Denisa D Wagner

Publications of Denisa D Wagner

• Extracellular DNA traps are associated with the pathogenesis of TRALI in humans and mice.

  Authors: Grace M Thomas, Carla Carbo, Brian R Curtis, Kimberly Martinod, Irina B Mazo, Daphne Schatzberg, Stephen M Cifuni, Tobias A Fuchs, Ulrich H von Andrian, John H Hartwig, Richard H Aster, Denisa D Wagner

  Blood. 05/2012;

  Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related death. The biological processes contributing to TRALI are poorly understood. All blood products can causeTransfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related death. The biological processes contributing to TRALI are poorly understood. All blood products can cause TRALI, and no specific treatment is available. A "two-event model" has been proposed as the trigger. The first event may include surgery, trauma or infection; the second involves the transfusion of anti-leukocyte antibodies or bioactive lipids within the blood product. Together, these events induce neutrophil activation in the lungs, causing endothelial damage and capillary leakage. Neutrophils, in response to pathogens or under stress, can release their chromatin coated with granule contents, thus forming neutrophil extracellular traps (NETs). Although protective against infection, these NETs are injurious to tissue. Here we show that NET biomarkers are present in TRALI patients' blood and that NETs are produced in vitro by primed human neutrophils when challenged with anti-HNA-3a antibodies previously implicated in TRALI. NETs are found in alveoli of mice experiencing antibody-mediated TRALI. DNase 1 inhalation prevents their alveolar accumulation and improves arterial oxygen saturation even when administered 90 minutes after TRALI onset. We suggest that NETs form in the lungs during TRALI, contribute to the disease process and thus could be targeted to prevent or treat TRALI.

• ARC15105 Is a Potent Antagonist of Von Willebrand Factor Mediated Platelet Activation and Adhesion.

  Authors: Jolanta M Siller-Matula, Yahye Merhi, Jean-François Tanguay, Daniel Duerschmied, Denisa D Wagner, Kathleen E McGinness, P Shannon Pendergrast, Jou-Ku Chung, Xianbin Tian, Robert G Schaub, Bernd Jilma

  Arteriosclerosis, thrombosis, and vascular biology. 01/2012;

  OBJECTIVE: We investigated the stability, pharmacokinetic, and pharmacodynamic profile of the 2(nd) generation anti-von Willeband factor aptamer ARC15105. METHODS AND RESULTS: Platelet plug formationOBJECTIVE: We investigated the stability, pharmacokinetic, and pharmacodynamic profile of the 2(nd) generation anti-von Willeband factor aptamer ARC15105. METHODS AND RESULTS: Platelet plug formation was measured by collagen/adenosine diphosphate-induced closure time with the platelet function analyzer-100 and platelet aggregation by multiple electrode aggregometry. Platelet adhesion was measured on denuded porcine aortas and in a flow chamber. Aptamer stability was assessed by incubation in nuclease rich human, monkey, and rat serum for up to 72 hours. Pharmacokinetic and pharmacodynamic profiles were tested in cynomolgus monkeys after IV and SC administration. The median IC(100) and IC(50) to prolong collagen/adenosine diphosphate-induced closure timewere 27 nmol/L and 12 nmol/L, respectively. ARC15105 (1.3 μmol/L) completely inhibited ristocetin-induced platelet aggregation in whole blood (P<0.001), but also diminished collagen, ADP, arachidonic acid, and thrombin receptor activating peptide-induced platelet aggregation to some extent (P<0.05). ARC15105 (40 nmol/L) decreased platelet adhesion by >90% on denuded porcine aortas (P<0.001), which was comparable to the degree of inhibition obtained with abciximab. ARC15105 (100 nmol/L) also inhibited platelet adhesion to collagen under arterial shear in a flow chamber by >90% (P<0.001). The IV and SC terminal half-lives in cynomolgus monkeys were 67 and 65 hours, respectively, and the SC bioavailability was ≈98%. Allometric scaling estimates the human T(1/2) would be ≈217 hours. Pharmacodynamic analysis confirms that ARC15105 inhibits von Willeband factor activity >90% in blood samples taken 300 hours after a 20 mg/kg IV or SC dose in monkeys. CONCLUSIONS: The potency, pharmacokinetic profile, and SC bioavailability of ARC15105 support its clinical investigation for chronic inhibition of von Willeband factor -mediated platelet activation.

