Platelet proteomics: from discovery to diagnosis

Introduction: Platelets are the smallest cells within the circulating blood with key roles in physiological hemostasis and pathological thrombosis regulated by the onset of activating/inhibiting processes via receptor responses and signaling cascades.

Areas covered: Proteomics as well as genomic approaches have been fundamental in identifying and quantifying potential targets for future diagnostic strategies in the prevention of bleeding and thrombosis, and uncovering the complexity of platelet functions in health and disease. In this article, we provide a critical overview on current functional tests used in diagnostics and the future perspectives for platelet proteomics in clinical applications.

Expert commentary: Proteomics represents a valuable tool for the identification of patients with diverse platelet associated defects. In-depth validation of identified biomarkers, e.g. receptors, signaling proteins, post-translational modifications, in large cohorts is decisive for translation into routine clinical diagnostics.     

Early signatures of bleeding and mortality in patients on left ventricular assist device support: novel methods for personalized risk-stratification

Background: Left ventricular assist devices (LVADs) provide support for patients with end-stage heart failure. The aims of this study were to determine whether baseline analysis and early trends in routine laboratory data, platelet activity, and thromboinflammatory biomarkers following LVAD implantation reveal trends that predict personalized risks of one-year gastrointestinal (GI) bleeding, stroke, pump thrombosis, drive-line infections and mortality in patients on LVAD support.

Methods: We performed an observational study at the University of Kentucky with 61 participants who underwent first-time LVAD implantation. Blood was collected at baseline and post-op days 0, 1, 3 and 6 as well as clinical follow-up. Demographics, clinical characteristics, one-year adverse events and routine laboratory data were collected from electronic medical records. Platelet function and plasma biomarkers were profiled.

Results: Evaluation of routine laboratory results revealed that sustained thrombocytopenia and increased mean platelet volume (MPV) were associated with development of GI bleeding and mortality. Platelet function at follow-up visit predicted one-year bleeding events. Thrombotic biomarker sCD40L strongly predicted one-year GI bleeding at baseline before implantation and within the first week following LVAD implant.

Conclusions: Early trends in routine bloodwork and platelet function may serve as novel signatures of patients at risk to experience adverse events.     

Platelet proteomics applied to the search for novel antiplatelet therapeutic targets

Introduction: Unwanted platelet activation is associated with numerous diseases, mainly thrombosis-related. In this context, proteomics has emerged as a novel tool with potential for drug target discovery and to scrutinize the effects of antiplatelet drugs.

Areas covered: The present review presents the main findings of platelet proteomic studies to date in the context of drug target discovery and perspectives for the future ahead. It includes data and evidences obtained from literature searches on PubMed as well as commentaries derived from the authors’ experience and opinions.

Expert commentary: Platelet proteomics applied to drug target discovery is a young field. Recent studies have shown promising data, especially in the context of coronary artery disease. However, challenges remain such as establishing definitive guidelines for blood collection and platelet isolation, essential to guarantee data reproducibility. Recent advances in quantitative platelet proteomics should lead to novel studies with higher clinical impact in the near future.     

Mutation in the beta3 subunit of Guanine nucleotide-binding protein (GNB3) gene is not associated with Type II diabetes mellitus risk: a case–control study of a North Indian population

Context: Genetics play a major role in development and pathophysiology of Type 2 diabetes mellitus (T2DM).

Objective: To asses the association of Guanine nucleotide-binding protein (GNB3) (C825T) gene's polymorphism with T2DM.

Materials and methods: A case–control study including 400 North Indians was performed using Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR-RFLP) approach to analyze genetic polymorphism.

Results: No significant difference was observed in genotype and allele frequencies of GNB3 gene on comparing cases with controls.

Discussion: Our study is in agreement with studies on Polish, Japanese, Hispanic-American and Danish populations who observed no significant association between GNB3 (C825T) polymorphism and T2DM.

Conclusion: GNB3 (C825T) polymorphism is not associated with T2DM.     

Beta-2-glycoprotein-1 and alpha-1-antitrypsin as urinary markers of renal cancer in von Hippel–Lindau patients

Context: Von Hippel–Lindau disease (VHLD) is a rare inherited neoplastic syndrome. Among all the VHLD-associated tumors, clear cell renal cell carcinoma (ccRCC) is the major cause of death.

Objective: The aim of this paper is the discovery of new non-invasive biomarker for the monitoring of VHLD patients.

Materials and methods: We compared the urinary proteome of VHLD patients, ccRCC patients and healthy volunteers.

Results: Among all differentially expressed proteins, alpha-1-antitrypsin (A1AT) and APOH (beta-2-glycoprotein-1) are strongly over-abundant only in the urine of VHLD patients with a history of ccRCC.

