TY - JOUR A1 - Ascheid, David A1 - Baumann, Magdalena A1 - Funke, Caroline A1 - Volz, Julia A1 - Pinnecker, Jürgen A1 - Friedrich, Mike A1 - Höhn, Marie A1 - Nandigama, Rajender A1 - Ergün, Süleyman A1 - Nieswandt, Bernhard A1 - Heinze, Katrin G. A1 - Henke, Erik T1 - Image-based modeling of vascular organization to evaluate anti-angiogenic therapy JF - Biology Direct N2 - In tumor therapy anti-angiogenic approaches have the potential to increase the efficacy of a wide variety of subsequently or co-administered agents, possibly by improving or normalizing the defective tumor vasculature. Successful implementation of the concept of vascular normalization under anti-angiogenic therapy, however, mandates a detailed understanding of key characteristics and a respective scoring metric that defines an improved vasculature and thus a successful attempt. Here, we show that beyond commonly used parameters such as vessel patency and maturation, anti-angiogenic approaches largely benefit if the complex vascular network with its vessel interconnections is both qualitatively and quantitatively assessed. To gain such deeper insight the organization of vascular networks, we introduce a multi-parametric evaluation of high-resolution angiographic images based on light-sheet fluorescence microscopy images of tumors. We first could pinpoint key correlations between vessel length, straightness and diameter to describe the regular, functional and organized structure observed under physiological conditions. We found that vascular networks from experimental tumors diverted from those in healthy organs, demonstrating the dysfunctionality of the tumor vasculature not only on the level of the individual vessel but also in terms of inadequate organization into larger structures. These parameters proofed effective in scoring the degree of disorganization in different tumor entities, and more importantly in grading a potential reversal under treatment with therapeutic agents. The presented vascular network analysis will support vascular normalization assessment and future optimization of anti-angiogenic therapy. KW - vascular structure KW - cancer KW - tumor microenvironment KW - optical clearing KW - light sheet fluorescence microscopy KW - 3D image analysis Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-357242 VL - 18 ER - TY - JOUR A1 - Göb, Vanessa A1 - Voll, Maximilian G. A1 - Zimmermann, Lena A1 - Hemmen, Katharina A1 - Stoll, Guido A1 - Nieswandt, Bernhard A1 - Schuhmann, Michael K. A1 - Heinze, Katrin G. A1 - Stegner, David T1 - Infarct growth precedes cerebral thrombosis following experimental stroke in mice JF - Scientific Reports N2 - Ischemic stroke is among the leading causes of disability and death worldwide. In acute ischemic stroke, successful recanalization of occluded vessels is the primary therapeutic aim, but even if it is achieved, not all patients benefit. Although blockade of platelet aggregation did not prevent infarct progression, cerebral thrombosis as cause of secondary infarct growth has remained a matter of debate. As cerebral thrombi are frequently observed after experimental stroke, a thrombus-induced impairment of the brain microcirculation is considered to contribute to tissue damage. Here, we combine the model of transient middle cerebral artery occlusion (tMCAO) with light sheet fluorescence microscopy and immunohistochemistry of brain slices to investigate the kinetics of thrombus formation and infarct progression. Our data reveal that tissue damage already peaks after 8 h of reperfusion following 60 min MCAO, while cerebral thrombi are only observed at later time points. Thus, cerebral thrombosis is not causative for secondary infarct growth during ischemic stroke. KW - cerebrovascular disorders KW - thrombosis Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265791 VL - 11 IS - 1 ER - TY - JOUR A1 - Kollikowski, Alexander M. A1 - Schuhmann, Michael K. A1 - Nieswandt, Bernhard A1 - Müllges, Wolfgang A1 - Stoll, Guido A1 - Pham, Mirko T1 - Local Leukocyte Invasion during Hyperacute Human Ischemic Stroke JF - Annals of Neurology N2 - Objective Bridging the gap between experimental stroke and patients by ischemic blood probing during the hyperacute stage of vascular occlusion is crucial to assess the role of inflammation in human stroke and for the development of adjunct treatments beyond recanalization. Methods We prospectively observed 151 consecutive ischemic stroke patients with embolic large vessel occlusion of the anterior circulation who underwent mechanical thrombectomy. In all these patients, we attempted microcatheter aspiration of 3 different arterial blood samples: (1) within the core of the occluded vascular compartment and controlled by (2) carotid and (3) femoral samples obtained under physiological flow conditions. Subsequent laboratory analyses comprised leukocyte counting and differentiation, platelet counting, and the