Agneta Siegbahn

Prognostic Influence of Increased Fibrinogen and C-Reactive Protein Levels in Unstable Coronary Artery Disease

BACKGROUND The prognostic influences of fibrinogen and C-reactive protein levels and their relations to myocardial damage in unstable coronary artery syndromes have not been well described. METHODS AND RESULTS Fibrinogen and C-reactive protein were determined at inclusion and related to outcome after 5 months in 965 patients with unstable angina or non-Q-wave myocardial infarction randomized to 5 weeks with low-molecular-weight heparin or placebo. The probabilities of death were 1.6%, 4.6%, and 6.9% (P=.005) and the probabilities of death and/or myocardial infarction were 9.3%, 14.2%, and 19.1% (P=.002), respectively, in patients stratified by tertiles of fibrinogen (< 3.38, 3.38 to 3.99, and > or = 4.0 g/L). The probabilities of death were 2.2%, 3.6%, and 7.5% (P=.003) after stratification of patient data by tertiles of C-reactive protein level (< 2, 2 to 10, and > 10 mg/L). In logistic multiple regression analysis, increased fibrinogen levels were independently associated with the incidence of death and/or myocardial infarction (P=.013), and elevated C-reactive protein level was associated with the incidence of death (P=.012). The increased relative risk of subsequent death or myocardial infarction in individuals with an elevated fibrinogen level was consistent in most subgroups evaluated; although significantly so only in patients with signs of myocardial damage. CONCLUSIONS Increased levels of both fibrinogen and C-reactive protein are associated with a worse outcome in patients with unstable coronary artery disease. The increased risk associated with elevated fibrinogen levels is independent of, and additive to, the prognostic influence of myocardial damage.

Vitamin K antagonists in heart disease: Current status and perspectives (Section III)

Oral anticoagulants are a mainstay of cardiovascular therapy, and for over 60 years vitamin K antagonists (VKAs) were the only available agents for long-term use. VKAs interfere with the cyclic inter-conversion of vitamin K and its 2,3 epoxide, thus inhibiting γ-carboxylation of glutamate residues at the amino-termini of vitamin K-dependent proteins, including the coagulation factors (F) II (prothrombin), VII, IX and X, as well as of the anticoagulant proteins C, S and Z. The overall effect of such interference is a dose-dependent anticoagulant effect, which has been therapeutically exploited in heart disease since the early 1950s. In this position paper, we review the mechanisms of action, pharmacological properties and side effects of VKAs, which are used in the management of cardiovascular diseases, including coronary heart disease (where their use is limited), stroke prevention in atrial fibrillation, heart valves and/or chronic heart failure. Using an evidence-based approach, we describe the results of completed clinical trials, highlight areas of uncertainty, and recommend therapeutic options for specific disorders. Although VKAs are being increasingly replaced in most patients with non-valvular atrial fibrillation by the new oral anticoagulants, which target either thrombin or FXa, the VKAs remain the agents of choice for patients with atrial fibrillation in the setting of rheumatic valvular disease and for those with mechanical heart valves.

Non-vitamin K antagonist oral anticoagulants (NOACs): No longer new or novel.

Note: The editorial process for this paper was fully handled by Prof Christian Weber, Editor in Chief.

Sensitive Plasma Protein Analysis by Microparticle-based Proximity Ligation Assays

Detection of proteins released in the bloodstream from tissues damaged by disease can promote early detection of pathological conditions, differential diagnostics, and follow-up of therapy. Despite these prospects and a plethora of candidate biomarkers, efforts in recent years to establish new protein diagnostic assays have met with limited success. One important limiting factor has been the challenge of detecting proteins present at trace levels in complex bodily fluids. To achieve robust, sensitive, and specific detection, we have developed a microparticle-based solid-phase proximity ligation assay, dependent on simultaneous recognition of target proteins by three antibody molecules for added specificity. After capture on a microparticle, solid-phase pairs of proximity probes are added followed by washes, enabling detection and identification of rare protein molecules in blood while consuming small amounts of sample. We demonstrate that single polyclonal antibody preparations raised against target proteins of interest can be readily used to establish assays where detection depends on target recognition by three individual antibody molecules, recognizing separate epitopes. The assay was compared with state-of-the-art sandwich ELISAs for detection of vascular endothelial growth factor, interleukin-8 and interleukin-6, and it was found to be superior both with regard to dynamic range and minimal numbers of molecules detected. Furthermore, the assays exhibited excellent performance in undiluted plasma and serum as well as in whole blood, producing comparable results for nine different antigens. We thus show that solid-phase proximity ligation assay is suitable for validation of a variety of protein biomarkers over broad dynamic ranges in clinical samples.