Discussion and conclusion: A1AT and APOH could be promising non-invasive biomarkers.     

Circulating adiponectin as a biomarker in renal cell carcinoma: a systematic review and meta-analysis

Context: Metabolic imbalance in renal cell carcinoma (RCC) can lead to abnormal adiponectin levels.

Objective: To evaluate circulating adiponectin as a detection or predictive marker for RCC.

Methods: A comprehensive literature search and meta-analysis was performed on studies reporting circulating adiponectin levels and RCC. The meta-analysis was performed using RevMan.

Results: Seven studies compared the circulating adiponection levels between RCC cases and controls. Adiponectin level was significantly lower in RCC cases compared to controls at pre-diagnosis and pre-operative time-points. RCC stage, grade and subtype did not affect adiponectin levels.

Conclusion: Low circulating adiponectin could be a predictive or risk factor for RCC.     

Infant sitting postural control appears robust across changes in surface context

Aim of the study: Independent sitting requires the control of the involved body segments over the base of support using information obtained from the three sensory systems (visual, vestibular, and somatosensory). The contribution of somatosensory information in infant sitting has not been explored. To address this gap, we altered the context of the sitting support surface and examined the infants’ immediate postural responses.

Materials and methods: Ten 7-month-old typically developing infants sat on compliant and firm surfaces in one session. Spatial, frequency, and temporal measures of postural control were obtained using center of pressure data.

Results Our results suggest that infants’ postural sway is not immediately affected by the different types of foam surface while sitting.

Conclusions: It seems that mature sitter infants are able to adapt to different environmental constraints by disregarding the distorted somatosensory information from the support surface and relying more on their remaining senses (visual and vestibular) to control their sitting posture.     

Riboflavin as an independent and accurate biomarker for adherence in a randomized double-blind and placebo-controlled clinical trial

Background: Medication adherence is critical for success of clinical trials.

Objective: To assess oral riboflavin is an adherence marker.

Methods: Riboflavin was incorporated into active treatment and placebo pills for a clinical trial lasting for 2 years.

Results: The accuracy (area under the receiver operating curve) of urinary riboflavin was 0.91 as a binary classifier of adherence, and was similar or better than for two active study ingredients daidzein (0.92) and genistein (0.87) (all p?<?0.0001). Decreased adherence over time was similar in the two study groups.

Conclusion: Riboflavin is an accurate and useful biomarker for study pill ingestion.     

Validation of a protein panel for the noninvasive detection of recurrent non-muscle invasive bladder cancer

Context: About 50–70% of patients with non-muscle invasive bladder cancer (NMIBC) experience relapse of disease.

Objective: To establish a panel of protein biomarkers incorporated in a multiplexed microarray (BCa chip) and a classifier for diagnosing recurrent NMIBC.

Materials and methods: Urine samples from 45 patients were tested. Diagnostic performance was evaluated by receiver operating characteristic (ROC) analysis.

Results: A multi biomarker panel (ECadh, IL8, MMP9, EN2, VEGF, past recurrences, BCG therapies and stage at diagnosis) was identified yielding an area under the curve of 0.96.

Discussion and conclusion: This biomarker panel represents a potential diagnostic tool for noninvasive diagnosis of recurrent NMIBC.     

Circulating levels of apoptotic markers and oxidative stress parameters in women with polycystic ovary syndrome: a case-controlled descriptive study

Context: Apoptotic dysregulation plays a role in the pathogenesis of polycystic ovary syndrome (PCOS).

Objective: To evaluate circulatory apoptotic markers and oxidative stress in patients with PCOS.

Materials and methods: Forty-four women with PCOS, and 44 healthy women as controls were enrolled in the study. Oxidative stress parameters and caspases levels were measured in serum.

Results: The caspase 9 level was significantly lower and related with oxidant status in patients with PCOS, while the circulating levels of caspases 3 and 7 were statistically similar in both groups.

Discussion: This study is the first report demonstrating the circulating levels of apoptotic markers and their relationship with oxidant status in PCOS.

Conclusion: The circulating caspase 9 and oxidant status might contribute to apoptotic dysregulation in PCOS.     

Inhibitory effect of Tanshinone IIA on inverted formin-2 protects HaCaT cells against oxidative injury via regulating mitochondrial stress

Context: Epidermal cells play an important role in regulating the regeneration of skin after burns and wounds.

Objective: The aim of our study is to explore the role of Tanshinone IIA (Tan IIA) in the apoptosis of epidermal HaCaT cells induced by H₂O₂, with a focus on mitochondrial homeostasis and inverted formin-2 (INF2).

