Diana Hernández-Romero

Plasma von Willebrand Factor Levels Are an Independent Risk Factor for Adverse Events Including Mortality and Major Bleeding in Anticoagulated Atrial Fibrillation Patients

OBJECTIVES The purpose of this study was to evaluate the prognostic value of plasma von Willebrand factor (vWF) levels and fibrin d-dimer in a large cohort of anticoagulated permanent atrial fibrillation (AF) patients. BACKGROUND In nonanticoagulated AF patients, plasma vWF levels have been related to stroke and vascular events. There are limited data on the prognostic role of biomarkers in anticoagulated AF patients in relation to adverse events (including thromboembolism), mortality, and major bleeding. METHODS We studied 829 patients (50% male; median age 76 years) with permanent AF who were stabilized (for at least 6 months) on oral anticoagulation therapy (international normalized ratio: 2.0 to 3.0). Plasma d-dimer and vWF levels were quantified by enzyme-linked immunosorbent assay. Patients were followed for 2 years, and adverse events (thrombotic and vascular events, mortality, and major bleeding) were recorded. RESULTS Patients were followed for a median of 828 days (range 18 to 1,085 days). On multivariate analysis, age 75 years and older, previous stroke, heart failure, and high plasma vWF levels (≥ 221 IU/dl) were associated with future adverse cardiovascular events (all p values <0.05). High plasma vWF levels, elderly patients, diabetes, hypercholesterolemia, and current smoking were associated with mortality (all p values <0.05). High plasma vWF levels were also an independent predictor of major bleeding (hazard ratio: 4.47, 95% confidence interval: 1.86 to 10.75; p < 0.001). High plasma vWF levels were able to refine clinical risk stratification schema for stroke (CHADS₂ [Congestive heart failure, Hypertension, Age ≥ 75, Diabetes mellitus, and prior Stroke or transient ischemic attack (doubled)], CHA₂DS₂-VASc [Congestive heart failure, Hypertension, Age ≥ 75 years, Diabetes mellitus, Stroke, Vascular disease, Age 65 to 74 years, Sex category]) and bleeding (HAS-BLED [Hypertension, Abnormal renal/liver function, Stroke, Bleeding history or predisposition, Labile International Normalized Ratio, Elderly, Drugs/alcohol concomitantly]). d-dimer did not show any significant impact on adverse events. CONCLUSIONS High plasma vWF levels (≥221 IU/dl) are an independent risk factor for adverse events in anticoagulated permanent AF patients. This biomarker may potentially be used to refine stroke and bleeding clinical risk stratification in AF.

A tyrosinase with an abnormally high tyrosine hydroxylase/dopa oxidase ratio

The sequencing of the genome of Ralstonia solanacearum[Salanoubat M, Genin S, Artiguenave F, et al. (2002) Nature415, 497–502] revealed several genes that putatively code for polyphenol oxidases (PPOs). This soil‐borne pathogenic bacterium withers a wide range of plants. We detected the expression of two PPO genes (accession numbers NP_518458 and NP_519622) with high similarity to tyrosinases, both containing the six conserved histidines required to bind the pair of type‐3 copper ions at the active site. Generation of null mutants in those genes by homologous recombination mutagenesis and protein purification allowed us to correlate each gene with its enzymatic activity. In contrast with all tyrosinases so far studied, the enzyme NP_518458 shows higher monophenolase than o‐diphenolase activity and its initial activity does not depend on the presence of l‐dopa cofactor. On the other hand, protein NP_519622 is an enzyme with a clear preference to oxidize o‐diphenols and only residual monophenolase activity, behaving as a catechol oxidase. These catalytic characteristics are discussed in relation to two other characteristics apart from the six conserved histidines. One is the putative presence of a seventh histidine which interacts with the carboxy group on the substrate and controls the preference for carboxylated and decarboxylated substrates. The second is the size of the residue isosteric with the aromatic F261 reported in sweet potato catechol oxidase which acts as a gate to control accessibility to CuA at the active site.

