Ildiko Konrad

Publications (18)206.02 Total impact

• Article: The dimeric platelet collagen receptor GPVI-Fc reduces platelet adhesion to activated endothelium and preserves myocardial function after transient ischemia in mice.

  Tanja Schönberger, Melanie Ziegler, Oliver Borst, Ildiko Konrad, Bernhard Nieswandt, Steffen Massberg, Carmen Ochmann, Tobias Jürgens, Peter Seizer, Harald Langer, Götz Münch, Martin Ungerer, Klaus T Preissner, Margitta Elvers, Meinrad Gawaz
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  ABSTRACT: Platelets play a critical role in the pathophysiology of reperfusion, sepsis, and cardiovascular diseases. In a multiple step process, they adhere to activated endothelium and release proinflammatory cytokines thereby promoting the inflammatory process. Glycoprotein VI (GPVI) is the major collagen receptor on the platelet surface and triggers platelet activation and primary hemostasis. Activation of GPVI leads to stable platelet adhesion and degranulation of platelet granules. However, GPVI is critically involved in platelet adhesion to activated endothelium without exposure of subendothelial matrix. Earlier studies show that the soluble GPVI-Fc binds to collagen and protects mice from atherosclerosis and decreases neointima proliferation after arterial injury. Here, we show for the first time that recombinant GPVI-Fc binds to activated endothelium mainly via vitronectin and prevents platelet/endothelial interaction. Administration of GPVI-Fc reduced infarct size and preserved cardiac function in a mouse model of myocardial infarction. This process was associated with reduced GPVI-induced platelet degranulation and release of proinflammatory cytokines in vitro and in vivo. Taken together, administration of GPVI-Fc offers a novel strategy to control platelet-mediated inflammation and to preserve myocardial function following myocardial infarction.
  AJP Cell Physiology 07/2012; 303(7):C757-66. · 3.54 Impact Factor
• Source

  Available from: Siegmund Braun

  Article: Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo.

  Marie-Luise von Brühl, Konstantin Stark, Alexander Steinhart, Sue Chandraratne, Ildiko Konrad, Michael Lorenz, Alexander Khandoga, Anca Tirniceriu, Raffaele Coletti, Maria Köllnberger, [......], Martina Rudelius, Christian Schulz, Katrin Echtler, Volker Brinkmann, Markus Schwaiger, Klaus T Preissner, Denisa D Wagner, Nigel Mackman, Bernd Engelmann, Steffen Massberg
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  ABSTRACT: Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features.
  Journal of Experimental Medicine 03/2012; 209(4):819-35. · 13.85 Impact Factor
• Article: Impact of glycoprotein VI and platelet adhesion on atherosclerosis--a possible role of fibronectin.

  Andreas Bültmann, Zhongmin Li, Silvia Wagner, Mario Peluso, Tanja Schönberger, Carla Weis, Ildiko Konrad, Konstantinos Stellos, Steffen Massberg, Bernhard Nieswandt, Meinrad Gawaz, Martin Ungerer, Götz Münch
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  ABSTRACT: Glycoprotein VI (GPVI) mediates binding of platelets to subendothelial collagen during acute arterial thrombosis. GPVI interactions with the activated atherosclerotic vascular endothelium during early atherosclerosis, however, are not well understood. In ApoE-/- mice, platelet adhesion to atherosclerotic arteries was increased, as measured by intravital microscopy. This platelet adhesion was significantly inhibited by IV injection of GPVI-Fc (1 mg/kg body weight). Atherosclerosis in ApoE-/- mice was attenuated both after 7 and 10 weeks of treatment with the anti-GPVI antibody JAQ1 (2 mg/kg body weight i.p. twice weekly). Binding of GPVI-Fc (1 mg/kg IV) occurred to deeper layers, but also to the luminal site of plaques in atherosclerotic rabbits, but not to the vessel wall of healthy littermates. Gene transfer of GPVI-Fc to the carotid vascular wall significantly attenuated athero-progression and endothelial dysfunction in atherosclerotic rabbits in vivo. Specific binding of the soluble GPVI receptor (GPVI-Fc) to fibronectin was found in vitro to coated ELISA plates. Platelet adhesion to fibronectin was significantly inhibited both by GPVI-Fc and by the anti-GPVI antibody 5C4 ex vivo in flow chamber experiments. GPVI plays a role in platelet adhesion to atherosclerotic endothelium in the absence of plaque rupture. Inhibition of GPVI both via GPVI-Fc and anti-GPVI-antibodies results in protection against atherosclerosis in both cholesterol-fed rabbits and ApoE-/- mice. This novel mechanism of GPVI-mediated platelet adhesion-possibly via fibronectin-could relevantly contribute to platelet-triggered atheroprogression.
  Journal of Molecular and Cellular Cardiology 09/2010; 49(3):532-42. · 5.17 Impact Factor
• Source

