Oliver Pötz

Influence of platelet count on the expression of platelet collagen receptor glycoprotein VI (GPVI) in patients with acute coronary syndrome

Platelets play a key role in the development of an acute coronary syndrome (ACS) and contribute to cardiovascular events. Platelet collagen receptor glycoprotein VI (GPVI) contributes significantly to platelet adhesion and thrombus formation in ACS. We consecutively investigated both the platelet count and the platelet surface expression of GPVI in 843 patients with a symptomatic coronary artery disease verified by coronary angiography. Four hundred fourteen patients presented with stable angina pectoris and 429 patients with ACS. Platelet surface expression of GPVI and CD62P was determined by flow cytometry and platelet count with a coulter counter, plasmatic soluble GPVI was measured by ELISA. Platelet GPVI expression in patients with ACS was compared to platelet count. Patients with ACS showed significantly elevated GPVI expression levels in the first and second quartiles of platelet count compared to patients with higher platelet count [mean fluorescence intensity (MFI) +/- standard deviation): 1(st) vs. 4(th): 20.44 +/- 6.1 vs. 18.62 +/- 3.7; p=0.012; 2(nd)vs.3(rd):21.2+/-8.5vs.18.76+/-3.7;P=0.03; 2(nd)vs.4(th): 21.2+/-8.5vs.18.62+/-3.7;P=0.004], which was paralleled in trend for the CD62P expression [MFI: 1(st) vs. 4(th): 11.2 +/- 6.8 vs. 12.3 +/- 9; p=0.057; 2(nd) vs. 3(rd): 16.3 +/- 16 vs.12.7 +/- 5.3; p=0.138; 2(nd) vs. 4(th): 16.3 +/- 16 vs.11 +/- 4.4; p=0.043]. In a subgroup of 48 patients with ACS, determination of soluble GPVI showed similar results [plasma GPVI (ng/ml): 1(st)vs.4(th): 1.6 +/- 0.6 vs. 1.2 +/- 0.4; p=0.046; 1(st) vs. 3(rd): 1.6 +/- 0.6 vs. 1.1 +/- 0.5; p=0.038; 2(nd) vs. 3(rd): 1.9 +/- 0.8 vs. 1.1 +/- 0.5; p=0.04; 2(nd) vs. 4(th): 1.9 +/- 0.8 vs. 1.2 +/- 0.4; p=0.056]. Thus, a lower platelet count comes along with a higher GPVI surface expression and plasma concentration in patients with ACS, which potentially reflects increased activation and enhanced recruitment of platelets to the site of vascular injury.

Concurrent epigenetic silencing of wnt/β-catenin pathway inhibitor genes in B cell chronic lymphocytic leukaemia

BackgroundThe Wnt/β-catenin signalling is aberrantly activated in primary B cell chronic lymphocytic leukaemia (CLL). Epigenetic silencing of pathway inhibitor genes may be a mechanism for its activation. In this study, we investigated systematically and quantitatively the methylation status of 12 Wnt/β-catenin pathway inhibitor genes – CDH1, DACT1, DKK1, DKK2, DKK3, DKK4, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5 and WIF1 – in the cell lines EHEB and MEC-1 as well as patient samples.MethodsQuantification of DNA methylation was performed by means of bisulphite pyrosequencing and confirmed by bisulphite Sanger sequencing. Gene expression was analysed by qPCR using GAPDH as internal control. E-cadherin and β-catenin protein quantification was carried out by microsphere-based immunoassays. Methylation differences observed between the patient and control groups were tested using generalised least squares models.ResultsFor 10 genes, a higher methylation level was observed in tumour material. Only DKK4 exhibited similarly high methylation levels in both tumour and normal specimens, while DACT1 was always essentially unmethylated. However, also for these inhibitors, treatment of cells with the demethylating agent 5-aza-2´-deoxycytidine resulted in an induction of their expression, as shown by quantitative PCR, suggesting an indirect epigenetic control of activity. While the degree of demethylation and its transcriptional consequences differed between the genes, there was an overall high correlation of demethylation and increased activity. Protein expression studies revealed that no constitutive Wnt/β-catenin signalling occurred in the cell lines, which is in discrepancy with results from primary CLL. However, treatment with 5-aza-2´-deoxycytidine caused accumulation of β-catenin. Simultaneously, E-cadherin expression was strongly induced, leading to the formation of a complex with β-catenin and thus demonstrating its epigenetically regulated inhibition effect.ConclusionsThe results suggest an epigenetic silencing mechanism of the Wnt/β-catenin pathway inhibitor genes in CLL. Hypermethylation and silencing of functionally related genes may not be completely stochastic but result from the tumour epigenome reprogramming orchestrated by Polycomb-group repressive complexes. The data are of interest in the context of epigenetic-based therapy.

Bulk cell density and Wnt/TGFbeta signalling regulate mesendodermal patterning of human pluripotent stem cells.

