Ulrich Walter

Publications of Ulrich Walter

• Low angle light scattering analysis: a novel quantitative method for functional characterization of human and murine platelet receptors.

  Authors: Igor Mindukshev, Stepan Gambaryan, Linda Kehrer, Claudia Schuetz, Anna Kobsar, Natalia Rukoyatkina, Viacheslav O Nikolaev, Alexander Krivchenko, Steve P Watson, Ulrich Walter, Joerg Geiger

  Clinical chemistry and laboratory medicine : CCLM / FESCC. 12/2011;

  Abstract Background: Determinations of platelet receptor functions are indispensable diagnostic indicators of cardiovascular and hemostatic diseases including hereditary and acquired receptor defectsAbstract Background: Determinations of platelet receptor functions are indispensable diagnostic indicators of cardiovascular and hemostatic diseases including hereditary and acquired receptor defects and receptor responses to drugs. However, presently available techniques for assessing platelet function have some disadvantages, such as low sensitivity and the requirement of large sample sizes and unphysiologically high agonist concentrations. Our goal was to develop and initially characterize a new technique designed to quantitatively analyze platelet receptor activation and platelet function on the basis of measuring changes in low angle light scattering. Methods: We developed a novel technique based on low angle light scattering registering changes in light scattering at a range of different angles in platelet suspensions during activation. Results: The method proved to be highly sensitive for simultaneous real time detection of changes in size and shape of platelets during activation. Unlike commonly-used methods, the light scattering method could detect platelet shape change and aggregation in response to nanomolar concentrations of extracellular nucleotides. Furthermore, our results demonstrate that the advantages of the light scattering method make it a choice method for platelet receptor monitoring and for investigation of both murine and human platelets in disease models. Conclusions: Our data demonstrate the suitability and superiority of this new low angle light scattering method for comprehensive analyses of platelet receptors and functions. This highly sensitive, quantitative, and online detection of essential physiological, pathophysiological and pharmacological-response properties of human and mouse platelets is a significant improvement over conventional techniques.

• The preanalytical influence of two different mechanical transport systems on laboratory analysis.

  Authors: Juergen Koessler, Anna L Kobsar, Kirsten Brunner, Herbert Stolz, Bettina Dossler, Ulrich Walter, Udo Steigerwald

  Clinical chemistry and laboratory medicine : CCLM / FESCC. 05/2011; 49(8):1379-82.

• Identification of SPRED2 (sprouty-related protein with EVH1 domain 2) as a negative regulator of the hypothalamic-pituitary-adrenal axis.

  Authors: Melanie Ullrich, Karin Bundschu, Peter M Benz, Marco Abesser, Ruth Freudinger, Tobias Fischer, Julia Ullrich, Thomas Renné, Ulrich Walter, Kai Schuh

  The Journal of biological chemistry. 01/2011; 286(11):9477-88.

  Sprouty-related proteins with EVH1 (enabled/vasodilator-stimulated phosphoprotein homology 1) domain (SPREDs) are inhibitors of MAPK signaling. To elucidate SPRED2 in vivo function, we characterizedSprouty-related proteins with EVH1 (enabled/vasodilator-stimulated phosphoprotein homology 1) domain (SPREDs) are inhibitors of MAPK signaling. To elucidate SPRED2 in vivo function, we characterized body homeostasis in SPRED2(-/-) mice. They showed a doubled daily water uptake, induced by elevated serum osmolality, originating from increased blood salt load. Accordingly, serum aldosterone was doubled, accompanied by augmented adrenal aldosterone synthase (AS) expression. Surprisingly, serum vasopressin (AVP) was unaltered, and, as evidenced by halved angiotensin II (Ang II) levels, the renin angiotensin system (RAS) was down-regulated. Adrenocorticotropic hormone (ACTH) was significantly elevated in SPRED2(-/-) mice, together with its secretagogue corticotropin-releasing hormone (CRH) and its downstream target corticosterone. ERK phosphorylation in brains was augmented, and hypothalamic CRH mRNA levels were elevated, both contributing to the increased CRH release. Our data were supported by CRH promoter reporter assays in hypothalamic mHypoE-44 cells, revealing a SPRED-dependent inhibition of Ets (ERK/E-twenty-six)-dependent transcription. Furthermore, SPRED suppressed CRH production in these cells. In conclusion, our study suggests that SPRED2 deficiency leads to an increased MAPK signaling, which results in an augmented CRH promoter activity. The subsequent CRH overproduction causes an up-regulation of downstream hypothalamic-pituitary-adrenal (HPA) hormone secretion. This constitutes a possible trigger for the observed compulsive grooming in SPRED2(-/-) mice and may, together with hyperplasia of aldosterone-producing cells, contribute to the hyperaldosteronism and homeostatic imbalances.