  Available from: Siegmund Braun

  Article: Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases.

  Steffen Massberg, Lenka Grahl, Marie-Luise von Bruehl, Davit Manukyan, Susanne Pfeiler, Christian Goosmann, Volker Brinkmann, Michael Lorenz, Kiril Bidzhekov, Avinash B Khandagale, Ildiko Konrad, Elisabeth Kennerknecht, Katja Reges, Stefan Holdenrieder, Siegmund Braun, Christoph Reinhardt, Michael Spannagl, Klaus T Preissner, Bernd Engelmann
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  ABSTRACT: Blood neutrophils provide the first line of defense against pathogens but have also been implicated in thrombotic processes. This dual function of neutrophils could reflect an evolutionarily conserved association between blood coagulation and antimicrobial defense, although the molecular determinants and in vivo significance of this association remain unclear. Here we show that major microbicidal effectors of neutrophils, the serine proteases neutrophil elastase and cathepsin G, together with externalized nucleosomes, promote coagulation and intravascular thrombus growth in vivo. The serine proteases and extracellular nucleosomes enhance tissue factor- and factor XII-dependent coagulation in a process involving local proteolysis of the coagulation suppressor tissue factor pathway inhibitor. During systemic infection, activation of coagulation fosters compartmentalization of bacteria in liver microvessels and reduces bacterial invasion into tissue. In the absence of a pathogen challenge, neutrophil-derived serine proteases and nucleosomes can contribute to large-vessel thrombosis, the main trigger of myocardial infarction and stroke. The ability of coagulation to suppress pathogen dissemination indicates that microvessel thrombosis represents a physiological tool of host defense.
  Nature medicine 08/2010; 16(8):887-96. · 27.14 Impact Factor
• Article: Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via tissue factor activation.

  Christoph Reinhardt, Marie-Luise von Brühl, Davit Manukyan, Lenka Grahl, Michael Lorenz, Berid Altmann, Silke Dlugai, Sonja Hess, Ildiko Konrad, Lena Orschiedt, Nigel Mackman, Lloyd Ruddock, Steffen Massberg, Bernd Engelmann
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  ABSTRACT: The activation of initiator protein tissue factor (TF) is likely to be a crucial step in the blood coagulation process, which leads to fibrin formation. The stimuli responsible for inducing TF activation are largely undefined. Here we show that the oxidoreductase protein disulfide isomerase (PDI) directly promotes TF-dependent fibrin production during thrombus formation in vivo. After endothelial denudation of mouse carotid arteries, PDI was released at the injury site from adherent platelets and disrupted vessel wall cells. Inhibition of PDI decreased TF-triggered fibrin formation in different in vivo murine models of thrombus formation, as determined by intravital fluorescence microscopy. PDI infusion increased - and, under conditions of decreased platelet adhesion, PDI inhibition reduced - fibrin generation at the injury site, indicating that PDI can directly initiate blood coagulation. In vitro, human platelet-secreted PDI contributed to the activation of cryptic TF on microvesicles (microparticles). Mass spectrometry analyses indicated that part of the extracellular cysteine 209 of TF was constitutively glutathionylated. Mixed disulfide formation contributed to maintaining TF in a state of low functionality. We propose that reduced PDI activates TF by isomerization of a mixed disulfide and a free thiol to an intramolecular disulfide. Our findings suggest that disulfide isomerases can act as injury response signals that trigger the activation of fibrin formation following vessel injury.
  Journal of Clinical Investigation 04/2008; 118(3):1110-22. · 15.39 Impact Factor
• Article: Effect of chronic treatment with acetylsalicylic acid and clopidogrel on atheroprogression and atherothrombosis in ApoE-deficient mice in vivo.