Use of a proximity extension assay proteomics chip to discover new biomarkers for human atherosclerosis

BACKGROUND AND AIMS We used a proteomics array to simultaneously measure multiple proteins that have been suggested to be associated with atherosclerosis and related them to plaque prevalence in carotid arteries in a human population-based study. METHODS In the Prospective Study of the Vasculature in Uppsala Seniors (PIVUS; n = 931, 50% women, all aged 70 years), the number of carotid arteries with plaques was recorded by ultrasound. Levels of 82 proteins were assessed in plasma by a proximity extension assay (Proseek Multiplex CVD, Olink Bioscience, Uppsala, Sweden) and related to carotid measures in a regression framework. RESULTS Following adjustment for multiple testing with Bonferroni correction, seven of the proteins were significantly related to the number of carotid arteries affected by plaques in sex-adjusted models (osteoprotegrin, T-cell immunoglobulin and mucin domain (TIM)-1, growth/differentiation factor 15 (GDF-15), matrix metalloprotease-12 (MMP-12), renin, tumor necrosis factor ligand superfamily member 14 (TNFSF14) and growth hormone). Of these, renin (odds ratio [OR], 1.30; 95% confidence interval [CI], 1.13-1.49 per standard deviation increase), growth hormone (OR, 1.24; 95% CI, 1.08-1.43), osteoprotegerin (OR, 1.22; 95% CI, 1.05-1.43) and TNFSF14 (OR, 1.17; 95% CI, 1.01-1.35) were related to plaque prevalence independently of each other and traditional cardiovascular risk factors. CONCLUSION A novel targeted proteomics approach using the proximity extension technique discovered several new associations of candidate proteins with carotid artery plaque prevalence in a large human sample.

Microparticles during long-term follow-up after acute myocardial infarction

Microparticles (MPs) are formed from platelets (PMPs), endothelial cells (EMPs) and monocytes (MMPs), and in acute myocardial infarction (MI), there is an increase of MPs in the culprit artery. In this study MPs were evaluated in whole blood in 105 patients with MI at five time-points during a two-year follow-up (FU). Patients with non-ST-elevated MI had higher concentrations of CD41+MPs compared to ST-elevated MI patients (p=0.024). The concentrations of PMPs in whole blood increased during the time period (p<0.001), but no significant change over time was found for EMPs and MMPs. CD62P+MP counts were higher in MI patients with diabetes (p=0.020), and patients with hypertension had increased levels of CD14+MPs (p=0.004). The amount of CD62P+TF+MPs increased significantly during FU (p<0.001). Patients with atherosclerosis in three arterial beds, i. e. coronary, carotid and peripheral arteries, had lower concentrations of CD62P+TF+MPs (p=0.035) and CD144+TF+MPs (p=0.004) compared to patients with atherosclerosis in one or two arterial beds. Higher concentrations of CD62P+MPs early after MI were associated with an increased risk of cardiovascular events during FU, hazard ratio 3.32 (95 %CI1.20-9.31). Only small variations in PMP, EMP and MMP concentrations were found during long-term FU after MI and their levels seem to reflect the underlying cardiovascular disease rather than the acute MI. PMPs expressing P-selectin might be a promising biomarker for predicting future cardiovascular events, but further studies are needed to confirm these results.

Decreased levels of stem cell factor in subjects with incident coronary events.

It has been proposed that vascular progenitor cells play an important role in vascular repair, but their possible clinical importance in cardiovascular disease has not been fully characterized. Vascular endothelial growth factor A, placental growth factor and stem cell factor (SCF) are three growth factors that are important in recruiting vascular progenitor cells. In this study, we investigated the association between the plasma levels of these growth factors and incident coronary events (CEs).