• The new INNOVANCE® PFA P2Y cartridge is sensitive to the detection of the P2Y₁₂ receptor inhibition.

  Authors: Juergen Koessler, Anna L Kobsar, Mirjana S Rajkovic, Andreas Schafer, Ulrike Flierl, Stephanie Pfoertsch, Johann Bauersachs, Udo Steigerwald, Andreas R Rechner, Ulrich Walter

  Platelets. 01/2011; 22(1):19-25.

  Insufficient response on antiplatelet medication has become an intensively discussed issue because of the risk factor of recurrent adverse cardiovascular events. However, the monitoring ofInsufficient response on antiplatelet medication has become an intensively discussed issue because of the risk factor of recurrent adverse cardiovascular events. However, the monitoring of antiplatelet therapy requires appropriate, robust and reliable test methods. For the measurement of thienopyridine effects, the manufacturer of the PFA-100® System provides the INNOVANCE® PFA P2Y * cartridge. We tested this cartridge for its capacity to detect the inhibition of the P2Y₁₂ receptor, which is the target for thienopyridine medication (e.g. clopidogrel). We compared the INNOVANCE® PFA P2Y * results with those obtained by the receptor specific flow cytometric vasodilator stimulated phosphoprotein (VASP) assay that expresses the status of the P2Y₁₂ receptor as "platelet reactivity index" (PRI). The in vitro addition of the P2Y₁₂ receptor antagonist cangrelor (AR-C69931MX) to citrated human whole blood resulted in a dose-dependent prolongation of closure times (CTs) of the INNOVANCE® PFA P2Y * cartridge correlating with decreased PRI levels. In volunteers, the intake of a 600 mg clopidogrel loading dose caused an increase of the CTs in all volunteers, although some of these volunteers were identified as "poor responders" by the VASP assay (no significant reduction of PRI levels). In 50 patients with stable coronary artery disease undergoing percutaneous coronary intervention (PCI) and under dual antiplatelet therapy, the new cartridge had a detection rate of 84% (CT 106 s as cut-off) for clopidogrel medication. After dividing the 50 patients into two groups according to their response to clopidogrel INNOVANCE® PFA P2Y * recognized all "responders" (defined by a PRI > 50%) using >106 s as cut-off but the specificity for a "good response" was only 42% because several "poor responders" (defined by a PRI > 50%) also showed CTs above the cut-off. The best correlation (substantial agreement) between the results of INNOVANCE® PFA P2Y * and of the VASP phosphorylation assay was achieved using CT > 200 s and PRI < 55% as cut-offs. Then, the sensitivity of INNOVANCE® PFA P2Y * was 97% and the specificity for a "good response" 65%. In summary, INNOVANCE® PFA P2Y * showed a high sensitivity for the detection of P2Y₁₂ receptor blockade, but had only a limited specificity for a "good response" to clopidogrel. Therefore, this new cartridge is a useful tool to rule out P2Y₁₂ receptor inhibition, if normal or only slightly prolonged CTs are obtained. Its predictive value for risk assessment of thromboembolic events, e.g. after coronary stent implantation, needs to be evaluated in clinical trials.

• A protein phosphorylation-based assay for screening and monitoring of drugs modulating cyclic nucleotide pathways.