  Christian Schulz, Ildiko Konrad, Susanne Sauer, Lena Orschiedt, Maria Koellnberger, Reinhard Lorenz, Ulrich Walter, Steffen Massberg
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  ABSTRACT: Acetylsalicylic acid (ASA) and the thienopyridine clopidogrel are established anti-platelet drugs that significantly reduce secondary cardiovascular events in patients with manifest atherosclerosis. However, their impact on atherosclerotic lesion development remains controversial. Four-week-old ApoE-deficient mice were randomly assigned to four groups receiving a cholesterol diet together with either ASA (5 mg/kg), or clopidogrel (25 mg/kg), or a combination of both ASA and clopidogrel, or vehicle for 8-12 weeks. Using intravital microscopy we found that daily administration of ASA in combination with clopidogrel reduces platelet thrombus formation following rupture of atherosclerotic plaque in vivo by approximately 50%. However, therapy with ASA or clopidogrel alone, or in combination for a period of 8-12 weeks had no significant effect on adhesion of platelets to dysfunctional endothelial cells or on atherosclerotic lesion formation in the aortic root or the carotid artery. In conclusion, anti-platelet therapy is effective in reducing platelet adhesion and subsequent thrombus formation following rupture of atherosclerotic plaque in vivo. However, our data do not support a role of either drug in the primary prevention of atherosclerosis in ApoE-deficient mice.
  Thrombosis and Haemostasis 02/2008; 99(1):190-5. · 5.04 Impact Factor
• Article: EXP3179 inhibits collagen-dependent platelet activation via glycoprotein receptor-VI independent of AT1-receptor antagonism: potential impact on atherothrombosis.

  Christina Grothusen, Sumaira Umbreen, Ildiko Konrad, Konstantinos Stellos, Christian Schulz, Boris Schmidt, Elisabeth Kremmer, Omke Teebken, Steffen Massberg, Maren Luchtefeld, Bernhard Schieffer, Meinrad Gawaz
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  ABSTRACT: Thrombus formation after atherosclerotic plaque rupture critically involves the platelet collagen receptor glycoprotein (GP) VI. We investigated the impact of EXP3179, an active metabolite of the angiotensin II type 1 (AT1)-receptor antagonist Losartan (LOS) on GPVI-dependent platelet activation. EXP3179 and LOS but not EXP3174--the major AT1-receptor blocking metabolite of LOS--dose-dependently inhibited collagen-I (P<0.01) and GPVI-dependent platelet aggregation (P<0.01) analyzed by optical aggregometry. Platelet activation was further determined by flow cytometry measuring the expression of platelet PAC-1, an epitope of the activated fibrinogen-receptor complex. EXP3179 and LOS inhibited collagen-I (P<0.01) and GPVI-dependent PAC-1 expression (P<0.01). EXP3179 and LOS but not EXP3174 decreased the adhesion of GPVI-receptor expressing Chinese hamster ovarian cells on collagen-I under arterial shear conditions determined by flow chamber analysis (P<0.01 and P<0.05). EXP3179 also reduced human atherosclerotic plaque material-induced platelet aggregation (P<0.01) in vitro and murine platelet adhesion after acute vessel injury in vivo as determined by intravital microscopy (P<0.01). EXP3179 acts as a specific inhibitor of the platelet collagen receptor GPVI independent of AT1-receptor antagonism. Further investigations may clarify its individual potential as a novel pharmacological approach to specifically inhibit atherothrombotic events by GPVI-receptor blockade.
  Arteriosclerosis Thrombosis and Vascular Biology 05/2007; 27(5):1184-90. · 6.37 Impact Factor
• Article: IRAG mediates NO/cGMP-dependent inhibition of platelet aggregation and thrombus formation.