• von Willebrand factor: an emerging target in stroke therapy.

  Authors: Simon F De Meyer, Guido Stoll, Denisa D Wagner, Christoph Kleinschnitz

  Stroke; a journal of cerebral circulation. 12/2011; 43(2):599-606.

  Thrombus formation is of paramount importance in the pathophysiology of acute ischemic stroke. Current antithrombotics used to treat or prevent cerebral ischemia are only moderately effective or bearThrombus formation is of paramount importance in the pathophysiology of acute ischemic stroke. Current antithrombotics used to treat or prevent cerebral ischemia are only moderately effective or bear an increased risk of severe bleeding. von Willebrand factor (VWF) has long been known to be a key player in thrombus formation at sites of vascular damage. While the association between VWF and coronary heart disease has been well studied, knowledge about the role of VWF in stroke is much more limited. However, in recent years, an increasing amount of clinical and preclinical evidence has revealed the critical involvement of VWF in stroke development. This review summarizes the latest insights into the pathophysiologic role of VWF-related processes in ischemic brain injury under experimental conditions and in humans. Potential clinical merits of novel inhibitors of VWF-mediated platelet adhesion and activation as powerful and safe tools to combat thromboembolic disorders including ischemic stroke are discussed. Preclinical and clinical evidence illustrates an important role of VWF in ischemic stroke, suggesting that VWF could become a promising target in stroke therapy.

• Desialylation accelerates platelet clearance after refrigeration and initiates GPIbα metalloproteinase-mediated cleavage in mice.

  Authors: A J Gerard Jansen, Emma C Josefsson, Viktoria Rumjantseva, Qiyong Peter Liu, Hervé Falet, Wolfgang Bergmeier, Stephen M Cifuni, Robert Sackstein, Ulrich H von Andrian, Denisa D Wagner, John H Hartwig, Karin M Hoffmeister

  Blood. 11/2011; 119(5):1263-73.

  When refrigerated platelets are rewarmed, they secrete active sialidases, including the lysosomal sialidase Neu1, and express surface Neu3 that remove sialic acid from platelet von Willebrand factorWhen refrigerated platelets are rewarmed, they secrete active sialidases, including the lysosomal sialidase Neu1, and express surface Neu3 that remove sialic acid from platelet von Willebrand factor receptor (VWFR), specifically the GPIbα subunit. The recovery and circulation of refrigerated platelets is greatly improved by storage in the presence of inhibitors of sialidases. Desialylated VWFR is also a target for metalloproteinases (MPs), because GPIbα and GPV are cleaved from the surface of refrigerated platelets. Receptor shedding is inhibited by the MP inhibitor GM6001 and does not occur in Adam17(ΔZn/ΔZn) platelets expressing inactive ADAM17. Critically, desialylation in the absence of MP-mediated receptor shedding is sufficient to cause the rapid clearance of platelets from circulation. Desialylation of platelet VWFR therefore triggers platelet clearance and primes GPIbα and GPV for MP-dependent cleavage.

• Platelet glycoprotein Ibα is an important mediator of ischemic stroke in mice.

  Authors: Simon F De Meyer, Tobias Schwarz, Daphne Schatzberg, Denisa D Wagner

  Experimental & translational stroke medicine. 09/2011; 3:9.

  ABSTRACT: Platelets play an important role in ischemic stroke. GPIbα is a major platelet receptor that is critical for platelet adhesion to exposed subendothelial matrix components at sites ofABSTRACT: Platelets play an important role in ischemic stroke. GPIbα is a major platelet receptor that is critical for platelet adhesion to exposed subendothelial matrix components at sites of vascular damage. In this study, we used transgenic mice in which the extracellular part of GPIbα is replaced by human interleukin 4-receptor (GPIbα/IL4Rα). We observed normal brain vasculature in these mice. We compared infarct size in GPIbα/IL4Rα and wild-type (WT) mice 23 hours after 1-hour transient middle cerebral artery occlusion (tMCAO). In addition, the functional outcome was evaluated using a modified Bederson score. We found a significantly smaller infarct size in GPIbα/IL4Rα mice compared to WT mice (38.0 ± 6.5 mm3 vs. 74.2 ± 8.6 mm3, p < 0.001). The decrease in infarct size was functionally relevant as indicated by a significantly better functional Bederson score in GPIbα/IL4Rα mice compared to WT animals (1.3 ± 0.4 vs. 2.7 ± 0.3, p < 0.05). Our data illustrate and further confirm the important role of platelet GPIbα in ischemic stroke, suggesting that targeted inhibition of this receptor may open new avenues in stroke treatment.