In vitro differentiation of human pluripotent stem cells (hPSCs) recapitulates early aspects of human embryogenesis, but the underlying processes are poorly understood and controlled. Here we show that modulating the bulk cell density (BCD: cell number per culture volume) deterministically alters anteroposterior patterning of primitive streak (PS)-like priming. The BCD in conjunction with the chemical WNT pathway activator CHIR99021 results in distinct paracrine microenvironments codifying hPSCs towards definitive endoderm, precardiac or presomitic mesoderm within the first 24 h of differentiation, respectively. Global gene expression and secretome analysis reveals that TGFß superfamily members, antagonist of Nodal signalling LEFTY1 and CER1, are paracrine determinants restricting PS progression. These data result in a tangible model disclosing how hPSC-released factors deflect CHIR99021-induced lineage commitment over time. By demonstrating a decisive, functional role of the BCD, we show its utility as a method to control lineage-specific differentiation. Furthermore, these findings have profound consequences for inter-experimental comparability, reproducibility, bioprocess optimization and scale-up.

Sandwich Immunoassay for Soluble Glycoprotein VI in Patients with Symptomatic Coronary Artery Disease

BACKGROUND Platelet glycoprotein VI (pGPVI) expression is increased in acute coronary syndrome (ACS), reflecting platelet activation. There is no reliable method available to measure pGPVI. Our aim was to develop a bead-based sandwich immunoassay to measure soluble GPVI (sGPVI). METHODS Based on antibodies for sGPVI developed earlier, we established and validated a bead-based sandwich immunoassay in 2438 consecutive patients with stable angina pectoris (SAP; n = 1371), non-ST-elevation myocardial infarction (NSTEMI; n = 724), and ST-elevation MI (STEMI; n = 343). In a subgroup (n = 1011), we measured surface expression of pGPVI using flow cytometry. RESULTS The assay revealed a working range of 8-500 ng/L. Intra- and interassay imprecision was <7% and <14%, respectively. Patients with NSTEMI and STEMI showed significantly lower mean sGPVI concentrations than patients with SAP [mean (SD), 8.4 (3.6) μg/L and 8.6 (4.1) μg/L vs 9.8 (4.8) μg/L; P = 0.002], whereas subgroup analysis revealed significantly enhanced pGPVI in NSTEMI (n = 276) and STEMI (n = 80) patients compared with SAP (n = 655) [mean fluorescence intensity (SD), 21.2 (8.1) and 19.8 (6.8) vs 18.5 (7.7); P = 0.002 and P = 0.018]. pGPVI and sGPVI were inversely correlated (r = -0.076; P = 0.023). Area under the ROC curve was 0.716, 95% CI 0.681-0.751, for sGPVI, distinguishing patients with SAP from those with ACS, and was superior (P = 0.044) to the curve of subgroup analysis for pGPVI (0.624, 95% CI 0.586-0.662). sGPVI (P = 0.023) and pGPVI (P = 0.028) had better association with the development of ACS than troponin I (P = 0.055) in the very early stage of disease, based on logistic regression analysis. CONCLUSIONS This sandwich immunoassay reliably measures sGPVI and may help to identify patients with ACS earlier than other laboratory markers.

Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition

Adenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein-coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin αIIbβ3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (1) critical hubs in platelet signaling toward aggregation and (2) novel molecular targets for antiplatelet treatment strategies. We applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution. Furthermore, to mimic the antagonistic efficacy of endothelial-derived prostacyclin, we determined how Iloprost reverses ADP-mediated signaling events. We provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal reorganization, but also in less well-understood pathways, involving ubiquitin ligases and GTPase exchange factors/GTPase-activating proteins (GEF/GAP). Our data demonstrate that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes. For a set of phosphorylation sites (eg, PDE3ASer312, CALDAG-GEFISer587, ENSASer109), we demonstrate an inverse regulation by ADP and Iloprost, suggesting that these are central modulators of platelet homeostasis. This study demonstrates an extensive spectrum of human platelet protein phosphorylation in response to ADP and Iloprost, which inversely overlap and represent major activating and inhibitory pathways.

Snapshots of Protein Dynamics and Post-translational Modifications In One Experiment—β-Catenin and Its Functions

β-catenin plays multiple roles in the canonical Wnt signaling pathway and in cell-cell adhesion complexes. In addition, β-catenin is a proto-oncogene and activating β-catenin mutations are relevant in the genesis of colorectal, hepatocellular and other common cancers. Different functions of β-catenin as transcriptional co-activator or cell adhesion molecule are orchestrated by changes in concentration and phosphorylation as well as its ability to complex with proteins such as cadherins or transcription factors. Detailed quantitative and time-resolved analysis of β-catenin, based on the evaluation of the changes in the Wnt pathway, enable greater insights into health- and disease-related β-catenin function. The present paper describes a novel suspension bead array assay panel for β-catenin, which requires minimal amounts of sample and is able to relatively quantify total β-catenin, the extent of phosphorylation at multiple sites and the ratio of complexed and free β-catenin. This is the first study to combine three biochemical methods—sandwich immunoassay, co-immunoprecipitation, and protein-protein interaction assay—in one suspension bead assay panel. The assay was used to measure changes in the concentration of eight different β-catenin forms in HEK293 cells in a time-resolved manner. In contrast to the general consensus, our study demonstrates an increase in β-catenin phosphorylated at Ser-45 upon treatment of cells with rWnt3a or a GSK3 inhibition; we also link C-terminal phosphorylation of β-catenin on Ser-552 and Ser-675 with canonical Wnt signaling.