  Melanie Antl, Marie-Luise von Brühl, Christina Eiglsperger, Matthias Werner, Ildiko Konrad, Thomas Kocher, Matthias Wilm, Franz Hofmann, Steffen Massberg, Jens Schlossmann
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  ABSTRACT: Defective regulation of platelet activation/aggregation is a predominant cause for arterial thrombosis, the major complication of atherosclerosis triggering myocardial infarction and stroke. A central regulatory pathway conveying inhibition of platelet activation/aggregation is nitric oxide (NO)/cyclic GMP (cGMP) signaling by cGMP-dependent protein kinase I (cGKI). However, the regulatory cascade downstream of cGKI mediating platelet inhibition is still unclear. Here, we show that the inositol-1,4,5-trisphosphate receptor-associated cGMP kinase substrate (IRAG) is abundantly expressed in platelets and assembled in a macrocomplex together with cGKIbeta and the inositol-1,4,5-trisphosphate receptor type I (InsP3RI). cGKI phosphorylates IRAG at Ser664 and Ser677 in intact platelets. Targeted deletion of the IRAG-InsP3RI interaction in IRAGDelta12/Delta12 mutant mice leads to a loss of NO/cGMP-dependent inhibition of fibrinogen-receptor activation and platelet aggregation. Intracellular calcium transients were not affected by DEA/NO or cGMP in mutant platelets. Furthermore, intravital microscopy shows that NO fails to prevent arterial thrombosis of the injured carotid artery in IRAGDelta12/Delta12 mutants. These findings reveal that interaction between IRAG and InsP3RI has a central role in NO/cGMP-dependent inhibition of platelet aggregation and in vivo thrombosis.
  Blood 02/2007; 109(2):552-9. · 9.90 Impact Factor
• Source

  Available from: Matthias Schiemann

  Article: Platelets secrete stromal cell-derived factor 1alpha and recruit bone marrow-derived progenitor cells to arterial thrombi in vivo.

  Steffen Massberg, Ildiko Konrad, Katrin Schürzinger, Michael Lorenz, Simon Schneider, Dietlind Zohlnhoefer, Katharina Hoppe, Matthias Schiemann, Elisabeth Kennerknecht, Susanne Sauer, [......], Stefan Seidl, Falko Sorge, Harald Langer, Mario Peluso, Pankaj Goyal, Dietmar Vestweber, Nikla R Emambokus, Dirk H Busch, Jon Frampton, Meinrad Gawaz
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  ABSTRACT: The accumulation of smooth muscle and endothelial cells is essential for remodeling and repair of injured blood vessel walls. Bone marrow-derived progenitor cells have been implicated in vascular repair and remodeling; however, the mechanisms underlying their recruitment to the site of injury remain elusive. Here, using real-time in vivo fluorescence microscopy, we show that platelets provide the critical signal that recruits CD34+ bone marrow cells and c-Kit+ Sca-1+ Lin- bone marrow-derived progenitor cells to sites of vascular injury. Correspondingly, specific inhibition of platelet adhesion virtually abrogated the accumulation of both CD34+ and c-Kit+ Sca-1+ Lin- bone marrow-derived progenitor cells at sites of endothelial disruption. Binding of bone marrow cells to platelets involves both P-selectin and GPIIb integrin on platelets. Unexpectedly, we found that activated platelets secrete the chemokine SDF-1alpha, thereby supporting further primary adhesion and migration of progenitor cells. These findings establish the platelet as a major player in the initiation of vascular remodeling, a process of fundamental importance for vascular repair and pathological remodeling after vascular injury.
  Journal of Experimental Medicine 06/2006; 203(5):1221-33. · 13.85 Impact Factor
• Source