  Authors: Jörg Geiger, Tobias Brandmann, Katharina Hubertus, Budy Tjahjadi, Reinhard Schinzel, Ulrich Walter

  Analytical biochemistry. 12/2010; 407(2):261-9.

  Cyclic nucleotide regulation is an important target for drug development, particularly for treatment and prophylaxis of cardiovascular diseases. Determination of cyclic nucleotide levels forCyclic nucleotide regulation is an important target for drug development, particularly for treatment and prophylaxis of cardiovascular diseases. Determination of cyclic nucleotide levels for screening and monitoring of cyclic nucleotide modulating drug action is necessary, yet the techniques available are cumbersome and not sufficiently accurate. Here we present an approach based on the detection of cyclic nucleotide-dependent protein phosphorylation. By use of a common substrate of cyclic nucleotide-dependent protein kinases, the protein vasodilator-stimulated phosphoprotein (VASP) featuring two phosphorylation sites specifically phosphorylated by these kinases, an assay was developed for the monitoring of intracellular cyclic nucleotide levels. The assay was tested with human platelets ex vivo treated with stimulants of nucleotide cyclases, kinases, and phosphodiesterase inhibitors. Phosphorylation of the protein VASP correlates with intracellular cyclic nucleotide concentration (R(2)>0.90 for cGMP and cAMP); however, VASP phosphorylation is more sensitive to elevated cyclic nucleotide levels and significantly more stable over time. Quantification of VASP phosphorylation offers a reliable and robust tool for fast and easy monitoring of cyclic nucleotide levels and is also applicable to unprocessed biological matrices. Owing to these properties, VASP is a promising biomarker for screening and monitoring of cyclic nucleotide modulating drugs.

• Detection of serum free light chains: the problem with antigen excess.

  Authors: Markus Bosmann, Jürgen Kössler, Herbert Stolz, Ulrich Walter, Stefan Knop, Udo Steigerwald

  Clinical chemistry and laboratory medicine : CCLM / FESCC. 10/2010; 48(10):1419-22.

  In recent years, measurement of serum immunoglobulin free light chains (FLCs) has greatly facilitated diagnosis and monitoring of various plasma cells dyscrasias, including multiple myeloma.In recent years, measurement of serum immunoglobulin free light chains (FLCs) has greatly facilitated diagnosis and monitoring of various plasma cells dyscrasias, including multiple myeloma. Detection of FLCs by nephelometry depends on the formation of immune complexes. However, it is known that if the antigen (free light chain) is present in great excess, non-precipitating immune complexes can be formed and be detected poorly. This may lead to inaccurate test results. Serum samples from 91 patients were subjected prospectively to the detection of free κ and λ light chains by automated nephelometry. A standard dilution of 1:100 was paralleled by a 1:2000 dilution. In addition, samples with values below the effective range (1:100) were subjected to a 1:20 dilution in order to calculate the κ/λ ratio. Here, we report the incidence of antigen excess in a cohort of 91 patients with a high proportion of monoclonal abnormalities. A standard dilution (1:100) of free light κ chains from a patient with monoclonal immunoglobulin A κ gammopathy were missed repeatedly. In a second patient (with myeloproliferative disease and an apparent incidental FLC monoclonal gammopathy of undetermined significance) free λ chains were also substantially underestimated in the standard dilution. Hence, the incidence of erroneously low test results due to antigen excess was 2.20%. We found a significantly higher incidence of falsely low test results due to antigen excess than reported previously. Antigen excess may result in erroneous interpretations in sera subjected to nephelometric analysis. Therefore, clinical decisions should not be based solely on a single assay, especially if FLC testing includes only one dilution of the serum sample. Instead, several parallel dilutions should be recommended for screening of patients.

• Vasodilator-stimulated phosphoprotein regulates inside-out signaling of beta2 integrins in neutrophils.