• Histones induce rapid and profound thrombocytopenia in mice.

  Authors: Tobias A Fuchs, Ashish A Bhandari, Denisa D Wagner

  Blood. 06/2011; 118(13):3708-14.

  Histones are released from dying cells and contribute to antimicrobial defense during infection. However, extracellular histones are a double-edged sword because they also damage host tissue and mayHistones are released from dying cells and contribute to antimicrobial defense during infection. However, extracellular histones are a double-edged sword because they also damage host tissue and may cause death. We studied the interactions of histones with platelets. Histones bound to platelets, induced calcium influx, and recruited plasma adhesion proteins such as fibrinogen to induce platelet aggregation. Hereby fibrinogen cross-linked histone-bearing platelets and triggered microaggregation. Fibrinogen interactions with αIIbβ3 integrins were not required for this process but were necessary for the formation of large platelet aggregates. Infused histones associated with platelets in vivo and caused a profound thrombocytopenia within minutes after administration. Mice lacking platelets or αIIbβ3 integrins were protected from histone-induced death but not from histone-induced tissue damage. Heparin, at high concentrations, prevented histone interactions with platelets and protected mice from histone-induced thrombocytopenia, tissue damage, and death. Heparin and histones are evolutionary maintained. Histones may combine microbicidal with prothrombotic properties to fight invading microbes and maintain hemostasis after injury. Heparin may provide an innate counter mechanism to neutralize histones and diminish collateral tissue damage.

• Rap1a activation by CalDAG-GEFI and p38 MAPK is involved in E-selectin-dependent slow leukocyte rolling.

  Authors: Anika Stadtmann, Laura Brinkhaus, Helena Mueller, Jan Rossaint, Matteo Bolomini-Vittori, Wolfgang Bergmeier, Hugo Van Aken, Denisa D Wagner, Carlo Laudanna, Klaus Ley, Alexander Zarbock

  European journal of immunology. 04/2011; 41(7):2074-85.

  Rolling leukocytes are exposed to different adhesion molecules and chemokines. Neutrophils rolling on E-selectin induce integrin αLβ2-mediated slow rolling on ICAM-1 by activating a phospholipase CRolling leukocytes are exposed to different adhesion molecules and chemokines. Neutrophils rolling on E-selectin induce integrin αLβ2-mediated slow rolling on ICAM-1 by activating a phospholipase C (PLC)γ2-dependent and a separate PI3Kγ-dependent pathway. E-selectin-signaling cooperates with chemokine signaling to recruit neutrophils into inflamed tissues. However, the distal signaling pathway linking PLCγ2 (Plcg2) to αLβ2-activation is unknown. To identify this pathway, we used different Tat-fusion-mutants and gene-deficient mice in intravital microscopy, autoperfused flow chamber, peritonitis, and biochemical studies. We found that the small GTPase Rap1 is activated following E-selectin engagement and that blocking Rap1a in Pik3cg-/- mice by a dominant-negative Tat-fusion mutant completely abolished E-selectin-mediated slow rolling. We identified CalDAG-GEFI (Rasgrp2) and p38 MAPK as key signaling intermediates between PLCγ2 and Rap1a. Gαi-independent leukocyte adhesion to and transmigration through endothelial cells in inflamed postcapillary venules of the cremaster muscle were completely abolished in Rasgrp2-/- mice. The physiological importance of CalDAG-GEFI in E-selectin-dependent integrin activation is shown by complete inhibition of neutrophil recruitment into the inflamed peritoneal cavity of Rasgrp2-/- leukocytes treated with pertussis toxin to block Gαi-signaling. Our data demonstrate that Rap1a activation by p38 MAPK and CalDAG-GEFI is involved in E-selectin-dependent slow rolling and leukocyte recruitment.

• The development of inflammatory joint disease is attenuated in mice expressing the anticoagulant prothrombin mutant W215A/E217A.

