Markus Bender

Publications of Markus Bender

• Differentially regulated GPVI ectodomain shedding by multiple platelet-expressed proteinases.

  Authors: Markus Bender, Sebastian Hofmann, David Stegner, Athena Chalaris, Michael Bösl, Attila Braun, Jürgen Scheller, Stefan Rose-John, Bernhard Nieswandt

  Blood. 10/2010; 116(17):3347-55.

  Glycoprotein VI (GPVI) mediates platelet activation on exposed subendothelial collagens at sites of vascular injury and thereby contributes to normal hemostasis, but also to the occlusion of diseasedGlycoprotein VI (GPVI) mediates platelet activation on exposed subendothelial collagens at sites of vascular injury and thereby contributes to normal hemostasis, but also to the occlusion of diseased vessels in the setting of myocardial infarction or stroke. GPVI is an attractive target for antithrombotic therapy, particularly because previous studies have shown that anti-GPVI antibodies induce irreversible down-regulation of the receptor in circulating platelets by internalization and/or ectodomain shedding. Metalloproteinases of the a disintegrin and metalloproteinase (ADAM) family have been proposed to mediate this ectodomain shedding, but direct evidence for this is lacking. Here, we studied GPVI shedding in vitro and in vivo in newly generated mice with a megakaryocyte-specific ADAM10 deficiency and in Adam17(ex/ex) mice, which lack functional ADAM17. We demonstrate that GPVI cleavage in vitro can occur independently through either ADAM10 or ADAM17 in response to distinct stimuli. In contrast, antibody (JAQ1)-induced GPVI shedding in vivo occurred in mice lacking both ADAM10/ADAM17 in their platelets, suggesting the existence of a third GPVI cleaving platelet enzyme. This was supported by in vitro studies on ADAM10/ADAM17 double-deficient platelets. These results reveal that ectodomain shedding of GPVI can be mediated through multiple differentially regulated platelet-expressed proteinases with obvious therapeutic implications.

• 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.

• 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.

• Orai1 (CRACM1) is the platelet SOC channel and essential for pathological thrombus formation.

  Authors: Attila Braun, David Varga-Szabo, Christoph Kleinschnitz, Irina Pleines, Markus Bender, Madeleine Austinat, Michael Bosl, Guido Stoll, Bernhard Nieswandt

  Blood. 11/2008;

  Platelet activation and aggregation at sites of vascular injury is essential for primary hemostasis, but is also a major pathomechanism underlying myocardial infarction and stroke. Changes inPlatelet activation and aggregation at sites of vascular injury is essential for primary hemostasis, but is also a major pathomechanism underlying myocardial infarction and stroke. Changes in [Ca(2+)]i are a central step in platelet activation. In non-excitable cells receptor-mediated depletion of intracellular Ca(2+) stores triggers Ca(2+) entry through store-operated calcium (SOC) channels. STIM1 has been identified as an endoplasmic reticulum (ER)-resident Ca(2+) sensor that regulates store-operated calcium entry (SOCE) in immune cells and platelets, but the identity of the platelet SOC channel has remained elusive. Orai1 (CRACM1) is the recently discovered SOC (CRAC) channel in T-cells and mast cells but its role in mammalian physiology is unknown. Here we report that Orai1 is strongly expressed in human and mouse platelets. To test its role in blood clotting, we generated Orai1-deficient mice and found that their platelets display severely defective SOCE, agonist-induced Ca(2+) responses, and impaired activation and thrombus formation under flow in vitro. As a direct consequence, Orai1-deficiency in mice results in resistance to pulmonary thromboembolism, arterial thrombosis and ischemic brain infarction, but only a mild bleeding time prolongation. These results establish Orai1 as the long-sought platelet SOC channel and a crucial mediator of ischemic cardio- and cerebrovascular events.

• The calcium sensor STIM1 is an essential mediator of arterial thrombosis and ischemic brain infarction.

  Authors: David Varga-Szabo, Attila Braun, Christoph Kleinschnitz, Markus Bender, Irina Pleines, Mirko Pham, Thomas Renné, Guido Stoll, Bernhard Nieswandt

  The Journal of experimental medicine. 08/2008; 205(7):1583-91.

