Margitta Elvers

Publications of Margitta Elvers

• A novel role for phospholipase D as an endogenous negative regulator of platelet sensitivity.

  Authors: Margitta Elvers, Magnus Grenegård, Hanieh Khoshjabinzadeh, Patrick Münzer, Oliver Borst, Huasong Tian, Gilbert Di Paolo, Florian Lang, Meinrad Gawaz, Tomas L Lindahl, Knut Fälker

  Cellular signalling. 05/2012;

  Platelet aggregation, secretion and thrombus formation play a critical role in primary hemostasis to prevent excessive blood loss. On the other hand, uncontrolled platelet activation leads toPlatelet aggregation, secretion and thrombus formation play a critical role in primary hemostasis to prevent excessive blood loss. On the other hand, uncontrolled platelet activation leads to pathological thrombus formation resulting in myocardial infarction or stroke. Stimulation of heterotrimeric G-proteins by soluble agonists or immunoreceptor tyrosine based activation motif-coupled receptors that interact with immobilized ligands such as the collagen receptor glycoprotein (GP) VI leads to the activation of phospholipases that cleave membrane phospholipids to generate soluble second messengers. Platelets contain the phospholipases (PL) D1 and D2 which catalyze the hydrolysis of phosphatidylcholine to generate the second messenger phosphatidic acid (PA). The production of PA is abrogated by primary alcohols that have been widely used for the analysis of PLD-mediated processes. However, it is not clear if primary alcohols effectively reduce PA generation or if they induce PLD-independent cellular effects. In the present study we made use of the specific PLD inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) and show for the first time, that FIPI enhances platelet dense granule secretion and aggregation of human platelets. Further, FIPI has no effect on cytosolic Ca(2+) activity but needs proper Rho kinase signaling to mediate FIPI-induced effects on platelet activation. Upon FIPI treatment the phosphorylation of the PKC substrate pleckstrin was prominently enhanced suggesting that FIPI affects PKC-mediated secretion and aggregation in platelets. Similar effects of FIPI were observed in platelets from mouse wild-type and Pld1(-/-) mice pointing to a new role for PLD2 as a negative regulator of platelet sensitivity.

• The Bispecific SDF1-GPVI Fusion Protein Preserves Myocardial Function After Transient Ischemia in Mice.

  Authors: Melanie Ziegler, Margitta Elvers, Yvonne Baumer, Christoph Leder, Carmen Ochmann, Tanja Schönberger, Tobias Jürgens, Tobias Geisler, Burkhard Schlosshauer, Oleg Lunov, Stefan Engelhardt, Thomas Simmet, Meinrad Gawaz

  Circulation. 02/2012; 125(5):685-96.

  Background CXCR4-positive bone marrow cells (BMCs) are critically involved in cardiac repair mechanisms contributing to preserved cardiac function. Stromal cell-derived factor-1 (SDF-1) is the mostBackground CXCR4-positive bone marrow cells (BMCs) are critically involved in cardiac repair mechanisms contributing to preserved cardiac function. Stromal cell-derived factor-1 (SDF-1) is the most prominent BMC homing factor known to augment BMC engraftment, which is a limiting step of stem cell-based therapy. After myocardial infarction, SDF-1 expression is rapidly upregulated and promotes myocardial repair. Methods and Results We have established a bifunctional protein consisting of an SDF-1 domain and a glycoprotein VI (GPVI) domain with high binding affinity to the SDF-1 receptor CXCR4 and extracellular matrix proteins that become exposed after tissue injury. SDF1-GPVI triggers chemotaxis of CXCR4-positive cells, preserves cell survival, enhances endothelial differentiation of BMCs in vitro, and reveals proangiogenic effects in ovo. In a mouse model of myocardial infarction, administration of the bifunctional protein leads to enhanced recruitment of BMCs, increases capillary density, reduces infarct size, and preserves cardiac function. Conclusions These results indicate that administration of SDF1-GPVI may be a promising strategy to treat myocardial infarction to promote myocardial repair and to preserve cardiac function.

• The GRAF family member Oligophrenin1 is a RhoGAP with a BAR domain and regulates Rho GTPases in platelets.