  Authors: Matthew J Flick, Anil K Chauhan, Malinda Frederick, Kathryn E Talmage, Keith W Kombrinck, Whitney Miller, Eric S Mullins, Joseph S Palumbo, Xunzhen Zheng, Naomi L Esmon, Charles T Esmon, Sherry Thornton, Ann Becker, Leslie A Pelc, Enrico Di Cera, Denisa D Wagner, Jay L Degen

  Blood. 03/2011; 117(23):6326-37.

  Thrombin is a positive mediator of thrombus formation through the proteolytic activation of protease-activated receptors (PARs), fibrinogen, factor XI (fXI), and other substrates, and a negativeThrombin is a positive mediator of thrombus formation through the proteolytic activation of protease-activated receptors (PARs), fibrinogen, factor XI (fXI), and other substrates, and a negative regulator through activation of protein C, a natural anticoagulant with anti-inflammatory/cytoprotective properties. Protease-engineering studies have established that 2 active-site substitutions, W215A and E217A (fII(WE)), result in dramatically reduced catalytic efficiency with procoagulant substrates while largely preserving thrombomodulin (TM)-dependent protein C activation. To explore the hypothesis that a prothrombin variant favoring antithrombotic pathways would be compatible with development but limit inflammatory processes in vivo, we generated mice carrying the fII(WE) mutations within the endogenous prothrombin gene. Unlike fII-null embryos, fII(WE/WE) mice uniformly developed to term. Nevertheless, these mice ultimately succumbed to spontaneous bleeding events shortly after birth. Heterozygous fII(WT/WE) mice were viable and fertile despite a shift toward an antithrombotic phenotype exemplified by prolonged tail-bleeding times and times-to-occlusion after FeCl₃ vessel injury. More interestingly, prothrombin(WE) expression significantly ameliorated the development of inflammatory joint disease in mice challenged with collagen-induced arthritis (CIA). The administration of active recombinant thrombin(WE) also suppressed the development of CIA in wild-type mice. These studies provide a proof-of-principle that pro/thrombin variants engineered with altered substrate specificity may offer therapeutic opportunities for limiting inflammatory disease processes.

• Increased efficacy of breast cancer chemotherapy in thrombocytopenic mice.

  Authors: Mélanie Demers, Benoit Ho-Tin-Noé, Daphne Schatzberg, Janie J Yang, Denisa D Wagner

  Cancer research. 01/2011; 71(5):1540-9.

  Platelets contribute to homeostasis of the tumor vasculature by helping prevent hemorrhage. Thus, we hypothesized that inducing thrombocytopenia would increase tumor vascular leakiness and facilitatePlatelets contribute to homeostasis of the tumor vasculature by helping prevent hemorrhage. Thus, we hypothesized that inducing thrombocytopenia would increase tumor vascular leakiness and facilitate the effective delivery of chemotherapeutic agents to tumors. In a mammary carcinoma murine model, platelet depletion induced bleeding specifically at the tumor site, favoring the accumulation of fluorescently labeled microspheres only in the tumor. Moreover, induction of thrombocytopenia in tumor-bearing mice before injection of paclitaxel increased its intratumoral accumulation and reduced growth of both slow- and fast-growing tumors, compared with mice with normal platelet counts that were treated only with paclitaxel. Histologic analysis confirmed the expectation of an increase in tumor apoptosis and a reduction in tumor proliferation in thrombocytopenic mice receiving chemotherapy. No increased toxicity was seen in other organs or blood cells. Taken together, our results indicate that low platelet count selectively induces leakiness of tumor vessels and favors the delivery of chemotherapy to tumor sites, enhancing its tumoricidal effects.

• von Willebrand factor-mediated platelet adhesion is critical for deep vein thrombosis in mouse models.

  Authors: Alexander Brill, Tobias A Fuchs, Anil K Chauhan, Janie J Yang, Simon F De Meyer, Maria Köllnberger, Thomas W Wakefield, Bernhard Lämmle, Steffen Massberg, Denisa D Wagner

  Blood. 10/2010; 117(4):1400-7.