Small-size circulating microparticles in acute coronary syndromes: relevance to fibrinolytic status, reparative markers and outcomes.

BACKGROUND Recent evidence suggests that circulating microparticles (MPs) contribute to inflammation, coagulation and vascular injury. Majority of MPs have usually not been included into prior analyses due their small size and limited resolution of conventional equipment. Our aim was to assess levels of MPs of different cellular origin sized below 0.5 μm polystyrene beads, denoted as small-size microparticles (sMP), their relation to markers of cardiovascular repair and their impact on prognosis in patients with acute coronary syndromes (ACS). METHODS In a cross-sectional study, we initially compared levels of sMP between patients with ST-segment elevation myocardial infarction (STEMI, n = 50), non-STEMI (n = 47), stable coronary artery disease (CAD, n = 40) and healthy individuals (HC, n = 40). In a separate study, the prognostic value of sMP was assessed in patients with non-STEMI (n = 160). Annexin V-binding sMP (sAMP), platelet CD42b(+) sMPs (sPMP), endothelial CD144(+) sMP (sEMP) and monocyte CD14(+) sMP (sMMP) were quantified using high resolution flow cytometry. Endothelial progenitor cells (EPCs) and monocyte expression of scavenger receptors was quantified by flow cytometry. Fibrinolytic factors were measured by ELISA. RESULTS Counts of sAMP and sEMP were lower in STEMI after PCI (p < 0.001 and p = 0.025, respectively) but not in non-STEMI vs. CAD. sAMP was positively correlated with EPCs in non-STEMI (p < 0.001). Likewise, plasminogen activators negatively correlated with sAMP in non-STEMI and STEMI (p = 0.02 and p = 0.002, respectively). In non-STEMI patients, sEMP and sMMP were independently predictive for future admissions related to heart failure (p = 0.034 and 0.013, respectively) and sPMP for major bleedings (p = 0.002). The sAMP/EPCs ratio was higher in patients (before PCI) compared to STEMI patients. CONCLUSIONS Small-size MPs could be potentially implicated in the modulation of the post-ACS reparative response to injury, with prognostic implications. Besides, the sAMP/EPCs ratio could reflect a change in the apoptotic/reparative potential, being a putative indicator for vascular repair.

Biomarkers of pathophysiology in hypertrophic cardiomyopathy: implications for clinical management and prognosis

The study of biomarkers and their signalling pathways has allowed the development of new therapeutic strategies in a range of disorders. The aim of the present systematic review is to provide an overview of different biomarkers in patients with hypertrophic cardiomyopathy that could give some insight into the pathophysiologic mechanism(s) underlying the typical clinical and histological manifestations of the disease. Several pathophysiological models are presented and discussed, including studies that have investigated these biomarkers for diagnostic and prognostic reasons, in relation to disease progression and/or mortality.

Growth differentiation factor-15, a novel biomarker related with disease severity in patients with hypertrophic cardiomyopathy

BACKGROUND The growth differentiation factor 15 (GDF-15) has been shown up-regulated in stress conditions and to have regulatory actions in myocyte hypertrophy. We hypothesized that GDF-15 could be related to disease severity and functional status in patients with hypertrophic cardiomyopathy (HCM). METHODS AND RESULTS We performed a study which includes 102 consecutive outpatient HCM subjects, 73% males, aged 47.1±14.6 years. A complete history and clinical examination was performed, including 12-lead electrocardiogram, echocardiography, symptom-limited treadmill exercise, 24-hour ECG-Holter monitoring, and magnetic resonance with Gadolinium. Several biomarkers, associated with myocardial remodeling and damage, were compared to GDF-15 levels. The assays were performed with commercial ELISAs or standardized methods when available. There was a significant association between GDF-15 levels and comorbidities, being higher in hypertension (p=0.001), diabetes (p=0.030), atrial fibrillation (p=0.012), dyspnea (p=0.020) and NYHA≥II functional class (p=0.037). GDF-15 levels were positively correlated with clinical variables (age, worse exercise capacity and mild renal dysfunction) and biomarkers of interstitial remodeling, such as metalloproteinase-2 (r: 0.40; p=0.009), N-terminal pro-B-type natriuretic peptide (r: 0.28; p=0.049), high-sensitivity troponin T (r: 0.30; p=0.003) and von Willebrand factor (r: 0.33; p=0.001). Multivariate analysis was assessed to estimate the involvement of these different factors in the GDF-15 levels, confirming the independent implication of severe dyspnea and functional status. CONCLUSIONS The present results show that higher levels of GDF-15 are associated to conditions of severe disease in HCM. Hence, GDF-15 is suggested as a novel marker related to the severity and could represent a further useful tool in monitoring functional capacity of HCM patients.