  Authors: Margitta Elvers, Sandra Beck, Anna Fotinos, Melanie Ziegler, Meinrad Gawaz

  Cardiovascular research. 02/2012;

  AIMS: Platelet adhesion and aggregation is essential for hemostasis and thrombosis. Cytoskeletal reorganization of activated platelets plays a crucial role in these processes and implies activationAIMS: Platelet adhesion and aggregation is essential for hemostasis and thrombosis. Cytoskeletal reorganization of activated platelets plays a crucial role in these processes and implies activation of Rho GTPases. Rho GTPases are regulated by GTPase activating proteins (GAPs) that stimulate GTP hydrolysis to terminate Rho signaling. In this study we explored the regulation of Rho GTPases in platelets.Methods and ResultsOligophrenin1 (OPHN1) is a RhoGAP-regulating cytoplasmic protein that has been investigated in patients with X-linked mental retardation. Here we identified OPHN1 in mouse platelets where it co-localizes to actin-rich regions and Rho GTPases. OPHN1 exhibits strong GTPase-stimulating activity towards RhoA, Cdc42, and Rac1 and regulates cell adhesion and spreading. Furthermore, OPHN1 controls RhoA-mediated stress fibre and focal adhesion formation as well as filopodia and lamellipodia development. The analysis of different domains of OPHN1 revealed distinct functions in Rho hydrolysis. The C-terminus of OPHN1 displays an essential unit for Rho regulation, whereas the PH domain is a regulatory unit of OPHN1 controlling GAP function. The N-terminal BAR (Bin/amphiphysin/Rvs)-like domain is involved in GAP regulation but not in cytoskeleton rearrangements or Rho regulation and acts as a guidance domain to direct this GAP to its substrate. CONCLUSIONS: Our results suggest that OPHN1 is a powerful regulator of Rho GTPase activity in platelets that is critical for the reorganization of the cytoskeleton, which is a major process required for stable platelet adhesion and thrombus formation to occur.

• The serum- and glucocorticoid-inducible kinase 1 (SGK1) influences platelet calcium signaling and function by regulation of Orai1 expression in megakaryocytes.

  Authors: Oliver Borst, Eva-Maria Schmidt, Patrick Münzer, Tanja Schönberger, Syeda T Towhid, Margitta Elvers, Christina Leibrock, Evi Schmid, Anja Eylenstein, Dietmar Kuhl, Andreas E May, Meinrad Gawaz, Florian Lang

  Blood. 01/2012; 119(1):251-61.

  Platelets are activated on increase of cytosolic Ca2+ activity ([Ca2+](i)), accomplished by store-operated Ca2+ entry (SOCE) involving the pore-forming ion channel subunit Orai1. Here, we show, forPlatelets are activated on increase of cytosolic Ca2+ activity ([Ca2+](i)), accomplished by store-operated Ca2+ entry (SOCE) involving the pore-forming ion channel subunit Orai1. Here, we show, for the first time, that the serum- and glucocorticoid-inducible kinase 1 (SGK1) is expressed in platelets and megakaryocytes. SOCE and agonist-induced [Ca2+](i) increase are significantly blunted in platelets from SGK1 knockout mice (sgk1(-/-)). Similarly, Ca2+ -dependent degranulation, integrin α(IIb)β3 activation, phosphatidylserine exposure, aggregation, and in vitro thrombus formation were significantly impaired in sgk1(-/-) platelets, whereas tail bleeding time was not significantly enhanced. Platelet and megakaryocyte Orai1 transcript levels and membrane protein abundance were significantly reduced in sgk1(-/-) mice. In human megakaryoblastic cells (MEG-01), transfection with constitutively active (S422D)SGK1 but not with inactive (K127N)SGK1 significantly enhanced Orai1 expression and SOCE, while effects reversed by the SGK1 inhibitor GSK650394 (1μM). Transfection of MEG-01 cells with (S422D)SGK1 significantly increased phosphorylation of IκB kinase α/β and IκBα resulting in nuclear translocation of NF-κB subunit p65. Treatment of (S422D)SGK1-transfected MEG-01 cells with the IκB kinase inhibitor BMS-345541 (10μM) abolished SGK1-induced increase of Orai1 expression and SOCE. The present observations unravel SGK1 as novel regulator of platelet function, effective at least in part by NF-κB-dependent transcriptional up-regulation of Orai1 in megakaryocytes and increasing platelet SOCE.

• Platelet and soluble glycoprotein VI - novel applications in diagnosis and therapy.

  Authors: Boris Bigalke, Margitta Elvers, Tanja Schonberger, Meinrad Gawaz

  Current drug targets. 06/2011; 12(12):1821-30.