  Available from: Pankaj Goyal

  Article: Platelets secrete stromal cell–derived factor 1α and recruit bone marrow–derived progenitor cells to arterial thrombi in vivo

  Steffen Massberg, Ildiko Konrad, Katrin Schürzinger, Michael Lorenz, Simon Schneider, Dietlind Zohlnhoefer, Katharina Hoppe, Matthias Schiemann, Elisabeth Kennerknecht, Susanne Sauer, [......], Stefan Seidl, Falko Sorge, Harald Langer, Mario Peluso, Pankaj Goyal, Dietmar Vestweber, Nikla R. Emambokus, Dirk H. Busch, Jon Frampton, Meinrad Gawaz
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  ABSTRACT: The accumulation of smooth muscle and endothelial cells is essential for remodeling and repair of injured blood vessel walls. Bone marrow–derived progenitor cells have been implicated in vascular repair and remodeling; however, the mechanisms underlying their recruitment to the site of injury remain elusive. Here, using real-time in vivo fluorescence microscopy, we show that platelets provide the critical signal that recruits CD34+ bone marrow cells and c-Kit+ Sca-1+ Lin− bone marrow–derived progenitor cells to sites of vascular injury. Correspondingly, specific inhibition of platelet adhesion virtually abrogated the accumulation of both CD34+ and c-Kit+ Sca-1+ Lin− bone marrow–derived progenitor cells at sites of endothelial disruption. Binding of bone marrow cells to platelets involves both P-selectin and GPIIb integrin on platelets. Unexpectedly, we found that activated platelets secrete the chemokine SDF-1α, thereby supporting further primary adhesion and migration of progenitor cells. These findings establish the platelet as a major player in the initiation of vascular remodeling, a process of fundamental importance for vascular repair and pathological remodeling after vascular injury.
  Journal of Experimental Medicine 05/2006; 203(5):1221-1233. · 13.85 Impact Factor
• Source

  Available from: ahajournals.org

  Article: Retroviral infection and selection of culture-derived platelets allows study of the effect of transgenes on platelet physiology ex vivo and on thrombus formation in vivo.

  Angelika Gillitzer, Mario Peluso, Karl-Ludwig Laugwitz, Götz Münch, Steffen Massberg, Ildiko Konrad, Meinrad Gawaz, Martin Ungerer
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  ABSTRACT: Background- We recently reported the development of culture-derived (CD) platelets with the aim to express any protein of interest in these platelets. We now report a specific protocol of retroviral infection into the progenitor cells and subsequent selection, which allows to generate large amounts of highly homogenous transgene-expressing CD platelets and to study transgene function rapidly and reliably at large-scale ex vivo and in vivo settings. METHODS AND RESULTS: After retroviral infection and selection, the activation-dependent expression profile of surface markers, aggregation, and granule release were investigated. The function of transgene-expressing CD platelets, the precursor cells of which had been retrovirally infected, compared well to noninfected CD platelets or freshly isolated platelets. Hence, the retroviral infection protocol did not alter platelet physiology. In contrast, adenoviral infection of precursors to CD platelets resulted in marked functional alterations that obviated their use in analytic experiments. Additionally, sufficient amounts of selected CD platelets were generated to warrant intravenous injections into living mice. This approach permitted study of their adhesive profile at endothelial lesions and their effect on thrombus formation in vivo by intravital videofluorescence microscopy. CONCLUSIONS: The novel selection method allowed us to produce recombinant transgene-expressing platelets in sufficient amounts to study genetically modified platelets in vitro and in vivo.
  Arteriosclerosis Thrombosis and Vascular Biology 09/2005; 25(8):1750-5. · 6.37 Impact Factor
• Article: Platelet adhesion via glycoprotein IIb integrin is critical for atheroprogression and focal cerebral ischemia: an in vivo study in mice lacking glycoprotein IIb.

