Ingvild Birschmann

Analysis of SAGE data in human platelets: Features of the transcriptome in an anucleate cell

A comprehensive SAGE (serial analysis of gene expression) library of purified human platelets was established. Twenty-five thousand (25,000) tags were sequenced, and after removal of mitochondrial tags, 12,609 (51%) non-mitochondrial-derived tags remained, corresponding to 2,300 different transcripts with expression levels of up to 30,000 tags per million. This new, highly purified SAGE library of platelets is enriched in specific transcripts. The complexity in terms of tag distribution is similar to cells that are still able to replenish their mRNA pool by transcription. We show that our SAGE data are consistent with recently published microarray data but show further details of the platelet transcriptome, including (i) longer UTR regions and more stable folding in the enriched mRNAs, (ii) biologically interesting new candidate mRNAs that show regulatory elements, including elements for RNA stabilization or for translational control, and (iii) significant enrichment of specific, highly transcribed mRNAs compared to a battery of SAGE libraries from other tissues. Among several regulatory mRNA elements known to be involved in mRNA localization and translational control, CPE elements are in particular enriched in the platelet transcriptome. mRNAs previously reported to be translationally regulated were found to be present in the library and were validated by real-time PCR. Furthermore, specific molecular functions such as signal transduction activity were found to be significantly enriched in the platelet transcriptome. These findings emphasize the richness and diversity of the platelet transcriptome.

Dabigatran, rivaroxaban, apixaban, argatroban and fondaparinux and their effects on coagulation POC and platelet function tests.

Abstract Background: In recent years, several selectively acting anticoagulants, including the direct thrombin inhibitors (DTI; argatroban, dabigatran) and the factor Xa inhibitors (rivaroxaban, apixaban, fondaparinux), have been developed. With their clinical application increasing, it is of interest to evaluate their interference with classical haemostaseological point-of-care tests. Additionally, the effect of the investigated anticoagulants on platelet function tests will come increasingly more into focus for monitoring not only hereditary platelet dysfunction, but also antiplatelet therapy. Methods: Blood samples from healthy volunteers were spiked with therapeutic and supratherapeutic concentrations of the drugs listed above and investigated with regard to their effects on the following POCTs: activated clotting time (ACT), thromboelastometry with ROTEM®, PFA® and Multiplate®. Light-transmission aggregometry (LTA) was used for a platelet function assay. Results: At supratherapeutic concentrations, ACT and ROTEM® analysis were always influenced after administration of the drugs listed above (except fondaparinux in EXTEM-CT). Therapeutic concentrations showed differential effects on these assays. LTA measurements revealed a distinct decrease in α-thrombin-induced platelet aggregation for both DTIs (therapeutic and supratherapeutic concentrations), while argatroban reduced platelet function in supratherapeutic concentrations. None of the drugs seemed to have any influence on PFA® or Multiplate®. Conclusions: Selective thrombin and factor Xa inhibitors exhibit distinct effects on POCTs and platelet function tests. This must be considered in assessing assay results when taking medical decisions.

Platelet protein interactions: map, signaling components, and phosphorylation groundstate.

Objectives—Assembly of a comprehensive proteome and transcriptome database of human platelets, derivation of a model of the platelet-specific interactome, and generation of a functional interaction map of platelet phosphorylations and kinases. Methods and Results—Interactions are derived from literature-curated data from HPRD and yeast two hybrid (Y2H) and mapped to platelet-specific expression data (SAGE or proteome). From this a cell-type specific model of platelet proteins and protein–protein interactions is derived. The obtained inventory of platelet-specific proteins includes key domains, protein GO annotations, and receptors. Collected interactions point to new platelet signaling components, actin remodeling processes, and pharmacological targets and offer incentives for further studies (eg, on the IPP complex). Integration of platelet-specific phosphoproteins and the characterization of the platelet kinase repertoire sketch a first outline of kinase signaling in human platelets. Conclusions—A first view of the platelet interactome, platelet phosphorylation, and platelet kinome is available from the in silico data.

Ambient hemolysis and activation of coagulation is different between HeartMate II and HeartWare left ventricular assist devices