  Authors: Ravi K Deevi, Madhuri Koney-Dash, Adrien Kissenpfennig, James A Johnston, Kai Schuh, Ulrich Walter, Karim Dib

  Journal of immunology (Baltimore, Md. : 1950). 06/2010; 184(12):6575-84.

  The monomeric GTPase Rap1 controls functional activation of beta2 integrins in leukocytes. In this article, we describe a novel mechanism by which the chemoattractant fMLP activates Rap1 andThe monomeric GTPase Rap1 controls functional activation of beta2 integrins in leukocytes. In this article, we describe a novel mechanism by which the chemoattractant fMLP activates Rap1 and inside-out signaling of beta2 integrins. We found that fMLP-induced activation of Rap1 in human polymorphonuclear leukocytes or neutrophils and differentiated PLB-985 cells was blocked by inhibitors of the NO/guanosine-3',5'-cyclic monophosphate-dependent protein kinase (cGKI) pathway [N-(3-(aminomethyl)benzyl)acetamidine, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, DT-3 peptide, 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphothioate, Rp-isomer triethylammonium salt-guanosine-3',5'-cyclic monophosphate], indicating that the downstream signaling events in Rap1 activation involve the production of NO and guanosine-3',5'-cyclic monophosphate, as well as the activation of cGKI. Silencing the expression of vasodilator-stimulated phosphoprotein (VASP), a substrate of cGKI, in resting PLB-985 cells or mice neutrophils led to constitutive activation of Rap1. In parallel, silencing VASP in differentiated PLB-985 cells led to recruitment of C3G, a guanine nucleotide exchange factor for Rap1, to the plasma membrane. Expression of murine GFP-tagged phosphodeficient VASP Ser235Ala mutant (murine serine 235 of VASP corresponds to human serine 239) in PLB-985 cells blunted fMLP-induced translocation of C3G to the membrane and activation of Rap1. Thus, bacterial fMLP triggers cGKI-dependent phosphorylation of human VASP on serine 239 and, thereby, controls membrane recruitment of C3G, which is required for activation of Rap1 and beta2 integrin-dependent antibacterial functions of neutrophils.

• Characterization of a novel interaction between vasodilator-stimulated phosphoprotein and Abelson interactor 1 in human platelets: a concerted computational and experimental approach.

  Authors: Marcus Dittrich, Verena Strassberger, Marc Fackler, Piet Tas, Urs Lewandrowski, Albert Sickmann, Ulrich Walter, Thomas Dandekar, Ingvild Birschmann

  Arteriosclerosis, thrombosis, and vascular biology. 04/2010; 30(4):843-50.

  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 vitroThe 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. 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. 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.

• Thrombin and collagen induce a feedback inhibitory signaling pathway in platelets involving dissociation of the catalytic subunit of protein kinase A from an NFkappaB-IkappaB complex.

  Authors: Stepan Gambaryan, Anna Kobsar, Natalia Rukoyatkina, Sabine Herterich, Joerg Geiger, Albert Smolenski, Suzanne M Lohmann, Ulrich Walter

  The Journal of biological chemistry. 03/2010; 285(24):18352-63.

  Protein kinase A (PKA) activation by cAMP phosphorylates multiple target proteins in numerous platelet inhibitory pathways that have a very important role in maintaining circulating platelets in aProtein kinase A (PKA) activation by cAMP phosphorylates multiple target proteins in numerous platelet inhibitory pathways that have a very important role in maintaining circulating platelets in a resting state. Here we show that in thrombin- and collagen-stimulated platelets, PKA is activated by cAMP-independent mechanisms involving dissociation of the catalytic subunit of PKA (PKAc) from an NFkappaB-IkappaBalpha-PKAc complex. We demonstrate mRNA and protein expression for most of the NFkappaB family members in platelets. From resting platelets, PKAc was co-immunoprecipitated with IkappaBalpha, and conversely, IkappaBalpha was also co-immunoprecipitated with PKAc. This interaction was significantly reduced in thrombin- and collagen-stimulated platelets. Stimulation of platelets with thrombin- or collagen-activated IKK, at least partly by PI3 kinase-dependent pathways, leading to phosphorylation of IkappaBalpha, disruption of an IkappaBalpha-PKAc complex, and release of free, active PKAc, which phosphorylated VASP and other PKA substrates. IKK inhibitor inhibited thrombin-stimulated IkBalpha phosphorylation, PKA-IkBalpha dissociation, and VASP phosphorylation, and potentiated integrin alphaIIbbeta3 activation and the early phase of platelet aggregation. We conclude that thrombin and collagen not only cause platelet activation but also appear to fine-tune this response by initiating downstream NFkappaB-dependent PKAc activation, as a novel feedback inhibitory signaling mechanism for preventing undesired platelet activation.