Materials and methods: Cellular viability was determined using the MTT assay, TUNEL staining, western blot analysis and LDH release assay. Adenovirus-loaded INF2 was transfected into HaCaT cells to overexpress INF2 in the presence of Tan IIA treatment. Mitochondrial function was determined using JC-1 staining, mitochondrial ROS staining, immunofluorescence and western blotting.

Results: Oxidative stress promoted the death of HaCaT cells and this effect could be reversed by Tan IIA. At the molecular levels, Tan IIA treatment sustained mitochondrial energy metabolism, repressed mitochondrial ROS generation, stabilized mitochondrial potential, and blocked the mitochondrial apoptotic pathway. Furthermore, we demonstrated that Tan IIA modulated mitochondrial homeostasis via affecting INF2-related mitochondrial stress. Overexpression of INF2 could abolish the protective effects of Tan IIA on HaCaT cells viability and mitochondrial function. Besides, we also reported that Tan IIA regulated INF2 expression via the ERK pathway; inhibition of this pathway abrogated the beneficial effects of Tan IIA on HaCaT cells survival and mitochondrial homeostasis.

Conclusions: Overall, our results indicated that oxidative stress-mediated HaCaT cells apoptosis could be reversed by Tan IIA treatment via reducing INF2-related mitochondrial stress in a manner dependent on the ERK signaling pathway.     

Sleep-disordered breathing is associated with higher carboxymethyllysine level in elderly women but not elderly men in the cardiovascular health study

Context: Carboxymethyl-lysine (CML) results from oxidative stress and has been linked to cardiovascular disease.

Objective: The objective of this study is to investigate the association between sleep-disordered breathing (SDB) – a source of oxidative stress – and CML.

Materials and methods: About 1002 participants in the Cardiovascular Health Study (CHS) were studied.

Results: Women with SDB had significantly higher CML concentration compared with those without SDB (OR?=?1.63, 95%CI?=?1.03–2.58, p?=?0.04). The association was not significant among men.

Discussion: SDB was associated with CML concentration among elderly women but not men in the Cardiovascular Health Study.

Conclusion: Accumulation of CML may be an adverse health consequence of SDB     

Reliability of two-point discrimination thresholds using a 4-2-1 stepping algorithm

Purpose: A 4-2-1 stepping algorithm reliably captures light touch thresholds but has not been used to assess two-point discrimination (TPD) thresholds. Therefore, the purpose of this investigation was to determine the intra- and inter-rater reliability of a 4-2-1 stepping algorithm at determining TPD thresholds.

Materials and methods: Fifteen healthy, physically active young adults were assessed twice over a 1-week period using digital calipers and a 4-2-1 stepping algorithm. TPD thresholds were assessed by an expert and a novice examiner at each time point. Reliability was assessed on the plantar surface of the foot at the head of the first and base of the fifth metatarsal.

Results: Three intra-rater intraclass correlation coefficient (ICC) values exceeded 0.75 and were interpreted as good. The inter-rater reliability was good with ICC values ranging from 0.76 to 0.93 at both sites during both test sessions.

Conclusions: The 4-2-1 stepping algorithm demonstrates good intra- and inter-tester reliability at determining TPD thresholds on the plantar surface of the foot at the head of the first and base of the fifth metatarsal in young healthy adults.     

Differential expression of circulating vascular cell adhesion molecule-1 in subjects with coronary artery disease and cardiac syndrome X without known diabetes mellitus

Context: Inflammation is one of the mechanisms underlying cardiac syndrome X (CSX).

Objectives: Few studies have compared the expression of inflammatory or adhesion molecules between coronary artery disease (CAD) versus CSX.

Materials and methods: Ninety-two CSX and 145 CAD subjects without known diabetes mellitus underwent coronary angiogram for angina.

Results: Vascular cell adhesion molecule (VCAM)-1 (median, 507 versus 431?ng/ml, p?=?0.001) was significantly higher in the CAD group. In the binary regression, VCAM-1 was a significant differential factor for CAD versus CSX.

Discussion and conclusion: Adhesion molecules might be implicated in the differential expression of macro versus microvascular coronary disease.

Trial registration number: NCT01198730 at https://clinicaltrials.gov     

Propeptide big-endothelin,N-terminal-pro brain natriuretic peptide and mortality. The Ludwigshafen risk and cardiovascular health (LURIC) study

Context: The endothelin system (Big-ET-1) is a key regulator in cardiovascular (CV) disease and congestive heart failure (CHF).

Objectives: We have examined the incremental value of Big-ET-1 in predicting total and CV mortality next to the well-established CV risk marker N-Terminal Pro-B-Type Natriuretic Peptide (NT-proBNP).

Methods: Big-ET-1 and NT-proBNP were determined in 2829 participants referred for coronary angiography (follow-up 9.9 years).