BACKGROUND Thromboembolic and bleeding events in patients with a left ventricular assist device (LVAD) are still a major cause of complications. Therefore, the balance between anti-coagulant and pro-coagulant factors needs to be tightly controlled. The principle hypothesis of this study is that different pump designs may have an effect on hemolysis and activation of the coagulation system. Referring to this, the HeartMate II (HMII; Thoratec Corp, Pleasanton, CA) and the HeartWare HVAD (HeartWare International Inc, Framingham, MA) were investigated. METHODS For 20 patients with LVAD support (n = 10 each), plasma coagulation, full blood count, and clinical chemistry parameters were measured. Platelet function was monitored using platelet aggregometry, platelet function analyzer-100 system ( Siemens, Marburg, Germany), vasodilator-stimulated phosphoprotein phosphorylation assay, immature platelet fraction, platelet-derived microparticles, and von Willebrand diagnostic. RESULTS Acquired von Willebrand syndrome could be detected in all patients. Signs of hemolysis, as measured by lactate dehydrogenase levels (mean, 470 U/liter HMII, 250 U/liter HVAD; p < 0.001), were more pronounced in the HMII patients. In contrast, D-dimer analysis indicated a significantly higher activation of the coagulation system in HVAD patients (mean, 0.94 mg/liter HMII, 2.01 mg/liter HVAD; p < 0.01). The efficacy of anti-platelet therapy using clopidogrel was not sufficient in more than 50% of the patients. CONCLUSIONS Our results support the finding that all patients with rotary blood pumps suffered from von Willebrand syndrome. In addition, a distinct footprint of effects on hemolysis and the coagulation system can be attributed to different devices. As a consequence, the individual status of the coagulation system needs to be controlled in long-term patients.

PlateletWeb: a systems biologic analysis of signaling networks in human platelets

Understanding the cellular mechanisms of platelet activation and their pharmacologic modulation is of major interest for basic and clinical research. Here we introduce a comprehensive human platelet repository (PlateletWeb) for systems biologic analysis of platelets in the functional context of integrated networks. Functional, drug, and pathway associations provide a first systemic insight into various aspects of platelet functionality and pharmacologic regulation. Detailed manual curation of recent platelet proteome and transcriptome studies yielded more than 5000 platelet proteins. Integration of protein-protein interactions with kinase-substrate relationships unraveled the platelet signaling network involving more than 70% of all platelet proteins. Analysis of the platelet kinome in the context of the kinase phylogenetic background revealed an over-representation of tyrosine kinase substrates. The extraction and graphical visualization of specific subnetworks allow identification of all major signaling modules involved in activation and inhibition. An in-depth analysis of DOK1 signaling identifies putative signal modulators of the integrin network. Through integration of various information sources and high curation standards, the PlateletWeb knowledge base offers the systems biologic background for the investigation of signal transduction in human platelets (http://plateletweb.bioapps.biozentrum.uni-wuerzburg.de).

Understanding platelets Lessons from proteomics, genomics and promises from network analysis

New large-scale analysis techniques such as bioinformatics, mass spectrometry and SAGE data analysis will allow a new framework for understanding platelets. This review analyses some important options and tasks for these tools and examines an outline of the new, refined picture of the platelet outlined by these new techniques. Looking at the platelet-specific building blocks of genome, (active) transcriptome and proteome (notably secretome and phospho-proteome), we summarize current bioinformatical and biochemical approaches, tasks as well as their limitations. Understanding the surprisingly complex platelet regarding compartmentalization, key cascades, and pathways including clinical implications will remain an exciting and hopefully fruitful challenge for the future.

ADAMTS13 activity is decreased in a septic porcine model. Significance for glomerular thrombus deposition.

During sepsis, the balance between abundantly secreted von Willebrand factor (VWF) and the activity of its size regulating protease ADAMTS13 is assumed to be involved in coagulation abnormalities. We aimed to establish a porcine model with haemorrhagic shock with consecutive sepsis and hypothesised that a decreased ADAMTS13-activity as well as an altered VWF multimer pattern is associated with renal failure. Animals (n=21) were subjected to haemorrhagic shock. After volume replacement, intraperitoneal Escherichia coli sepsis was induced. Blood samples were drawn at baseline, after haemorrhage and sepsis induction. Directly postmortem we examined renal tissue by JONES-silver, CD61, VWF and fibrin staining for characterisation of thrombi. Renal failure was analysed by scoring PAS-stained sections for acute tubular damage. Glomerular microthrombi were observed in six of 21 septic animals. Porcine ADAMTS13 activity declined significantly during sepsis, accompanied by a drop-off in platelet count. At 12 hours after sepsis induction, ADAMTS13 activity was significantly diminished compared to sham controls, and an elevated acute tubular damage score was associated with an increased proportion of high-molecular-weight VWF multimers. Compared to baseline the proportion of high-molecular-weight VWF multimers increased significantly in septic animals. Similar to human sepsis, diminished ADAMTS13 activity was observed in a septic porcine model associated with a shift to rather thrombogenic VWF multimers and deposition of microthrombi. Therefore, this porcine model seems to be appropriate for performing functional and therapeutic studies in sepsis-associated ADAMTS13 deficiency.

Glomerular mRNA expression of prothrombotic and antithrombotic factors in renal transplants with thrombotic microangiopathy.