• Platelet proinflammatory activity in clinically stable patients with CF starts in early childhood.

  Authors: Alexander Sturm, Helge Hebestreit, Corinne Koenig, Ulrich Walter, Ralf Grossmann

  Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society. 02/2010; 9(3):179-86.

  Early onset chronic inflammation is present in CF. Platelets may contribute to inflammation by cytokine release and interaction with leukocytes. Parameters of platelet proinflammatory functionEarly onset chronic inflammation is present in CF. Platelets may contribute to inflammation by cytokine release and interaction with leukocytes. Parameters of platelet proinflammatory function (soluble CD62P, soluble CD40L, the percentage of platelet-leukocyte aggregates, platelet CD62P) and platelet procoagulatory function (PAC-1-binding to activated integrin alpha(IIb)beta(3) and expression of integrin alpha(IIb)beta(3)=CD41a) were measured in patients and controls. Levels of sCD62P, sCD40L were increased in CF irrespective of age and activity of inflammation. The number of platelet-leukocyte aggregates was elevated in older CF patients. PAC-1-binding to platelets decreased with growing activity of inflammation. Exocytosis of CD41a upon platelet activation was reduced. In CF, platelet proinflammatory activity is increased at very young age already and might promote inflammation and tissue damage. On the other hand, platelets seem to downregulate the activation of their most important integrin (alpha(IIb)beta(3)) for clot formation.

• Deficiency of vasodilator-stimulated phosphoprotein (VASP) increases blood-brain-barrier damage and edema formation after ischemic stroke in mice.

  Authors: Peter Kraft, Peter Michael Benz, Madeleine Austinat, Marc Elmar Brede, Kai Schuh, Ulrich Walter, Guido Stoll, Christoph Kleinschnitz

  PloS one. 01/2010; 5(12):e15106.

  Stroke-induced brain edema formation is a frequent cause of secondary infarct growth and deterioration of neurological function. The molecular mechanisms underlying edema formation after stroke areStroke-induced brain edema formation is a frequent cause of secondary infarct growth and deterioration of neurological function. The molecular mechanisms underlying edema formation after stroke are largely unknown. Vasodilator-stimulated phosphoprotein (VASP) is an important regulator of actin dynamics and stabilizes endothelial barriers through interaction with cell-cell contacts and focal adhesion sites. Hypoxia has been shown to foster vascular leakage by downregulation of VASP in vitro but the significance of VASP for regulating vascular permeability in the hypoxic brain in vivo awaits clarification. Focal cerebral ischemia was induced in Vasp(-/-) mice and wild-type (WT) littermates by transient middle cerebral artery occlusion (tMCAO). Evan's Blue tracer was applied to visualize the extent of blood-brain-barrier (BBB) damage. Brain edema formation and infarct volumes were calculated from 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain slices. Both mouse groups were carefully controlled for anatomical and physiological parameters relevant for edema formation and stroke outcome. BBB damage (p<0.05) and edema volumes (1.7 mm(3)±0.5 mm(3) versus 0.8 mm(3)±0.4 mm(3); p<0.0001) were significantly enhanced in Vasp(-/-) mice compared to controls on day 1 after tMCAO. This was accompanied by a significant increase in infarct size (56.1 mm(3)±17.3 mm(3) versus 39.3 mm(3)±10.7 mm(3), respectively; p<0.01) and a non significant trend (p>0.05) towards worse neurological outcomes. Our study identifies VASP as critical regulator of BBB maintenance during acute ischemic stroke. Therapeutic modulation of VASP or VASP-dependent signalling pathways could become a novel strategy to combat excessive edema formation in ischemic brain damage.