  Steffen Massberg, Katrin Schürzinger, Michael Lorenz, Ildiko Konrad, Christian Schulz, Nikolaus Plesnila, Elisabeth Kennerknecht, Martina Rudelius, Susanne Sauer, Siegmund Braun, Elisabeth Kremmer, Nikla R Emambokus, Jon Frampton, Meinrad Gawaz
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  ABSTRACT: The platelet glycoprotein (GP) IIb/IIIa integrin binds to fibrinogen and thereby mediates platelet aggregation. Here, we addressed the role of GP IIb for platelet adhesion and determined the relevance of platelet GP IIb for the processes of atherosclerosis and cerebral ischemia-reperfusion (I/R) injury. GP IIb(-/-) mice were generated and bred with ApoE(-/-) animals to create GP IIb(-/-)ApoE(-/-) mice. Platelet adhesion to the mechanically injured or atherosclerotic vessel wall was monitored by in vivo video fluorescence microscopy. In the presence of GP IIb, vascular injury and early atherosclerosis induced platelet adhesion in the carotid artery (CA). In contrast, platelet adhesion was significantly reduced in the absence of GP IIb integrin (P<0.05). To address the contribution of platelet GP IIb to atheroprogression, we determined atherosclerotic lesion formation in the CA and aortic arch (AA) of GP IIb(+/+)ApoE(-/-) or GP IIb(-/-)ApoE(-/-) mice. Interestingly, the absence of GP IIb attenuated lesion formation in CA and AA, indicating that platelets, via GP IIb, contribute substantially to atherosclerosis. Next, we assessed the implication of GP IIb for cerebral I/R injury. We observed that after occlusion of the middle cerebral artery, the cerebral infarct size was drastically reduced in mice lacking GP IIb compared with wild-types. These findings show for the first time in vivo that GP IIb not only mediates platelet aggregation but also triggers platelet adhesion to exposed extracellular matrices and dysfunctional endothelial cells. In a process strictly involving GP IIb, platelets, which are among the first blood cells to arrive at the scene of endothelial dysfunction, contribute essentially to atherosclerosis and cerebral I/R injury.
  Circulation 08/2005; 112(8):1180-8. · 14.74 Impact Factor
• Article: Non-invasive imaging of glycoprotein VI binding to injured arterial lesions.

  Meinrad Gawaz, Ildiko Konrad, Andrea I Hauser, Suzanne Sauer, Zhongyan Li, Hans-Jürgen Wester, Frank M Bengel, Markus Schwaiger, Albert Schömig, Steffen Massberg, Roland Haubner
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  ABSTRACT: Glycoprotein VI (GPVI) is the major platelet collagen receptor and plays a critical role in the process of thrombosis at sites of atherosclerotic lesions. This study evaluates the feasibility of radiolabeled soluble GPVI to identify injured arterial lesions. Radiolabeling was carried out using the iodogen method and resulted in the radioiodinated GPVI in radiochemical yields between 97-100%. The biodistribution of [(125)I]GPVI was determined in normal mice and demonstrated a blood clearance halftime of approximately 5.5 hours. Vascular lesions were induced in the carotid artery in wild type and ApoE(-/-)mice. Immediately after injury radioiodinated GPVI was injected intravenously. Binding of [(123)I]GPVI to carotid lesions was assessed by szintigraphic in vivo imaging. Carotid arteries were explanted for ex vivo autoradiography and histological characterization of the lesion. In vivo and ex vivo imaging revealed substantial accumulation of radioiodinated GPVI in the injured artery wall, with a ratio of lesion to control vessel of 3:1 and 7:1, respectively. Because GPVI is the critical collagen receptor that mediates platelet adhesion to vascular lesions, soluble radiolabeled GPVI may be an agent for non-invasive imaging of thrombogenic thus, vulnerable atherosclerotic plaques.
  Thrombosis and Haemostasis 06/2005; 93(5):910-3. · 5.04 Impact Factor
• Article: Soluble glycoprotein VI dimer inhibits platelet adhesion and aggregation to the injured vessel wall in vivo.