  Deep vein thrombosis (DVT) and its complication, pulmonary embolism, are frequent causes of disability and mortality. Although blood flow disturbance is considered an important triggering factor, theDeep vein thrombosis (DVT) and its complication, pulmonary embolism, are frequent causes of disability and mortality. Although blood flow disturbance is considered an important triggering factor, the mechanism of DVT initiation remains elusive. Here we show that 48-hour flow restriction in the inferior vena cava (IVC) results in the development of thrombi structurally similar to human deep vein thrombi. von Willebrand factor (VWF)-deficient mice were protected from thrombosis induced by complete (stasis) or partial (stenosis) flow restriction in the IVC. Mice with half normal VWF levels were also protected in the stenosis model. Besides promoting platelet adhesion, VWF carries Factor VIII. Repeated infusions of recombinant Factor VIII did not rescue thrombosis in VWF(-/-) mice, indicating that impaired coagulation was not the primary reason for the absence of DVT in VWF(-/-) mice. Infusion of GPG-290, a mutant glycoprotein Ibα-immunoglobulin chimera that specifically inhibits interaction of the VWF A1 domain with platelets, prevented thrombosis in wild-type mice. Intravital microscopy showed that platelet and leukocyte recruitment in the early stages of DVT was dramatically higher in wild-type than in VWF(-/-) IVC. Our results demonstrate a pathogenetic role for VWF-platelet interaction in flow disturbance-induced venous thrombosis.

• Extracellular DNA traps promote thrombosis.

  Authors: Tobias A Fuchs, Alexander Brill, Daniel Duerschmied, Daphne Schatzberg, Marc Monestier, Daniel D Myers, Shirley K Wrobleski, Thomas W Wakefield, John H Hartwig, Denisa D Wagner

  Proceedings of the National Academy of Sciences of the United States of America. 09/2010; 107(36):15880-5.

  Neutrophil extracellular traps (NETs) are part of the innate immune response to infections. NETs are a meshwork of DNA fibers comprising histones and antimicrobial proteins. Microbes are immobilizedNeutrophil extracellular traps (NETs) are part of the innate immune response to infections. NETs are a meshwork of DNA fibers comprising histones and antimicrobial proteins. Microbes are immobilized in NETs and encounter a locally high and lethal concentration of effector proteins. Recent studies show that NETs are formed inside the vasculature in infections and noninfectious diseases. Here we report that NETs provide a heretofore unrecognized scaffold and stimulus for thrombus formation. NETs perfused with blood caused platelet adhesion, activation, and aggregation. DNase or the anticoagulant heparin dismantled the NET scaffold and prevented thrombus formation. Stimulation of platelets with purified histones was sufficient for aggregation. NETs recruited red blood cells, promoted fibrin deposition, and induced a red thrombus, such as that found in veins. Markers of extracellular DNA traps were detected in a thrombus and plasma of baboons subjected to deep vein thrombosis, an example of inflammation-enhanced thrombosis. Our observations indicate that NETs are a previously unrecognized link between inflammation and thrombosis and may further explain the epidemiological association of infection with thrombosis.

• A novel interaction between FlnA and Syk regulates platelet ITAM-mediated receptor signaling and function.

  Authors: Hervé Falet, Alice Y Pollitt, Antonija Jurak Begonja, Sarah E Weber, Daniel Duerschmied, Denisa D Wagner, Steve P Watson, John H Hartwig

  The Journal of experimental medicine. 08/2010; 207(9):1967-79.

  Filamin A (FlnA) cross-links actin filaments and connects the Von Willebrand factor receptor GPIb-IX-V to the underlying cytoskeleton in platelets. Because FlnA deficiency is embryonic lethal, miceFilamin A (FlnA) cross-links actin filaments and connects the Von Willebrand factor receptor GPIb-IX-V to the underlying cytoskeleton in platelets. Because FlnA deficiency is embryonic lethal, mice lacking FlnA in platelets were generated by breeding FlnA(loxP/loxP) females with GATA1-Cre males. FlnA(loxP/y) GATA1-Cre males have a macrothrombocytopenia and increased tail bleeding times. FlnA-null platelets have decreased expression and altered surface distribution of GPIbalpha because they lack the normal cytoskeletal linkage of GPIbalpha to underlying actin filaments. This results in approximately 70% less platelet coverage on collagen-coated surfaces at shear rates of 1,500/s, compared with wild-type platelets. Unexpectedly, however, immunoreceptor tyrosine-based activation motif (ITAM)- and ITAM-like-mediated signals are severely compromised in FlnA-null platelets. FlnA-null platelets fail to spread and have decreased alpha-granule secretion, integrin alphaIIbbeta3 activation, and protein tyrosine phosphorylation, particularly that of the protein tyrosine kinase Syk and phospholipase C-gamma2, in response to stimulation through the collagen receptor GPVI and the C-type lectin-like receptor 2. This signaling defect was traced to the loss of a novel FlnA-Syk interaction, as Syk binds to FlnA at immunoglobulin-like repeat 5. Our findings reveal that the interaction between FlnA and Syk regulates ITAM- and ITAM-like-containing receptor signaling and platelet function.