• Prostacyclin receptor stimulation facilitates detection of human platelet P2Y(12) receptor inhibition by the PFA-100 system.

  Authors: Anna L Kobsar, Juergen Koessler, Mirjana S Rajkovic, Kirsten P Brunner, Udo Steigerwald, Ulrich Walter

  Platelets. 01/2010; 21(2):112-6.

  The rationale for monitoring platelet inhibition by thienopyridines for the identification of patients at risk for future recurrent arterial thrombosis or ischemic events is intensively discussed, asThe rationale for monitoring platelet inhibition by thienopyridines for the identification of patients at risk for future recurrent arterial thrombosis or ischemic events is intensively discussed, as well as which monitoring systems are appropriate, robust and reliable. Flow cytometric measurement of phosphorylated VASP (vasodilator-stimulated phosphoprotein), expressed as platelet reactivity index (PRI), is presently "the gold standard method" for evaluating P2Y(12) receptor inhibition. The PFA-100 system, a commercially available and clinically widely used platelet test system, is based on a different principle, not that of VASP phosphorylation. The aim of the present study was to compare the two methods and evaluate whether the conventional PFA-100 collagen/ADP cartridge could be pharmacologically improved to enable its routine clinical use for detection of platelet P2Y(12) receptor inhibition. The effects of increasing concentrations of the competitive P2Y(12) receptor antagonist cangrelor (AR-C69931MX) and the time-dependent effects of a single oral loading dose of clopidogrel (600 mg) were analysed with human whole blood. P2Y(12) receptor inhibition was measured by the VASP/PRI assay and the PFA-100 collagen/ADP cartridge system, with and without preincubation with the prostacyclin analog iloprost (Ilomedin). In vitro addition of iloprost (0.5 nM) enabled PFA-100 collagen/ADP cartridge system detection of P2Y(12) receptor inhibition in whole blood by cangrelor in vitro or by clopidogrel treatment of volunteers. The addition of a prostacyclin analog facilitates PFA-100 collagen/ADP system detection of P2Y(12) receptor inhibition, achieving a sensitivity similar to that of the VASP/PRI reference method. Future studies should now evaluate whether this modified PFA-100 system, like the VASP assay, is a reliable test system for monitoring P2Y(12) receptor inhibition under clinical conditions.

• VASP phosphorylation at serine239 regulates the effects of NO on smooth muscle cell invasion and contraction of collagen.

  Authors: Olivier D Defawe, Sarah Kim, Lihua Chen, Daming Huang, Richard D Kenagy, Thomas Renné, Ulrich Walter, Günter Daum, Alexander W Clowes

  Journal of cellular physiology. 10/2009;

  Nitric oxide triggers cGMP-dependent kinase-mediated phosphorylation of the actin regulator vasodilator-stimulated phosphoprotein (VASP) at residue serine239. The function of this phosphorylation forNitric oxide triggers cGMP-dependent kinase-mediated phosphorylation of the actin regulator vasodilator-stimulated phosphoprotein (VASP) at residue serine239. The function of this phosphorylation for smooth muscle cell (SMC) adhesion, spreading, matrix contraction, and invasion is not well understood. We reconstituted VASP deficient SMC with wild-type VASP (wt-VASP) or VASP mutants that mimic "locked" serine239 phosphorylation (S239D-VASP) or "blocked" serine239 phosphorylation (S239A-VASP). Collagen gel contraction was reduced in S239D-VASP compared to S239A-VASP and wt-VASP expressing cells and nitric oxide (NO) stimulation decreased gel contraction of wt-VASP reconstituted SMC. Invasion of collagen was enhanced in S239D-VASP and NO-stimulated wild-type SMCs compared to S239A-VASP expressing cells. Expression of S239D-VASP impaired SMC attachment to collagen, reduced the number of membrane protrusions, and caused cell rounding compared to expression of S239A-VASP. Treatment of wt-VASP reconstituted SMCs with NO exerted similar effects as expression of S239D-VASP. As unstimulated cells were spreading on collagen S239A-VASP and wt-VASP localized to actin fibers whereas S239D-VASP was enriched in the cytosol. NO interferes with SMC invasion and contraction of collagen matrices. This requires phosphorylation of VASP on serine239, which reduces VASP binding to actin fibers. These findings support the conclusion that VASP phosphorylation at serine239 regulates cytoskeleton remodeling. J. Cell. Physiol. (c) 2009 Wiley-Liss, Inc.