  One of the key receptors involved in the prothrombotic stage of an acute coronary syndrome (ACS) is platelet glycoprotein VI (GPVI). This constitutively expressed collagen receptor isOne of the key receptors involved in the prothrombotic stage of an acute coronary syndrome (ACS) is platelet glycoprotein VI (GPVI). This constitutively expressed collagen receptor is platelet-specific and has shown to be a useful biomarker tool for the early detection of atheroslerotic diseases such as ACS and ischemic stroke. In a multimarker panel of several biomarkers, platelet GPVI may contribute to risk stratification and prediction of clinical outcome in patients with symptomatic atherosclerotic diseases. Moreover, the soluble receptor of GPVI may also be an interesting and prospective target for molecular imaging to identify sites of vulnerable plaques as the results of preclinical studies suggest. Apart from the diagnostic use of platelet as well as soluble, plasmatic GPVI, therapeutical implications may be the blockade of collagen binding sides of GPVI, as platelet GPVI is not able to bind to collagen where the binding sides were already saturated with soluble GPVI. This therapeutic aspect could serve as a promising strategy for antithrombotic and antiatherosclerotic therapy in future.

• ADF/n-cofilin-dependent actin turnover determines platelet formation and sizing.

  Authors: Markus Bender, Anita Eckly, John H Hartwig, Margitta Elvers, Irina Pleines, Shuchi Gupta, Georg Krohne, Elisabeth Jeanclos, Antje Gohla, Christine Gurniak, Christian Gachet, Walter Witke, Bernhard Nieswandt

  Blood. 09/2010; 116(10):1767-75.

  The cellular and molecular mechanisms orchestrating the complex process by which bone marrow megakaryocytes form and release platelets remain poorly understood. Mature megakaryocytes generate longThe cellular and molecular mechanisms orchestrating the complex process by which bone marrow megakaryocytes form and release platelets remain poorly understood. Mature megakaryocytes generate long cytoplasmic extensions, proplatelets, which have the capacity to generate platelets. Although microtubules are the main structural component of proplatelets and microtubule sliding is known to drive proplatelet elongation, the role of actin dynamics in the process of platelet formation has remained elusive. Here, we tailored a mouse model lacking all ADF/n-cofilin-mediated actin dynamics in megakaryocytes to specifically elucidate the role of actin filament turnover in platelet formation. We demonstrate, for the first time, that in vivo actin filament turnover plays a critical role in the late stages of platelet formation from megakaryocytes and the proper sizing of platelets in the periphery. Our results provide the genetic proof that platelet production from megakaryocytes strictly requires dynamic changes in the actin cytoskeleton.

• Multiple alterations of platelet functions dominated by increased secretion in mice lacking Cdc42 in platelets.

  Authors: Irina Pleines, Anita Eckly, Margitta Elvers, Ina Hagedorn, Sandra Eliautou, Markus Bender, Xunwei Wu, Francois Lanza, Christian Gachet, Cord Brakebusch, Bernhard Nieswandt

  Blood. 02/2010; 115(16):3364-73.

  Platelet activation at sites of vascular injury is crucial for hemostasis, but it may also cause myocardial infarction or stroke. Cytoskeletal reorganization is essential for platelet activation andPlatelet activation at sites of vascular injury is crucial for hemostasis, but it may also cause myocardial infarction or stroke. Cytoskeletal reorganization is essential for platelet activation and secretion. The small GTPase Cdc42 has been implicated as an important mediator of filopodia formation and exocytosis in various cell types, but its exact function in platelets is not established. Here, we show that the megakaryocyte/platelet-specific loss of Cdc42 leads to mild thrombocytopenia and a small increase in platelet size in mice. Unexpectedly, Cdc42-deficient platelets were able to form normally shaped filopodia and spread fully on fibrinogen upon activation, whereas filopodia formation upon selective induction of GPIb signaling was reduced compared with wild-type platelets. Furthermore, Cdc42-deficient platelets showed enhanced secretion of alpha granules, a higher adenosine diphosphate (ADP)/adenosine triphosphate (ATP) content, increased aggregation at low agonist concentrations, and enhanced aggregate formation on collagen under flow. In vivo, lack of Cdc42 resulted in faster occlusion of ferric chloride-injured arterioles. The life span of Cdc42-deficient platelets was markedly reduced, suggesting increased clearing of the cells under physiologic conditions. These data point to novel multiple functions of Cdc42 in the regulation of platelet activation, granule organization, degranulation, and a specific role in GPIb signaling.

• Impaired alpha(IIb)beta(3) integrin activation and shear-dependent thrombus formation in mice lacking phospholipase D1.

  Authors: Margitta Elvers, David Stegner, Ina Hagedorn, Christoph Kleinschnitz, Attila Braun, Marijke E J Kuijpers, Michael Boesl, Qin Chen, Johan W M Heemskerk, Guido Stoll, Michael A Frohman, Bernhard Nieswandt

  Science signaling. 01/2010; 3(103):ra1.

