypes 3 and 6, haptoglobin isotypes 3,4,5 and 5, haemopexin isotypes 1 and 2, and Rho/Rac guanine nucleotide exchange factor 2. Moreover, apolipoprotein J plasma levels were found lower in participants that died by cardiovascular cause. Association between plasma levels of proteins and CVD death was independent of age, gender, conventional risk factors and plasma C‐reactive protein levels. Several protein plasma levels and protein isotypes related to inflammation and thrombo‐coagulating phenomena were independently associated with the risk of future CVD death.

Effects of Platelets on the Protein Expression in Aortic Segments: A Proteomic Approach

It is well known the effects of the vascular wall on platelet activity but little is known about the effects of platelets on the proteins expression in the vascular wall. We analyzed whether platelets may modify the protein expression in the vascular wall. We used an in vitro model coincubating human platelet rich plasma (PRP) with control and 10 ng/ml tumor necrosis factor‐α (TNF‐α)‐preincubated bovine aortic segments. 2DE, mass spectrometry and Western blot analysis were used to determine changes in the expression of proteins associated with the cytoskeleton and energetic metabolism in the aortic segments. In control healthy vascular wall, only the cytoskeleton‐related proteins expression was modified by PRP. However, when PRP was coincubated with TNF‐α pre‐stimulated aortic segments lesser number of cytoskeleton‐related proteins were modified. With respect to energetic metabolism, in control segments, PRP failed to modify any of the analyzed energetic‐related proteins. However, in TNF‐α‐preincubated segments the presence of PRP upexpressed glyceraldehyde‐3‐phosphate dehydrogenase. Moreover, by western blot experiments it was observed that in TNF‐α‐preincubated segments the expression of fructose 1,6‐bisphosphate aldolase was downregulated by platelets. However, no differences were found in the expression of triosephosphate isomerase and ATP synthase α‐chain. In addition, the activity of fructose 1,6‐bisphosphate aldolase and piruvate content was significantly reduced without modification on triosephosphate isomerase activity. In conclusion, the crosstalk between platelets and vascular wall is bidirectional and platelets regulated in the vascular wall the expression of proteins associated with the cytoskeleton and energetic metabolism, particularly in the healthy vascular wall. J. Cell. Biochem. 111: 889–898, 2010.

Effects of hormones on platelet aggregation.

Abstract Platelets and their activation/inhibition mechanisms play a central role in haemostasis. It is well known agonists and antagonists of platelet activation; however, during the last years novel evidences of hormone effects on platelet activation have been reported. Platelet functionality may be modulated by the interaction between different hormones and their platelet receptors, contributing to sex differences in platelet function and even in platelet-mediated vascular damage. It has suggested aspects that apparently are well established should be reviewed. Hormones effects on platelet activity are included among them. This article tries to review knowledge about the involvement of hormones in platelet biology and activity.

BRCA2 gene mutations and coagulation-associated biomarkers

Thromboembolic events are the second cause of death in cancer patients, although the mechanisms underlying this increased thromboembolic risk remain unclear. The aims of this study were to examine whether BRCA2 gene mutations may modify the circulating levels of thrombocoagulation biomarkers and whether breast cancer development may influence changes in such circulating biomarkers. The study was performed in 25 women with mutations in the BRCA2 gene (n=12 breast cancer, n=13 breast cancer-free) and in 13 BRCA2 non-mutant controls. Results revealed that plasma levels of fibrinogen gamma chain isotypes 2 and 3, haptoglobin isotypes 4 and 5, serotransferrin isotypes 3 and 4 and convertase C3/C5 isotypes 4 and 5 were significantly higher in BRCA2 mutation carriers compared to controls. However, plasma levels of vitamin D binding protein isotype 1 and alpha1-antitrypsin isotypes 2, 3 and 4 were significantly decreased in BRCA2 mutation carriers compared to controls. Plasma expression of PF4 and P-selectin was significantly higher in BRCA2 mutations carriers than in controls. BRCA2 truncated mutations conserving a binding region for RAD51 were associated with increased plasma levels of alpha1-antitrypsin isotypes 3 and 4 with respect to women showing BRCA2 mutations that loss the binding RD51 region to BRCA2. Only plasma levels of vitamin D binding protein isotypes 1 and 3 were significantly reduced and alpha 1-antitrypsin isotype 1 was increased in cancer-free BRCA2 mutation carriers compared to BRCA2 mutation carriers with breast cancer. The presence of BRCA2 mutations is associated with increased plasma levels of thrombo-coagulating-related proteins, which are independent to breast cancer development.

Vardenafil Improves Penile Erection in Type 2 Diabetes Mellitus Patients with Erectile Dysfunction: Role of Tropomyosin

INTRODUCTION Evidences have been suggested that phosphodiesterase type 5 (PDE5) inhibition promotes vasculoprotective benefits in patients with cardiovascular diseases. AIM The aim of this study is to analyze the systemic effect of PDE5 inhibition in type 2 diabetes mellitus patients with erectile dysfunction (ED) determining changes in the expression levels of plasma proteins. METHODS Seventeen patients with controlled type 2 diabetes mellitus and ED were included in the study. Patients received vardenafil hydrochloride 20 mg on demand during 12 weeks. At the beginning and 12 weeks after vardenafil administration, plasma samples were collected and analyzed using proteomics. MAIN OUTCOME MEASURES International Index of Erectile Function-Erectile Function Domain (IIEF-EFD) and plasma protein expression before and after vardenafil administration. Nitrate/nitrite release, PDE5, and soluble guanylate cyclase (sGC) expression and cyclic guanosine monophosphate (cGMP) content in cultured bovine aortic endothelial cells (BAECs). RESULTS The IIEF-EFD score was markedly improved after 12 weeks of vardenafil administration. Plasma levels of alpha 1-antitrypsin isotypes 4 and 6 and β-tropomyosin were decreased, whereas apolipoprotein AI isoype 5 was increased 12 weeks after vardenafil administration. Only β-tropomyosin plasma levels were inversely correlated with IIEF-EFD score. Tropomyosin has been added to cultured BAECs and after 24 hours reduced the protein expression level of sGC-β1 subunit and decreased the cGMP content. Tropomyosin did not modify PDE5 expression and nitric oxide release in BAECs as compared with control BAECs. Vardenafil (10 μg/mL) did not modify sGC-β1 subunit expression in tropomyosin + vardenafil-incubated BAECs; however, vardenafil significantly reversed the reduction of cGMP content induced by tropomyosin. CONCLUSION Vardenafil administration improved erectile functionality in controlled type 2 diabetes mellitus patients with ED, which was associated with reduction of circulating plasma β-tropomyosin levels. Tropomyosin affected by itself the cGMP generating system suggesting a possible new mechanism involved in ED. Vardenafil reversed the reduction effect of cGMP content elicited by tropomyosin in BAECs.