• Platelet membrane proteomics: a novel repository for functional research.

  Authors: Urs Lewandrowski, Stefanie Wortelkamp, Katharina Lohrig, Rene P Zahedi, Dirk A Wolters, Ulrich Walter, Albert Sickmann

  Blood. 06/2009;

  Being central players in thrombosis and hemostasis, platelets react in manifold and complex ways to extracellular stimuli. Cell-matrix and cell-cell interactions are mandatory for initial adhesion asBeing central players in thrombosis and hemostasis, platelets react in manifold and complex ways to extracellular stimuli. Cell-matrix and cell-cell interactions are mandatory for initial adhesion as well as for final development of stable plugs. Primary interfaces for interactions are plasma membrane proteins, of which many have been identified over the past decades in individual studies. However, due to their enucleate structure, platelets are not accessible to large-scale genomic screens and thus a comprehensive inventory of membrane proteins is still missing. For this purpose, we here present an advanced proteomic setup for the detailed analysis of enriched platelet plasma membranes and the so far most complete collection of platelet membrane proteins. In summary, 1282 proteins were identified of which more than half are termed to be of membrane origin. This study provides a brief overview of gene ontology subcellular and functional classification, as well as interaction network analysis. In addition, the mass spectrometric data was used to assemble a first tentative relative quantification of large scale data on the protein level. We therefore estimate the presented data to be of major interest to the platelet research field and to support rational design of functional studies.

• cGMP and cGMP-Dependent Protein Kinase in Platelets and Blood Cells.

  Authors: Ulrich Walter, Stepan Gambaryan

  Handbook of experimental pharmacology. 02/2009;

  Platelets are specialized adhesive cells that play a key role in normal and pathological hemostasis through their ability to rapidly adhere to subendothelial matrix proteins (platelet adhesion) andPlatelets are specialized adhesive cells that play a key role in normal and pathological hemostasis through their ability to rapidly adhere to subendothelial matrix proteins (platelet adhesion) and to other activated platelets (platelet aggregation). NO plays a crucial role in preventing platelet adhesion and aggregation. In platelets, cGMP synthesis is catalyzed by sGC, whereas PDE2, PDE3 and PDE5 are responsible for cGMP degradation. Stimulation of cGK by cGMP leads to phosphorylation of multiple target substrates. These substrates inhibit elevation of intracellular calcium, integrin activation, cytoskeletal reorganization, and platelet granule secretion, events normally associated with platelet activation. The NO/cGMP pathway also plays a significant role in many other blood cell types in addition to platelets. In leukocytes, depending on the specific cell type, cGMP signaling regulates gene expression, differentiation, migration, cytokine production, and apoptosis.

• NO inhibits platelet apoptosis by cGMP-dependent and-independent pathways

  Authors: Natalia Rukoyatkina, Ulrich Walter, Stepan Gambaryan

  BMC Pharmacology. 01/2009;

• Cross-talk of inhibitory and stimulatory signalling pathways of human platelets

  Authors: Ulrich Walter, Stepan Gambaryan

  BMC Pharmacology. 01/2009;

• Phosphatidylserine surface expression and integrin alphaIIbbeta3 activity on thrombin/convulxin stimulated platelets/particles of different sizes.