quantification of 13 proinflammatory human chemokines/cytokines. Results Forty patients meeting all clinical, imaging, interventional, and laboratory inclusion criteria could be analyzed, showing that the total number of leukocytes significantly increased under the occlusion condition. This increase was predominantly driven by neutrophils. Significant increases were also apparent for lymphocytes and monocytes, accompanied by locally elevated plasma levels of the T‐cell chemoattractant CXCL‐11. Finally, we found evidence that short‐term clinical outcome (National Institute of Health Stroke Scale at 72 hours) was negatively associated with neutrophil accumulation. Interpretation We provide the first direct human evidence that neutrophils, lymphocytes, and monocytes, accompanied by specific chemokine upregulation, accumulate in the ischemic vasculature during hyperacute stroke and may affect outcome. These findings strongly support experimental evidence that immune cells contribute to acute ischemic brain damage and indicate that ischemic inflammation initiates already during vascular occlusion. Ann Neurol 2020;87:466–479 KW - neurology Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-212168 VL - 87 IS - 3 ER - TY - JOUR A1 - Elgheznawy, Amro A1 - Öftering, Patricia A1 - Englert, Maximilian A1 - Mott, Kristina A1 - Kaiser, Friederike A1 - Kusch, Charly A1 - Gbureck, Uwe A1 - Bösl, Michael R. A1 - Schulze, Harald A1 - Nieswandt, Bernhard A1 - Vögtle, Timo A1 - Hermanns, Heike M. T1 - Loss of zinc transporters ZIP1 and ZIP3 augments platelet reactivity in response to thrombin and accelerates thrombus formation in vivo JF - Frontiers in Immunology N2 - Zinc (Zn2+) is considered as important mediator of immune cell function, thrombosis and haemostasis. However, our understanding of the transport mechanisms that regulate Zn2+ homeostasis in platelets is limited. Zn2+ transporters, ZIPs and ZnTs, are widely expressed in eukaryotic cells. Using mice globally lacking ZIP1 and ZIP3 (ZIP1/3 DKO), our aim was to explore the potential role of these Zn2+ transporters in maintaining platelet Zn2+ homeostasis and in the regulation of platelet function. While ICP-MS measurements indicated unaltered overall Zn2+ concentrations in platelets of ZIP1/3 DKO mice, we observed a significantly increased content of FluoZin3-stainable free Zn2+, which, however, appears to be released less efficiently upon thrombin-stimulated platelet activation. On the functional level, ZIP1/3 DKO platelets exhibited a hyperactive response towards threshold concentrations of G protein-coupled receptor (GPCR) agonists, while immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptor agonist signalling was unaffected. This resulted in enhanced platelet aggregation towards thrombin, bigger thrombus volume under flow ex vivo and faster in vivo thrombus formation in ZIP1/3 DKO mice. Molecularly, augmented GPCR responses were accompanied by enhanced Ca2+ and PKC, CamKII and ERK1/2 signalling. The current study thereby identifies ZIP1 and ZIP3 as important regulators for the maintenance of platelet Zn2+ homeostasis and function. KW - platelets KW - zinc KW - ZIP KW - thrombin KW - signaling KW - thrombosis Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-320154 VL - 14 ER - TY - JOUR A1 - Wolf, Karen A1 - Braun, Attila A1 - Haining, Elizabeth J. A1 - Tseng, Yu-Lun A1 - Kraft, Peter A1 - Schuhmann, Michael K. A1 - Gotru, Sanjeev K. A1 - Chen, Wenchun A1 - Hermanns, Heike M. A1 - Stoll, Guido A1 - Lesch, Klaus-Peter A1 - Nieswandt, Bernhard T1 - Partially Defective Store Operated Calcium Entry and Hem(ITAM) Signaling in Platelets of Serotonin Transporter Deficient Mice JF - PLoS One N2 - Background Serotonin (5-hydroxytryptamin, 5-HT) is an indolamine platelet agonist, biochemically derived from tryptophan. 5-HT is secreted from the enterochromaffin cells into the gastrointestinal tract and blood. Blood 5-HT has been proposed to regulate hemostasis by acting as a vasoconstrictor and by triggering platelet signaling through 5-HT receptor 2A (5HTR2A). Although platelets do not synthetize 5-HT, they take 5-HT up from the blood and store it in their dense granules which are secreted upon platelet activation. Objective To identify the molecular composite of the 5-HT uptake system in platelets and elucidate the role of platelet released 5-HT in thrombosis and ischemic stroke. Methods: 5-HT transporter knockout mice (5Htt\(^{-/-}\)) were analyzed in different in vitro and in vivo assays and in a model of ischemic stroke. Results In 5Htt\(^{-/-}\) platelets, 5-HT uptake from the blood was completely abolished and agonist-induced Ca2+ influx through store operated Ca\(^{2+}\) entry (SOCE), integrin activation, degranulation and aggregation responses to glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2) were reduced. These observed in vitro defects in 5Htt\(^{-/-}\) platelets could be normalized by