Background Thrombotic microangiopathy (TMA) in renal transplants (rTx-TMA) is a serious complication and is usually either recurrent TMA (RecTMA) due to humoral rejection (HR-TMA) or due to calcineurin inhibitor toxicity (CNI-TMA). Although the triggers are known, our knowledge about the thrombogenic transcriptome changes in the microvessels is rudimentary. Methods We examined the expression of several prothrombotic and antithrombotic genes in 25 biopsies with rTx-TMA (6 RecTMA, 9 HR-TMA, and 10 CNI-TMA) and 8 controls. RNA from microdissected glomeruli of paraffin-embedded tissue was isolated and mRNA transcripts were quantified with real-time polymerase chain reaction after preamplification. Results were correlated with clinicopathologic parameters. Results Glomerular mRNA expression of KLF2, KLF4, and tPA was lower and that of PAI-1 was higher in rTx-TMA than in the controls. Glomerular mRNA expression of KLF2 and KLF4 correlated with that of tPA and inversely with that of PAI-1 in rTx-TMA. The mRNA expression of complement regulators CD46 and CD59 were higher in rTx-TMA than in the controls. Only in HR-TMA were glomerular ADAMTS13 and CD55 down-regulated. Conclusions The glomerular capillary bed seems to contribute to all subtypes of rTx-TMA by down-regulation of the endothelial transcription factors KLF2 and KLF4, indicating dedifferentiation with subsequent up-regulation of PAI-1 and down-regulation of tPA, resulting in inhibition of local fibrinolysis. Decreased glomerular expression of ADAMTS13 and CD55 could be an additional pathway toward microthrombosis exclusively in HR-TMA.

Characterization of a Novel Interaction Between Vasodilator-Stimulated Phosphoprotein and Abelson Interactor 1 in Human Platelets: A Concerted Computational and Experimental Approach

Objective—The goal of this study was systematic profiling of vasodilator-stimulated phosphoprotein (VASP)-Ena/VASP homology 1 (EVH1) interactors in human platelets using a combined in silico and in vitro approach. Methods and Results—Exploiting the information of the comprehensive proteome catalogue in the PlateletWeb database (http://plateletweb.bioapps.biozentrum.uni-wuerzburg.de/PlateletWeb.php), we performed a motif search of all sequences and identified potential target sites of class I EVH1 domains in human platelet proteins. Performing affinity purification with VASP-EVH1 domain and the lysates of platelets, we examined complex partners by mass spectrometry. Combining the results of both analyses, we identified Abelson interactor 1 (Abi-1) as a novel EVH1 domain–specific interaction partner of VASP in human platelets and investigated this interaction by yeast 2-hybrid mutational studies and immunoprecipitation. Immunofluorescence microscopy indicated colocalization of both proteins at the lamellipodia of spread human platelets, suggesting a role in reorganizing the cytoskeleton during spreading. Conclusion—The combination of experimental and computational interactome research has emerged as a valuable tool for the analysis of protein-protein interaction networks and facilitates the discovery and characterization of novel interactions as detailed here for Abi-1 and VASP in human platelets. System biological approaches can be expected to play an important role in basic and clinical platelet research, as they offer the potential to analyze signal transduction beyond the scope of established pathways.

Arteriolar vascular smooth muscle cell differentiation in benign nephrosclerosis

BACKGROUND Benign nephrosclerosis (bN) is the most prevalent form of hypertensive damage in kidney biopsies. It is defined by early hyalinosis and later fibrosis of renal arterioles. Despite its high prevalence, very little is known about the contribution of arteriolar vascular smooth muscle cells (VSMCs) to bN. We examined classical and novel candidate markers of the normal contractile and the pro-fibrotic secretory phenotype of VSMCs in arterioles in bN. METHODS Sixty-three renal tissue specimens with bN and eight control specimens were examined by immunohistochemistry for the contractile markers caldesmon, alpha-smooth muscle actin (alpha-SMA), JunB, smoothelin and the secretory marker S100A4 and by double stains for caldesmon or smoothelin with S100A4. RESULTS Smoothelin immunostaining showed an inverse correlation with hyalinosis and fibrosis scores, while S100A4 correlated with fibrosis scores only. Neither caldesmon, alpha-SMA nor JunB correlated with hyalinosis or fibrosis scores. Cells in the arteriolar wall were exclusively positive either for caldesmon/smoothelin or S100A4. CONCLUSIONS This is the first systematic analysis of VSMC differentiation in bN. The results suggest that smoothelin is the most sensitive marker for the contractile phenotype and that S100A4 could be a novel marker for the secretory phenotype in vivo. The other markers did not seem to differentiate these phenotypes in bN. Thus, VSMC phenotype markers should be defined in the context of the vessel segment and disease under examination. S100A4 could not only be a marker of pro-fibrotic secretory VSMCs in bN but also an important mediator of arteriolar fibrosis.