Proteomic Biomarkers for Incident Aortic Stenosis Requiring Valvular Replacement

Background: Aortic valve stenosis (AS) is the most common indication for cardiac valve surgery; untreated AS is linked to high mortality. The etiological background of AS is unknown. Previous human studies were typically based on case-control studies. Biomarkers identified in prospective studies could lead to novel mechanistic insights. Methods: Within a large population survey with blood samples obtained at baseline, 334 patients were identified who later underwent surgery for AS (median age [interquartile range], 59.9 [10.4] years at survey and 68.3 [12.7] at surgery; 48% female). For each case, 2 matched referents were allocated. Plasma was analyzed with the multiplex proximity extension assay for screening of 92 cardiovascular candidate proteins. Conditional logistic regression models were used to assess associations between each protein and AS, with correction for multiple testing. A separate set of 106 additional cases with 212 matched referents was used in a validation study. Results: Six proteins (growth differentiation factor 15, galectin-4, von Willebrand factor, interleukin 17 receptor A, transferrin receptor protein 1, and proprotein convertase subtilisin/kexin type 9) were associated with case status in the discovery cohort; odds ratios ranged from 1.25 to 1.37 per SD increase in the protein signal. Adjusting the multivariable models for classical cardiovascular risk factors at baseline yielded similar results. Subanalyses of case-referent triplets (n=133) who showed no visible coronary artery disease at the time of surgery in the index person supported associations between AS and growth differentiation factor 15 (odds ratio, 1.40; 95% confidence interval, 1.10-1.78) and galectin-4 (odds ratio, 1.27; 95% confidence interval, 1.02-1.59), but these associations were attenuated after excluding individuals who donated blood samples within 5 years before surgery. In triplets (n=201), which included index individuals with concurrent coronary artery disease at the time of surgery, all 6 proteins were robustly associated with case status in all sensitivity analyses. In the validation study, the association of all but 1 (interleukin 17 receptor A) of these proteins were replicated in patients with AS with concurrent coronary artery disease but not in patients with AS without coronary artery disease. Conclusions: We provide evidence that 5 proteins were altered years before AS surgery and that the associations seem to be driven by concurrent atherosclerotic disease.

Cross-talk between the Tissue Factor/coagulation factor VIIa complex and the tyrosine kinase receptor EphA2 in cancer

BackgroundTissue Factor (TF) forms a proteolytically active complex together with coagulation factor VIIa (FVIIa) and functions as the trigger of blood coagulation or alternatively activates cell signaling. We recently described that EphA2 of the Eph tyrosine kinase receptor family is cleaved directly by the TF/FVIIa complex. The aim of the present study was to further characterize the cross-talk between TF/FVIIa and EphA2 using in vitro model systems and human cancer specimens.MethodsCleavage and phosphorylation of EphA2 was studied by Western blot. Subcellular localization of TF and EphA2 was investigated by a proximity ligation assay and confocal microscopy. Phalloidin staining of the actin cytoskeleton was used to study cell rounding and retraction fiber formation. Expression of TF and EphA2 in human colorectal cancer specimens was examined by immunohistochemistry.ResultsTF and EphA2 co-localized constitutively in MDA-MB-231 cells, and addition of FVIIa resulted in cleavage of EphA2 by a PAR2-independent mechanism. Overexpression of TF in U251 glioblastoma cells lead to co-localization with EphA2 at the leading edge and FVIIa-dependent cleavage of EphA2. FVIIa potentiated ephrin-A1-induced cell rounding and retraction fiber formation in MDA-MB-231 cells through a RhoA/ROCK-dependent pathway that did not require PAR2-activation. TF and EphA2 were expressed in colorectal cancer specimens, and were significantly correlated.ConclusionsThese results suggest that TF/FVIIa-EphA2 cross-talk might potentiate ligand-dependent EphA2 signaling in human cancers, and provide initial evidence that it is possible for this interaction to occur in vivo.

Sensitive and Specific Detection of Platelet-Derived and Tissue Factor–Positive Extracellular Vesicles in Plasma Using Solid-Phase Proximity Ligation Assay

Extracellular vesicles (EVs) derived from blood cells are promising biomarkers for various diseases. However, they are difficult to measure accurately in plasma due to their small size. Here, we demonstrate that platelet-derived EVs in plasma can be measured using solid-phase proximity ligation assay with high sensitivity and specificity using very small sample volume of biological materials. The results correlate well with high-sensitivity flow cytometry with the difference that the smallest EVs are detected. Briefly, the EVs are first captured on a solid phase, using lactadherin binding, and detection requires recognition with two antibodies followed by qPCR. The assay, using cholera toxin subunit-B or lactadherin as capture agents, also allowed detection of the more rare population of tissue factor (TF)-positive EVs at a concentration similar to sensitive TF activity assays. Thus, this assay can detect different types of EVs with high specificity and sensitivity, and has the potential to be an attractive alternative to flow cytometric analysis of preclinical and clinical samples. Improved techniques for measuring EVs in plasma will hopefully contribute to the understanding of their role in several diseases.