Results: Big-ET-1 is an independent predictor of total, CV mortality and death due to CHF.

Discussion: The conjunct use of Big-ET-1 and NT-proBNP improves the risk stratification of patients with intermediate to high risk of CV death and CHF.

Conclusions: Big-ET-1improves risk stratification in patients referred for coronary angiography.     

Biomarker discovery for drug-induced phospholipidosis: phenylacetylglycine to hippuric acid ratio in urine and plasma as potential markers

Context: Drug-induced phospholipidosis is one of the significant concerns in drug development, especially in safety assessment and noninvasive diagnostic tool is highly desirable.

Objective: The objective of this study is to explored novel biomarkers for phospholipidosis using a metabolomic approach.

Method: NMR spectrometry and LC/MS/MS analyses were applied to urine and plasma of rats administrated cationic amphiphilic drugs.

Results: The phenylacetylglycine to hippuric acid ratio in plasma was increased in time and dose-dependent manners; and it was well correlated with histopathological observation.

Conclusion: The plasma phenylacetylglycine to hippuric acid ratio is a potential marker in monitoring drug-induced phospholipidosis.     

Matrix metalloproteinase multiplex screening identifies increased MMP-2 urine concentrations in women predicted to develop preeclampsia

Background: Preeclampsia, a pregnancy disorder characterized by hypertension and proteinuria, represents the leading cause of fetal and maternal morbidity and mortality in developing countries. The identification of novel and accurate biomarkers that are predictive of preeclampsia is necessary to improve the prognosis of patients with preeclampsia.

Objective: The objective of this study is to evaluate the usefulness of nine urinary metalloproteinases to predict the risk of preeclampsia development.

Methods: MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12 and MMP-13 were analyzed in urine (early-pregnancy) from 17 women predicted to develop preeclampsia and 48 controls using the Bio-Plex Pro-Human MMP panel (Bio-Rad, Hercules, CA).

Results: Urinary MMP-2 showed differences between groups which allowed us to calculate an increased risk for PE development of up to 20 times among the study population.

Conclusion: Increased urinary concentration of MMP-2 at 12 and 16 weeks of gestation predicted an increased risk of developing preeclampsia in the study population.     

Release kinetics of high-sensitivity cardiac troponins I and T and troponin T upstream open reading frame peptide (TnTuORF) in clinically induced acute myocardial infarction

Context: Troponin T upstream open reading frame peptide (TnTuORF) may be useful as a novel biomarker in acute cardiac syndromes.

Objective: The study examined the early release kinetics of TnTuORF.

Materials and methods: We analyzed the time course of the release of cardiac troponins I and T and TnTuORF in patients (n?=?31) with hypertrophic obstructive cardiomyopathy undergoing transcoronary ablation of septal hypertrophy (TASH).

Results: Fifteen minutes after TASH, the levels of both troponins increased significantly (cTnT median: 18?ng/L versus 27?ng/L; cTnI median: 15?ng/L versus 25?ng/L). TnTuORF showed no variation.

Discussion: We observed a significantly greater increase in cTnI compared with cTnT.

Conclusion: Our results demonstrate that troponin assays allow early detection of myocardial injury, whereas TnTuORF levels remain unchanged in this setting.     

Soluble CD40 ligand in haemodialysis patients: survival impact and cardiovascular prognostic role

Context: Soluble CD40 ligand (sCD40l) can predict cardiovascular events (CVE) and mortality in haemodialysis (HD) patients (short-, medium-term follow-up studies).

Objective: To evaluate the relationship between sCD40l and survival, CVE and mortality in HD patients on long-term follow-up.

Methods: We registered 46?HD patients’ baseline characteristics, mortality and CVE for 108 months.

Results: SCD40l correlated positively with C-reactive protein, was higher in survivors, but had no impact on survival and was not predictive for CVE or CV mortality.

Conclusion: The levels of sCD40l have no influence on survival or CVE and mortality in HD patients in a long-term follow-up.     

Blood markers of inflammation and endothelial dysfunction in cardioembolic stroke: systematic review and meta-analysis

Context: Various processes including inflammation and endothelial dysfunction have been implicated in the pathogenesis of cardioembolic (CE) strokes.

Objective: To review the evidence and investigate the association between immune-inflammatory biomarkers and CE strokes versus other stroke subtypes.

Methods: We systematically reviewed the literature (sources: MEDLINE, web-based register http://stroke-biomarkers.com, reference lists) with quality assessment and meta-analysis of selected articles.

Results: The most consistent association was found between C-reactive protein (CRP) and CE strokes when compared to other stroke subtypes (standardized mean difference 0.223 (0.116, 0.343); p?<?0.001)

Conclusions: Our findings confirm a possible association between selected inflammatory biomarkers and CE stroke.