  Steffen Massberg, Ildiko Konrad, Andreas Bültmann, Christian Schulz, Götz Münch, Mario Peluso, Michael Lorenz, Simon Schneider, Felicitas Besta, Iris Müller, Bin Hu, Harald Langer, Elisabeth Kremmer, Martina Rudelius, Ulrich Heinzmann, Martin Ungerer, Meinrad Gawaz
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  ABSTRACT: Platelet-collagen interactions play a fundamental role in the process of arterial thrombosis. The major platelet collagen receptor is the glycoprotein VI (GPVI). Here, we determined the effects of a soluble dimeric form of GPVI on platelet adhesion in vitro and in vivo. We fused the extracellular domain of GPVI with the human immunoglobulin Fc domain. The soluble dimeric form of GPVI (GPVI-Fc) specifically bound to immobilized collagen. Binding of GPVI-Fc to collagen was inhibited competitively by soluble GPVI-Fc, but not control Fc lacking the external GPVI domain. GPVI-Fc inhibited the adhesion of CHO cells that stably express human GPVI and of platelets on collagen and attenuated thrombus formation under shear conditions in vitro. To test the effects of GPVI-Fc in vivo, arterial thrombosis was induced in the mouse carotid artery, and platelet-vessel wall interactions were visualized by intravital fluorescence microscopy. Infusion of GPVI-Fc but not of control Fc virtually abolished stable arrest and aggregation of platelets following vascular injury. Importantly, GPVI-Fc but not control Fc, was detected at areas of vascular injury. These findings further substantiate the critical role of the collagen receptor GPVI in the initiation of thrombus formation at sites of vascular injury and identify soluble GPVI as a promising antithrombotic strategy.
  The FASEB Journal 03/2004; 18(2):397-9. · 5.71 Impact Factor
• Article: Enhanced in vivo platelet adhesion in vasodilator-stimulated phosphoprotein (VASP)-deficient mice.

  Steffen Massberg, Sabine Grüner, Ildiko Konrad, Maisa I Garcia Arguinzonis, Martin Eigenthaler, Kathrin Hemler, Julia Kersting, Christian Schulz, Iris Muller, Felicitas Besta, Bernhard Nieswandt, Ulrich Heinzmann, Ulrich Walter, Meinrad Gawaz
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  ABSTRACT: Platelet adhesion and activation at the vascular wall are the initial steps leading to arterial thrombosis and vascular occlusion. Prostacyclin and nitric oxide inhibit platelet adhesion, acting via cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-dependent protein kinases. A major downstream target for both cAMP- and cGMP-dependent protein kinases is the vasodilator-stimulated phosphoprotein (VASP). To test the significance of VASP for the regulation of platelet adhesion in vivo, we studied platelet-vessel wall interactions using VASP-deficient (VASP-/-) mice. Under physiologic conditions, platelet adhesion to endothelial cells was significantly enhanced in VASP null mutants when compared with wild-type mice (P <.05). Platelet recruitment in VASP null mice involved P-selectin and the fibrinogen receptor glycoprotein IIb-IIIa (GPIIb-IIIa). Under pathophysiologic conditions, the loss of VASP increased platelet adhesion to the postischemic intestinal microvasculature, to the atherosclerotic endothelium of ApoE-deficient mice, and to the subendothelial matrix following endothelial denudation (P <.05 vs wild type). Importantly, platelet adhesion in VASP null mutants was unresponsive to nitric oxide. These data show for the first time in vivo that VASP is involved in down-regulation of platelet adhesion to the vascular wall under both physiologic and pathophysiologic conditions.
  Blood 01/2004; 103(1):136-42. · 9.90 Impact Factor
• Article: G13 is an essential mediator of platelet activation in hemostasis and thrombosis.