  Platelet aggregation is essential for hemostasis but can also cause myocardial infarction and stroke. A key but poorly understood step in platelet activation is the shift of the principal adhesivePlatelet aggregation is essential for hemostasis but can also cause myocardial infarction and stroke. A key but poorly understood step in platelet activation is the shift of the principal adhesive receptor, alpha(IIb)beta(3) integrin, from a low- to high-affinity state for its ligands, a process that enables adhesion and aggregation. In response to stimulation of heterotrimeric guanosine triphosphate-binding protein or immunoreceptor tyrosine-based activation motif-coupled receptors, phospholipases cleave membrane phospholipids to generate lipid and soluble second messengers. An essential role in platelet activation has been established for phospholipase C (PLC) but not for PLD and its product phosphatidic acid. Here, we report that platelets from Pld1(-/-) mice displayed impaired alpha(IIb)beta(3) integrin activation in response to major agonists and defective glycoprotein Ib-dependent aggregate formation under high shear conditions. These defects resulted in protection from thrombosis and ischemic brain infarction without affecting tail bleeding times. These results indicate that PLD1 may be a critical regulator of platelet activity in the setting of ischemic cardiovascular and cerebrovascular events.

• CLEC-2 is an essential platelet activating receptor in hemostasis and thrombosis.

  Authors: Frauke May, Ina Hagedorn, Irina Pleines, Markus Bender, Timo Vogtle, Johannes Eble, Margitta Elvers, Bernhard Nieswandt

  Blood. 08/2009;

  Damage to the integrity of the vessel wall leads to exposure of the subendothelial extracellular matrix (ECM) which triggers platelet activation and aggregation. This process is essential for primaryDamage to the integrity of the vessel wall leads to exposure of the subendothelial extracellular matrix (ECM) which triggers platelet activation and aggregation. This process is essential for primary hemostasis but it may also lead to arterial thrombosis. While the mechanisms underlying platelet activation on the ECM are well explored, it is less clear which receptors mediate cellular activation in a growing thrombus. Here we studied the role of the recently identified C-type lectin-like receptor 2 (CLEC-2) in this process. We show that anti-CLEC-2 antibody treatment of mice leads to complete and highly specific loss of CLEC-2 in circulating platelets for several days. CLEC-2-deficient platelets displayed normal adhesion under flow but subsequent aggregate formation was severely defective in vitro and in vivo. As a consequence, CLEC-2 deficiency was associated with increased bleeding times and profound protection from occlusive arterial thrombus formation. These results reveal an essential function of CLEC-2 in hemostasis and thrombosis.

• Rac1 is essential for phospholipase C-gamma2 activation in platelets.

  Authors: Irina Pleines, Margitta Elvers, Amrei Strehl, Miroslava Pozgajova, David Varga-Szabo, Frauke May, Anna Chrostek-Grashoff, Cord Brakebusch, Bernhard Nieswandt

  Pflugers Archiv : European journal of physiology. 09/2008;

  Platelet activation at sites of vascular injury is triggered through different signaling pathways leading to activation of phospholipase (PL) Cbeta or PLCgamma2. Active PLCs trigger Ca(2+)Platelet activation at sites of vascular injury is triggered through different signaling pathways leading to activation of phospholipase (PL) Cbeta or PLCgamma2. Active PLCs trigger Ca(2+) mobilization and entry, which is a prerequisite for adhesion, secretion, and thrombus formation. PLCbeta isoenzymes are activated downstream of G protein-coupled receptors (GPCRs), whereas PLCgamma2 is activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as the major platelet collagen receptor glycoprotein (GP) VI or CLEC-2. The mechanisms underlying PLC regulation are not fully understood. An involvement of small GTPases of the Rho family (Rho, Rac, Cdc42) in PLC activation has been proposed but this has not been investigated in platelets. We here show that murine platelets lacking Rac1 display severely impaired GPVI- or CLEC-2-dependent activation and aggregation. This defect was associated with impaired production of inositol 1,4,5-trisphosphate (IP(3)) and intracellular calcium mobilization suggesting inappropriate activation of PLCgamma2 despite normal tyrosine phosphorylation of the enzyme. Rac1 ( -/- ) platelets displayed defective thrombus formation on collagen under flow conditions which could be fully restored by co-infusion of ADP and the TxA(2) analog U46619, indicating that impaired GPVI-, but not G-protein signaling, was responsible for the observed defect. In line with this, Rac1 ( -/- ) mice were protected in two collagen-dependent arterial thrombosis models. Together, these results demonstrate that Rac1 is essential for ITAM-dependent PLCgamma2 activation in platelets and that this is critical for thrombus formation in vivo.