Polyphenol Oxidase Activity Expression in Ralstonia solanacearum

ABSTRACT Sequencing of the genome of Ralstonia solanacearum revealed several genes that putatively code for polyphenol oxidases (PPOs). To study the actual expression of these genes, we looked for and detected all kinds of PPO activities, including laccase, cresolase, and catechol oxidase activities, in cellular extracts of this microorganism. The conditions for the PPO assays were optimized for the phenolic substrate, pH, and sodium dodecyl sulfate concentration used. It was demonstrated that three different PPOs are expressed. The genes coding for the enzymes were unambiguously correlated with the enzymatic activities detected by generation of null mutations in the genes by using insertional mutagenesis with a suicide plasmid and estimating the changes in the levels of enzymatic activities compared to the levels in the wild-type strain. The protein encoded by the RSp1530 locus is a multicopper protein with laccase activity. Two other genes, RSc0337 and RSc1501, code for nonblue copper proteins exhibiting homology to tyrosinases. The product of RSc0337 has strong tyrosine hydroxylase activity, and it has been shown that this enzyme is involved in melanin synthesis by R. solanacearum. The product of the RSc1501 gene is an enzyme that shows a clear preference for oxidation of o-diphenols. Preliminary characterization of the mutants obtained indicated that PPOs expressed by R. solanacearum may participate in resistance to phenolic compounds since the mutants exhibited higher sensitivity to l-tyrosine than the wild-type strain. These results suggest a possible role in the pathogenic process to avoid plant resistance mechanisms involving the participation of phenolic compounds.

Biomarkers in atrial fibrillation: an overview

Atrial fibrillation (AF) confers a raised risk of stroke and death, and this risk of adverse events is increased by the coexistence of other cardiovascular risk factors. The pathophysiology of AF is complex, involving the role of inflammation, structural remodelling with apoptosis, inflammation or fibrosis. These changes confer a prothrombotic or hypercoagulable state in this arrhythmia. Despite being easy to use for decision‐making concerning oral anticoagulant therapy in AF, clinical risk scores used for stratification have shown modest capability in predicting thromboembolic events, and biomarkers may improve our identification of ‘high risk’ patients. Biomarkers, whether measured in the peripheral blood, urine or imaging‐based may improve our knowledge of the pathophysiology of AF. Importantly these biomarkers could help in the assessment of AF prognosis. The aim of this review was to summarise the published data about biomarkers studied in AF, with focus on data from randomised prospective clinical trials and large community‐based cohorts. We will also review the application of these biomarkers to prognosis on the main schemes used to help stratify risk in AF.

High-sensitivity troponin T as a biomarker for the development of atrial fibrillation after cardiac surgery.