• Integrin-independent role of CalDAG-GEFI in neutrophil chemotaxis.

  Authors: Carla Carbo, Daniel Duerschmied, Tobias Goerge, Hidenori Hattori, Jiro Sakai, Stephen M Cifuni, Gilbert C White, Magdalena Chrzanowska-Wodnicka, Hongbo R Luo, Denisa D Wagner

  Journal of leukocyte biology. 04/2010; 88(2):313-9.

  Chemotaxis and integrin activation are essential processes for neutrophil transmigration in response to injury. CalDAG-GEFI plays a key role in the activation of beta1, beta2, and beta3 integrins inChemotaxis and integrin activation are essential processes for neutrophil transmigration in response to injury. CalDAG-GEFI plays a key role in the activation of beta1, beta2, and beta3 integrins in platelets and neutrophils by exchanging a GDP for a GTP on Rap1. Here, we explored the role of CalDAG-GEFI and Rap1b in integrin-independent neutrophil chemotaxis. In a transwell assay, CalDAG-GEFI-/- neutrophils had a 46% reduction in transmigration compared with WT in response to a low concentration of LTB4. Visualization of migrating neutrophils in the presence of 10 mM EDTA revealed that CalDAG-GEFI-/- neutrophils had abnormal chemotactic behavior compared with WT neutrophils, including reduced speed and directionality. Interestingly, Rap1b-/- neutrophils had a similar phenotype in this assay, suggesting that CalDAG-GEFI may be acting through Rap1b. We investigated whether the deficit in integrin-independent chemotaxis in CalDAG-GEFI-/- neutrophils could be explained by defective cytoskeleton rearrangement. Indeed, we found that CalDAG-GEFI-/- neutrophils had reduced formation of F-actin pseudopodia after LTB4 stimulation, suggesting that they have a defect in polarization. Overall, our studies show that CalDAG-GEFI helps regulate neutrophil chemotaxis, independent of its established role in integrin activation, through a mechanism that involves actin cytoskeleton and cellular polarization.

• The lack of ADAM17 activity during embryonic development causes hemorrhage and impairs vessel formation.

  Authors: Matthias Canault, Kaan Certel, Daphne Schatzberg, Denisa D Wagner, Richard O Hynes

  PloS one. 01/2010; 5(10):e13433.

  ADAM17/TACE activity is important during embryonic development. We wished to investigate possible roles of this metalloprotease, focusing on vascular development. Mice mutant in the enzymaticADAM17/TACE activity is important during embryonic development. We wished to investigate possible roles of this metalloprotease, focusing on vascular development. Mice mutant in the enzymatic activity of ADAM17 were examined at various stages of embryonic development for vascular pattern and integrity using markers for vessel wall cells. We observed hemorrhage and edema starting at embryonic day E14.5 and becoming more severe as development proceeded; prior to embryonic day E14.5, embryos appeared normal. Staining for PECAM-1/CD31 revealed abnormalities in the patterns of branching of the embryonic vasculature at E14.5. These abnormalities preceded association of pericytes or monocyte/macrophage cells with the affected vessels and, therefore, presumably arise from defects in endothelial function consequent upon failure of ADAM17 to cleave one or more substrates involved in vascular development, such as Notch, Delta, VEGFR2 or JAM-A. Our study demonstrates a role for ADAM17 in modulating embryonic vessel development and function.

• p38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo.

  Authors: Matthias Canault, Daniel Duerschmied, Alexander Brill, Lucia Stefanini, Daphne Schatzberg, Stephen M Cifuni, Wolfgang Bergmeier, Denisa D Wagner

  Blood. 11/2009;

  Platelets undergo several modifications during storage which reduce their post transfusion survival and functionality. One important feature of these changes, which are known as platelet storagePlatelets undergo several modifications during storage which reduce their post transfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIbalpha and GPV. We recently demonstrated that TNF-alpha converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced post transfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37 degrees C or 22 degrees C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 MAPK in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, ERK MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved post transfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets.