  Alexandra Moers, Bernhard Nieswandt, Steffen Massberg, Nina Wettschureck, Sabine Grüner, Ildiko Konrad, Valerie Schulte, Barsom Aktas, Marie-Pierre Gratacap, Melvin I Simon, Meinrad Gawaz, Stefan Offermanns
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  ABSTRACT: Platelet activation at sites of vascular injury is essential for primary hemostasis, but also underlies arterial thrombosis leading to myocardial infarction or stroke. Platelet activators such as adenosine diphosphate, thrombin or thromboxane A(2) (TXA(2)) activate receptors that are coupled to heterotrimeric G proteins. Activation of platelets through these receptors involves signaling through G(q), G(i) and G(z) (refs. 4-6). However, the role and relative importance of G(12) and G(13), which are activated by various platelet stimuli, are unclear. Here we show that lack of Galpha(13), but not Galpha(12), severely reduced the potency of thrombin, TXA(2) and collagen to induce platelet shape changes and aggregation in vitro. These defects were accompanied by reduced activation of RhoA and inability to form stable platelet thrombi under high shear stress ex vivo. Galpha(13) deficiency in platelets resulted in a severe defect in primary hemostasis and complete protection against arterial thrombosis in vivo. We conclude that G(13)-mediated signaling processes are required for normal hemostasis and thrombosis and may serve as a new target for antiplatelet drugs.
  Nature Medicine 12/2003; 9(11):1418-22. · 22.46 Impact Factor
• Source

  Available from: PubMed Central

  Article: A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo.

  Steffen Massberg, Meinrad Gawaz, Sabine Grüner, Valerie Schulte, Ildiko Konrad, Dietlind Zohlnhöfer, Ulrich Heinzmann, Bernhard Nieswandt
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  ABSTRACT: Platelet adhesion and aggregation at sites of vascular injury is crucial for hemostasis but may lead to arterial occlusion in the setting of atherosclerosis and precipitate diseases such as myocardial infarction. A current hypothesis suggests that platelet glycoprotein (GP) Ib interaction with von Willebrand factor recruits flowing platelets to the injured vessel wall, where subendothelial fibrillar collagens support their firm adhesion and activation. However, so far this hypothesis has not been tested in vivo. Here, we demonstrate by intravital fluorescence microscopy of the mouse carotid artery that inhibition or absence of the major platelet collagen receptor, GPVI, abolishes platelet-vessel wall interactions after endothelial denudation. Unexpectedly, inhibition of GPVI by the monoclonal antibody JAQ1 reduced platelet tethering to the subendothelium by approximately 89%. In addition, stable arrest and aggregation of platelets was virtually abolished under these conditions. Using different models of arterial injury, the strict requirement for GPVI in these processes was confirmed in GPVI-deficient mice, where platelets also failed to adhere and aggregate on the damaged vessel wall. These findings reveal an unexpected role of GPVI in the initiation of platelet attachment at sites of vascular injury and unequivocally identify platelet-collagen interactions (via GPVI) as the major determinant of arterial thrombus formation.
  Journal of Experimental Medicine 02/2003; 197(1):41-9. · 13.85 Impact Factor
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  Available from: PubMed Central

  Article: A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation.

  Steffen Massberg, Korbinian Brand, Sabine Grüner, Sharon Page, Elke Müller, Iris Müller, Wolfgang Bergmeier, Thomas Richter, Michael Lorenz, Ildiko Konrad, Bernhard Nieswandt, Meinrad Gawaz
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  ABSTRACT: The contribution of platelets to the process of atherosclerosis remains unclear. Here, we show in vivo that platelets adhere to the vascular endothelium of the carotid artery in ApoE(-)(/)(-) mice before the development of manifest atherosclerotic lesions. Platelet-endothelial cell interaction involved both platelet glycoprotein (GP)Ibalpha and GPIIb-IIIa. Platelet adhesion to the endothelium coincides with inflammatory gene expression and preceded atherosclerotic plaque invasion by leukocytes. Prolonged blockade of platelet adhesion in ApoE(-)(/)(-) mice profoundly reduced leukocyte accumulation in the arterial intima and attenuated atherosclerotic lesion formation in the carotid artery bifurcation, the aortic sinus, and the coronary arteries. These findings establish the platelet as a major player in initiation of the atherogenetic process.
  Journal of Experimental Medicine 11/2002; 196(7):887-96. · 13.85 Impact Factor