OBJECTIVES Atrial fibrillation (AF) occurs in ∼ 30% of patients undergoing coronary artery bypass grafting (CABG) and in 40% of patients after valve surgery. High-sensitivity cardiac troponin T (hsTnT) is a specific and high-sensitivity marker of myocardial injury, while N-terminal proB-type natriuretic peptide (NT-proBNP) is an established biomarker for wall remodelling. We investigated whether hsTnT and NT-proBNP levels could be used as valuable biomarkers for AF occurrence after cardiac surgery. METHODS We included consecutive haemodynamically stable patients undergoing programmed cardiac surgery with cardiopulmonary bypass pump. We determined hsTnT and NT-proBNP levels before and after cardiac surgery and recorded AF development by prolonged electrocardiogram monitoring. RESULTS We included 100 patients with predominantly aortic valve (n = 42) or ischaemic heart (n = 58) diseases. Twenty-nine patients (29%) developed post-surgical AF. Patients developing AF had a longer hospital stay (P = 0.005). hsTnT levels increased after surgery [P < 0.001], indicating perioperative myocardial injury, with higher presurgery levels in patients who developed AF [P = 0.015]. Body mass index and EuroSCORE risk scale were independently associated with higher hsTnT levels presurgery. On univariate analysis, age (P = 0.048), male sex (P = 0.031), indexed left atrial volume (P = 0.042), β-blockers treatment (P = 0.024), type of surgery (valve surgery vs CABG; P = 0.034), EuroSCORE risk scale (P = 0.025) and higher preoperative hsTnT levels (P = 0.009) were predictors of AF development, but NT-proBNP did not reach statistical significance (P = 0.060). hsTnT levels in blood samples obtained the day after surgery were not associated with post-surgical AF development (P = 0.165). In a multivariate model, only higher hsTnT levels before cardiac surgery (>11.87 ng/l) [Odds Ratio, OR; (95% Confidence interval, CI) 4.27 (1.43-12.77), P = 0.009] and male sex [OR 5.10 (1.72-15.13), P = 0.003)] were independently associated with the occurrence of post-surgical AF. CONCLUSION High presurgical hsTnT levels were independently predictive of patients developing AF after cardiac surgery. hsTnT levels determined post-surgery suggest that cardiac perioperative myocardial injury is not associated with postoperative AF development. NT-proBNP did not reach statistical significance as a biomarker for AF prediction.

Small‐size platelet microparticles trigger platelet and monocyte functionality and modulate thrombogenesis via P‐selectin

This study aimed to examine the mechanisms of cellular activation by small‐size platelet microparticles (sPMP) and to present the performance of high‐resolution flow cytometry for the analysis of subcellular entities from different origins. Plasma counts of sPMP were analysed in coronary artery disease patients (n = 40) and healthy controls (n = 40). The effect of sPMP and platelet debris (PD) in pathophysiologically relevant doses on platelet and monocyte activation parameters and thrombogenesis was investigated via flow cytometry and thromboelastometry. New generation flow cytometry identifies differences in size, levels and surface molecules of sPMP derived in the absence of stimulus, thrombin activation and platelet disruption. Addition of sPMP resulted in platelet degranulation and P‐selectin redistribution to the membrane (P = 0·019) in a dose and time‐dependent manner. Blood clotting time decreased after addition of sPMP (P = 0·005), but was not affected by PD. Blocking P‐selectin (CD62P) in sPMP markedly reverted the effect on thrombus kinetics (P = 0·035). Exposure to sPMP stimulated monocyte expression of intercellular adhesion molecule‐1 (P < 0·03) and decreased monocyte interleukin‐6 receptor density (P < 0·01). These results implicate sPMP as a direct source of downstream platelet and monocyte activation. In pathological coronary artery disease conditions, higher levels of sPMP favour a prothrombotic state, partly through P‐selectin expression.

Left atrial remodelling in hypertrophic cardiomyopathy: relation with exercise capacity and biochemical markers of tissue strain and remodelling.

Background:  Left atrial remodelling, assessed as left atrial volume (LAV), has been proposed as a good marker of left ventricular diastolic dysfunction. The aim of this study was to analyse the influence of LAV on exercise performance in hypertrophic cardiomyopathy (HCM), and in a subset of subjects, assess the relation of LAV and exercise performance to four biomarkers of disease pathophysiology: matrix metalloproteinase‐2 (MMP‐2) and tissue inhibitor of matrix metalloproteinase‐1 (TIMP‐1) (as indices of tissue remodelling), N‐terminal portion of pro B‐type natriuretic peptide (NT‐pro‐BNP) (associated with ventricular dysfunction) and C‐reactive protein (CRP, an index of inflammation).