• Innate Immune Cells Induce Hemorrhage in Tumors during Thrombocytopenia.

  Authors: Benoit Ho-Tin-Noé, Carla Carbo, Mélanie Demers, Stephen M Cifuni, Tobias Goerge, Denisa D Wagner

  The American journal of pathology. 10/2009;

  Platelets are crucial regulators of tumor vascular homeostasis and continuously prevent tumor hemorrhage through secretion of their granules. However, the reason for tumor bleeding in the absence ofPlatelets are crucial regulators of tumor vascular homeostasis and continuously prevent tumor hemorrhage through secretion of their granules. However, the reason for tumor bleeding in the absence of platelets remains unknown. Tumors are associated with inflammation, a cause of hemorrhage in thrombocytopenia. Here, we investigated the role of the inflamed tumor microenvironment in the induction of tumor vessel injury in thrombocytopenic mice. Using s.c. injections of vascular endothelial growth factor or tumor necrosis factor-alpha combined with depletion of neutrophils, we demonstrate that enhancing the opening of endothelial cell junctions was not sufficient to cause bleeding in the absence of platelets; instead, induction of tissue hemorrhage in thrombocytopenia required recruitment of leukocytes. Immunohistology revealed that thrombocytopenia-induced tumor hemorrhage occurs at sites of macrophage and neutrophil accumulation. Mice deficient in beta2 or beta3 integrins, which have decreased neutrophil and/or macrophage infiltration in their tumor stroma, were protected from thrombocytopenia-induced tumor hemorrhage, indicating that, in the absence of platelets, stroma-infiltrating leukocytes induced tumor vessel injury. This injury was independent of reactive oxygen species generation and of complement activation, as suggested by the persistence of tumor hemorrhage in C3- and nicotinamide adenine dinucleotide phosphate oxidase-deficient thrombocytopenic mice. Our results show that platelets counteract tumor-associated inflammation and that the absence of this platelet function elicits vascular injuries by tumor-infiltrating innate immune cells.

• Von Willebrand factor-cleaving protease ADAMTS13 reduces ischemic brain injury in experimental stroke.

  Authors: Bing-Qiao Zhao, Anil K Chauhan, Matthias Canault, Ian S Patten, Janie J Yang, Michael Dockal, Friedrich Scheiflinger, Denisa D Wagner

  Blood. 09/2009;

  Stroke is a leading cause of death and disability. The only FDA-approved therapy available is recombinant tissue plasminogen activator, but side effects limit its use. Besides coagulation, plateletsStroke is a leading cause of death and disability. The only FDA-approved therapy available is recombinant tissue plasminogen activator, but side effects limit its use. Besides coagulation, platelets play a crucial role during stroke and the inflammatory reaction promotes neurodegeneration. von Willebrand factor (VWF), an adhesion molecule for platelets, is elevated in acute stroke patients. The activity of VWF is modulated by ADAMTS13 (A Disintegrin-like And Metalloprotease with Thrombospondin type I repeats-13) which cleaves VWF to smaller less active forms. We have recently documented that ADAMTS13 negatively regulates both thrombosis and inflammation. We now report that deficiency or reduction of VWF reduces infarct volume up to two-fold after focal cerebral ischemia in mice, thus demonstrating the importance of VWF in stroke injury. In contrast, ADAMTS13 deficiency results in larger infarctions, but only in mice that have VWF. Importantly, infusion of a high dose of recombinant human ADAMTS13 into a wild-type mouse immediately before reperfusion significantly reduces both infarct volume and improves functional outcome without producing cerebral hemorrhage. Furthermore, recombinant ADAMTS13 did not enhance bleeding in a hemorrhagic stroke model in thrombocytopenic mice. Our findings reveal the importance of VWF activity and levels in regulating infarction and suggest that recombinant ADAMTS13 could be considered as a new therapeutic agent for prevention and/or treatment of stroke.

• Platelets: guardians of tumor vasculature.

  Authors: Benoit Ho-Tin-Noé, Tobias Goerge, Denisa D Wagner

  Cancer research. 08/2009; 69(14):5623-6.