Release of Matrix Metalloproteinase-8 During Physiological Trafficking and Induced Mobilization of Human Hematopoietic Stem Cells

Previous studies indicate that the release of proteases, including the gelatinase matrix metalloproteinase (MMP)-9, from mature granulocytes plays a crucial role in cytokine-induced hematopoietic stem and progenitor cell (HSPC) mobilization. However, studies with MMP-9-deficient mice revealed that HSPC mobilization was normal in these animals, suggesting that additional proteases must be active at clinically relevant cytokine concentrations. In the present study, we provide evidence that the collagenase MMP-8 is involved in stem cell mobilization. A rapid release of MMP-8 from isolated neutrophil granulocytes can be observed during an in vitro culture. During granulocyte colony-stimulating factor-induced HSPC mobilization, highly elevated serum concentrations of MMP-8 were observed on days 4 to 6 of the mobilization regimen, concomitantly with elevated MMP-9 serum levels and higher numbers of circulating CD34(+) cells. Elevated serum concentrations of both proteases were also found in umbilical cord blood serum. In functional assays, adhesion of HSPC to osteoblasts as an essential component of the endosteal stem cell niche is negatively influenced by MMP-8. The chemokine CXCL12, which is critically involved in stem cell trafficking, can be proteolytically processed by MMP-8 treatment. This degradation has a strong inhibitory influence on HSPC migration. Taken together, our data strongly suggest that MMP-8 can be directly involved in hematopoietic stem cell mobilization and trafficking.

A dual array-based approach to assess the abundance and posttranslational modification state of signaling proteins.

Combining two strategies enables simultaneous quantification of multiple signaling proteins. A system-wide analysis of cell signaling requires detecting and quantifying many different proteins and their posttranslational modification states in the same cellular sample. Here, we present Protocols for two miniaturized, array-based methods, one of which provides detailed information on a central signaling protein and the other of which provides a broad characterization of the surrounding signaling network. We describe a bead-based array and its use in characterizing the different forms and functions of β-catenin, as well as lysate microarrays (reverse-phase protein arrays) and their use in detecting and quantifying proteins involved in the canonical and noncanonical Wnt signaling pathways. As an application of this dual approach, we characterized the state of β-catenin signaling in cell lysates and linked these molecule-specific data with pathway-wide changes in signaling. The Protocols described here provide detailed instructions for cell culture methods, bead arrays, and lysate microarrays and outline how to use these complementary approaches to obtain insight into a complex network at a systems level.

Systematic reference sample generation for multiplexed serological assays

Quality controls of serological assays have to contain defined amounts of human antibodies specific for the targeted antigen. A prevailing issue for array-based antigen assays is that dozens of antigens are targeted within the same assay. Commonly different patient sera are combined and optimal pools are empirically identified. Here, we report a mathematical approach how an optimal sample pool composition can be systematically calculated and accurately compiled. The approach was used to compose suitable quality controls for a 71 plex Tuberculosis antigen bead array using a limited number of positive human sera.

Combining Ultracentrifugation and Peptide Termini Group-specific Immunoprecipitation for Multiplex Plasma Protein Analysis

Blood plasma is a valuable source of potential biomarkers. However, its complexity and the huge dynamic concentration range of its constituents complicate its analysis. To tackle this problem, an immunoprecipitation strategy was employed using antibodies directed against short terminal epitope tags (triple X proteomics antibodies), which allow the enrichment of groups of signature peptides derived from trypsin-digested plasma. Isolated signature peptides are subsequently detected using MALDI-TOF/TOF mass spectrometry. Sensitivity of the immunoaffinity approach was, however, compromised by the presence of contaminant peaks derived from the peptides of nontargeted high abundant proteins. A closer analysis of the enrichment strategy revealed nonspecific peptide binding to the solid phase affinity matrix as the major source of the contaminating peptides. We therefore implemented a sucrose density gradient ultracentrifugation separation step into the procedure. This yielded a 99% depletion of contaminating peptides from a sucrose fraction containing 70% of the peptide-antibody complexes and enabled the detection of the previously undetected low abundance protein filamin-A. Assessment of this novel approach using 15 different triple X proteomics antibodies demonstrated a more consistent detection of a greater number of targeted peptides and a significant reduction in the intensity of nonspecific peptides. Ultracentrifugation coupled with immunoaffinity MS approaches presents a powerful tool for multiplexed plasma protein analysis without the requirement for demanding liquid chromatography separation techniques.