  Platelet activation and aggregation are essential to limit posttraumatic blood loss at sites of vascular injury but also contributes to arterial thrombosis, leading to myocardial infarction andPlatelet activation and aggregation are essential to limit posttraumatic blood loss at sites of vascular injury but also contributes to arterial thrombosis, leading to myocardial infarction and stroke. Agonist-induced elevation of [Ca(2+)](i) is a central step in platelet activation, but the underlying mechanisms are not fully understood. A major pathway for Ca(2+) entry in nonexcitable cells involves receptor-mediated release of intracellular Ca(2+) stores, followed by activation of store-operated calcium (SOC) channels in the plasma membrane. Stromal interaction molecule 1 (STIM1) has been identified as the Ca(2+) sensor in the endoplasmic reticulum (ER) that activates Ca(2+) release-activated channels in T cells, but its role in mammalian physiology is unknown. Platelets express high levels of STIM1, but its exact function has been elusive, because these cells lack a normal ER and Ca(2+) is stored in a tubular system referred to as the sarcoplasmatic reticulum. We report that mice lacking STIM1 display early postnatal lethality and growth retardation. STIM1-deficient platelets have a marked defect in agonist-induced Ca(2+) responses, and impaired activation and thrombus formation under flow in vitro. Importantly, mice with STIM1-deficient platelets are significantly protected from arterial thrombosis and ischemic brain infarction but have only a mild bleeding time prolongation. These results establish STIM1 as an important mediator in the pathogenesis of ischemic cardio- and cerebrovascular events.

• Store-operated Ca(2+) entry in platelets occurs independently of transient receptor potential (TRP) C1.

  Authors: David Varga-Szabo, Kalwant Authi, Attila Braun, Markus Bender, Archana Ambily, Sheila Hassock, Thomas Gudermann, Alexander Dietrich, Bernhard Nieswandt

  Pflugers Archiv : European journal of physiology. 07/2008;

  Changes in [Ca(2+)](i) are a central step in platelet activation. In nonexcitable cells, receptor-mediated depletion of intracellular Ca(2+) stores triggers Ca(2+) entry through store-operatedChanges in [Ca(2+)](i) are a central step in platelet activation. In nonexcitable cells, receptor-mediated depletion of intracellular Ca(2+) stores triggers Ca(2+) entry through store-operated calcium (SOC) channels. Stromal interaction molecule 1 (STIM1) has been identified as an endoplasmic reticulum (ER)-resident Ca(2+) sensor that regulates store-operated calcium entry (SOCE), but the identity of the SOC channel in platelets has been controversially debated. Some investigators proposed transient receptor potential (TRP) C1 to fulfil this function based on the observation that antibodies against the channel impaired SOCE in platelets. However, others could not detect TRPC1 in the plasma membrane of platelets and raised doubts about the specificity of the inhibiting anti-TRPC1 antibodies. To address the role of TRPC1 in SOCE in platelets, we analyzed mice lacking TRPC1. Platelets from these mice display fully intact SOCE and also otherwise unaltered calcium homeostasis compared to wild-type. Furthermore, platelet function in vitro and in vivo is not altered in the absence of TRPC1. Finally, studies on human platelets revealed that the presumably inhibitory anti-TRPC1 antibodies have no specific effect on SOCE and fail to bind to the protein. Together, these results provide evidence that SOCE in platelets is mediated by channels other than TRPC1.

• Diverging signaling events control the pathway of GPVI down-regulation in vivo.

  Authors: Tamer Rabie, David Varga-Szabo, Markus Bender, Rastislav Pozgaj, Francois Lanza, Takashi Saito, Stephen P Watson, Bernhard Nieswandt

  Blood. 08/2007; 110(2):529-35.

  Coronary artery thrombosis is often initiated by platelet activation on collagen-rich subendothelial layers in the disrupted atherosclerotic plaque. The activating platelet collagen receptorCoronary artery thrombosis is often initiated by platelet activation on collagen-rich subendothelial layers in the disrupted atherosclerotic plaque. The activating platelet collagen receptor glycoprotein VI (GPVI) noncovalently associates with the Fc receptor gamma-chain (FcRgamma), which signals through its immunoreceptor-tyrosine-based activation motif (ITAM) via the adaptor LAT leading to the activation of phospholipase Cgamma2 (PLCgamma2). GPVI is a promising antithrombotic target as anti-GPVI antibodies induce the irreversible loss of the receptor from circulating platelets by yet undefined mechanisms in humans and mice and long-term antithrombotic protection in the latter. However, the treatment is associated with transient but severe thrombocytopenia and reduced platelet reactivity to thrombin questioning its clinical usefulness. Here we show that GPVI down-regulation occurs through 2 distinct pathways, namely ectodomain shedding or internalization/intracellular clearing, and that both processes are abrogated in mice carrying a point mutation in the FcRgamma-associated ITAM. In mice lacking LAT or PLCgamma2, GPVI shedding is abolished, but the receptor is irreversibly down-regulated through internalization/intracellular clearing. This route of GPVI loss is not associated with thrombocytopenia or altered thrombin responses. These results reveal the existence of 2 distinct signaling pathways downstream of the FcRgamma-ITAM and show that it is possible to uncouple GPVI down-regulation from undesired side effects with obvious therapeutic implications.