  Solid tumors generate a prothrombotic environment capable of platelet activation. Recent findings indicate that the activated platelets are crucial regulators of tumor vascular homeostasis in thatSolid tumors generate a prothrombotic environment capable of platelet activation. Recent findings indicate that the activated platelets are crucial regulators of tumor vascular homeostasis in that they prevent tumor hemorrhage. Surprisingly, this effect is independent of platelets' capacity to form thrombi and instead relies on the secretion of their granule content. Thus, targeting platelet secretory activities may represent a new approach to specifically destabilize tumor vasculature.

• Oxidative stress activates ADAM17/TACE and induces its target receptor shedding in platelets in a p38-dependent fashion.

  Authors: Alexander Brill, Anil K Chauhan, Matthias Canault, Meghan T Walsh, Wolfgang Bergmeier, Denisa D Wagner

  Cardiovascular research. 07/2009;

  AIMS: Oxidative stress accompanies inflammatory and vascular diseases. The objective of this study was to explore whether reactive oxygen species can activate shedding of platelet receptors and thusAIMS: Oxidative stress accompanies inflammatory and vascular diseases. The objective of this study was to explore whether reactive oxygen species can activate shedding of platelet receptors and thus suppress platelet function. METHODS AND RESULTS: Hydrogen peroxide and glucose oxidase were chosen to model oxidative stress in vitro. We demonstrate that oxidative damage activated tumour necrosis factor-alpha-converting enzyme (TACE) and induced shedding of its targets, glycoprotein (GP) Ibalpha and GPV, in murine and human platelets. Also, 12-HpETE, a peroxide synthesized in the platelet lipoxygenase pathway, induced TACE-mediated receptor cleavage. The TACE activation was independent of platelet activation, as alpha-granule secretion, activation of alphaIIbbeta3, or phosphatidylserine expression was not observed. TACE activation induced by hydrogen peroxide was dependent on p38 mitogen-activated protein kinase signalling, whereas protein kinase C, phosphoinositide 3-kinase, and caspases were not involved. Inhibition of p38 cytoplasmic targets, phospholipase A(2) and heat shock protein 27, did not prevent shedding, whereas blocking 12-lipoxygenase or Src kinase slightly inhibited TACE activation. The loss of the GPIbalpha receptor induced by oxidative stress rendered platelets unable to incorporate into a growing thrombus in vivo. CONCLUSION: Oxidative stress can render platelets functionally less active by shedding key adhesion receptors via the activation of p38. This suggests that oxidative injury of platelets may attenuate their function.

• Inhibition of VEGF or TGF-{beta} Signaling Activates Endothelium and Increases Leukocyte Rolling.

  Authors: Tony E Walshe, Vandana S Dole, Arindel S R Maharaj, Ian S Patten, Denisa D Wagner, Patricia A D'Amore

  Arteriosclerosis, thrombosis, and vascular biology. 06/2009;

  OBJECTIVE: Motivated by the central roles that vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-beta play in the assembly and maintenance of the vasculature, we examinedOBJECTIVE: Motivated by the central roles that vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-beta play in the assembly and maintenance of the vasculature, we examined the impact of systemic VEGF or TGF-beta signal inhibition on endothelial activation as detected by leukocyte-endothelial interactions. METHODS AND RESULTS: VEGF or TGF-beta inhibition, accomplished using adenovirus expression of soluble Flt1 (Ad-sFlt1) or soluble endoglin (Ad-sEng), resulted in a significant increase in the number of leukocytes rolling along the mesenteric venous endothelium and a significant decrease in rolling velocity in Ad-sEng mice. Neutralization of VEGF or TGF-beta resulted in endothelial surface expression of P-selectin and impaired peripheral vasodilatation. Neither inhibition of VEGF nor TGF-beta was associated with platelet or leukocyte activation, as detected by the activation markers platelet P-selectin and the active integrin alphaIIbbetaIII, or by leukocyte expression of L-selectin. Soluble vascular cell adhesion molecule (VCAM)-1 and E-selectin were increased in sEng-expressing mice, indicating higher levels of these adhesion receptors. CONCLUSIONS: VEGF or TGF-beta neutralization results in impaired endothelium-mediated vasodilatation and elevated expression of surface adhesion molecules, resulting in increased leukocyte adhesion. These results indicate an essential role for both VEGF and TGF-beta in maintaining the endothelium in a nonactivated state and have implications for therapeutic approaches that neutralize VEGF or TGF-beta.