  Authors: Natalia Rukoyatkina, Antonija J Begonja, Jörg Geiger, Martin Eigenthaler, Ulrich Walter, Stepan Gambaryan

  British journal of haematology. 12/2008;

  Platelets stimulated by a combination of thrombin/convulxin have been shown to develop two to three populations characterized by different phosphatidylserine (PS) surface expression and integrinPlatelets stimulated by a combination of thrombin/convulxin have been shown to develop two to three populations characterized by different phosphatidylserine (PS) surface expression and integrin alphaIIbbeta3 activity. To determine how these markers are distributed on the surface of platelets/particles, we studied Annexin V and PAC-1 binding to platelets/particles of different sizes by flow cytometry analysis and evaluated influences of calpain and caspase inhibitors on thrombin/convulxin-activated platelets. Analysed platelets/particles were divided by their sizes, according to the standard size beads, into seven populations from 0.37 to 4.8 mum. PAC-1 binding/mum(2) was almost equal in platelets/particles ranging from 1.2 to 4.8 mum and was significantly lower on smaller-sized particles sizes (0.37-0.7 mum). PS surface exposure/mum(2) was high in the particles of 0.37-1.2 mum and very low in platelets (2.6-4.8 mum). Upon thrombin/convulxin stimulation caspase inhibitors prevented microparticle (MP) formation, while a calpain inhibitor stimulated MP formation. It was also shown that stimulated platelets are heterogeneous not only in their ability to activate alphaIIbbeta3 integrin complex and expose PS on their surface, but also in the distribution of activation markers, which strongly depends on platelet/particle size and that platelets/particles of different sizes provide different responses to the same stimulus.

• ADP-induced platelet aggregation frequently fails to detect impaired clopidogrel-responsiveness in patients with coronary artery disease compared to a P2Y(12)-specific assay.

  Authors: Andreas Schäfer, Sarah Weinberger, Ulrike Flierl, Martin Eigenthaler, Stefan Störk, Ulrich Walter, Georg Ertl, Johann Bauersachs

  Thrombosis and haemostasis. 10/2008; 100(4):618-25.

  Incomplete P2Y(12)-inhibition during clopidogrel treatment is associated with increased cardiovascular events and mortality after coronary intervention. We investigated the incidence of impairedIncomplete P2Y(12)-inhibition during clopidogrel treatment is associated with increased cardiovascular events and mortality after coronary intervention. We investigated the incidence of impaired individual clopidogrel-responsiveness using a P2Y(12)-specific and pre-treatment-independent assay in a real world situation. One hundred consecutive patients with coronary artery disease (CAD) on combined acetylsalicylic acid and clopidogrel treatment (75 mg/d) and 33 patients on aspirin only were screened for platelet ADP-induced signalling by conventional aggregometry, platelet P-selectin expression and the platelet reactivity index (PRI). Impaired P2Y(12)-specific inhibition by clopidogrel was defined as a PRI>50%. Functional platelet reactivity was significantly lower in clopidogrel-treated patients compared to controls. Impaired individual response to treatment was diagnosed in 69% of clopidogrel-treated patients. Conventional assessment of maximum ADP-induced platelet aggregation failed to detect impaired P2Y(12) inhibition in 36% of patients identified by PRI to have an impaired clopidogrel response. Impaired clopidogrel response was associated with lower HDL levels and a history of hyperlipidaemia. In conclusion, PRI as a P2Y(12)-specific assay to evaluate the treatment effect of clopidogrel in patients with CAD revealed insufficient P2Y(12)-inhibition in two thirds of patients in a real-world scenario indicating a markedly higher incidence than previously assumed. PRI detected significantly more patients with impaired response than conventional platelet aggregation.

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

  Authors: Marcus Dittrich, Ingvild Birschmann, Silke Mietner, Albert Sickmann, Ulrich Walter, Thomas Dandekar

  Arteriosclerosis, thrombosis, and vascular biology. 08/2008; 28(7):1326-31.

  OBJECTIVE: 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 interactionOBJECTIVE: 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.