the addition of exogenous 5-HT. Moreover, reduced 5-HT levels in the plasma, an increased bleeding time and the formation of unstable thrombi were observed ex vivo under flow and in vivo in the abdominal aorta and carotid artery of 5Htt\(^{-/-}\) mice. Surprisingly, in the transient middle cerebral artery occlusion (tMCAO) model of ischemic stroke 5Htt\(^{-/-}\) mice showed nearly normal infarct volume and the neurological outcome was comparable to control mice. Conclusion Although secreted platelet 5-HT does not appear to play a crucial role in the development of reperfusion injury after stroke, it is essential to amplify the second phase of platelet activation through SOCE and plays an important role in thrombus stabilization. KW - platelets KW - serotonin KW - integrins KW - blood flow KW - collagens KW - platelet activation KW - platelet aggregation KW - ischemic stroke Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146399 VL - 11 IS - 1 ER - TY - JOUR A1 - Chillo, Omary A1 - Kleinert, Eike Christian A1 - Lautz, Thomas A1 - Lasch, Manuel A1 - Pagel, Judith-Irina A1 - Heun, Yvonn A1 - Troidl, Kerstin A1 - Fischer, Silvia A1 - Caballero-Martinez, Amelia A1 - Mauer, Annika A1 - Kurz, Angela R. M. A1 - Assmann, Gerald A1 - Rehberg, Markus A1 - Kanse, Sandip M. A1 - Nieswandt, Bernhard A1 - Walzog, Barbara A1 - Reichel, Christoph A. A1 - Mannell, Hanna A1 - Preissner, Klaus T. A1 - Deindl, Elisabeth T1 - Perivascular Mast Cells Govern Shear Stress-Induced Arteriogenesis by Orchestrating Leukocyte Function JF - Cell Reports N2 - The body has the capacity to compensate for an occluded artery by creating a natural bypass upon increased fluid shear stress. How this mechanical force is translated into collateral artery growth (arteriogenesis) is unresolved. We show that extravasation of neutrophils mediated by the platelet receptor GPIbα and uPA results in Nox2-derived reactive oxygen radicals, which activate perivascular mast cells. These c-kit+/CXCR-4+ cells stimulate arteriogenesis by recruiting additional neutrophils as well as growth-promoting monocytes and T cells. Additionally, mast cells may directly contribute to vascular remodeling and vascular cell proliferation through increased MMP activity and by supplying growth-promoting factors. Boosting mast cell recruitment and activation effectively promotes arteriogenesis, thereby protecting tissue from severe ischemic damage. We thus find that perivascular mast cells are central regulators of shear stress-induced arteriogenesis by orchestrating leukocyte function and growth factor/cytokine release, thus providing a therapeutic target for treatment of vascular occlusive diseases. KW - Mast cells Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164800 VL - 16 IS - 8 ER - TY - JOUR A1 - Viera, Jonathan Trujillo A1 - El-Merahbi, Rabih A1 - Nieswandt, Bernhard A1 - Stegner, David A1 - Sumara, Grzegorz T1 - Phospholipases D1 and D2 Suppress Appetite and Protect against Overweight JF - PLoS ONE N2 - Obesity is a major risk factor predisposing to the development of peripheral insulin resistance and type 2 diabetes (T2D). Elevated food intake and/or decreased energy expenditure promotes body weight gain and acquisition of adipose tissue. Number of studies implicated phospholipase D (PLD) enzymes and their product, phosphatidic acid (PA), in regulation of signaling cascades controlling energy intake, energy dissipation and metabolic homeostasis. However, the impact of PLD enzymes on regulation of metabolism has not been directly determined so far. In this study we utilized mice deficient for two major PLD isoforms, PLD1 and PLD2, to assess the impact of these enzymes on regulation of metabolic homeostasis. We showed that mice lacking PLD1 or PLD2 consume more food than corresponding control animals. Moreover, mice deficient for PLD2, but not PLD1, present reduced energy expenditure. In addition, deletion of either of the PLD enzymes resulted in development of elevated body weight and increased adipose tissue content in aged animals. Consistent with the fact that elevated content of adipose tissue predisposes to the development of hyperlipidemia and insulin resistance, characteristic for the pre-diabetic state, we observed that Pld1\(^{-/-}\) and Pld2\(^{-/-}\) mice present elevated free fatty acids (FFA) levels and are insulin as well as glucose intolerant. In conclusion, our data suggest that deficiency of PLD1 or PLD2 activity promotes development of overweight and diabetes. KW - enzyme regulation KW - insulin resistance KW - body weight KW - mouse models KW - bioenergetics KW - insulin KW - hypothalamus KW - adipose tissue Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-179729 VL - 11 IS - 6 ER - TY - JOUR A1 - Schuhmann, Michael K. A1 - Bieber, Michael A1 - Franke, Maximilian A1 - Kollikowski, Alexander M. A1 - Stegner, David A1 - Heinze, Katrin G. A1 - Nieswandt, Bernhard A1 - Pham, Mirko A1 - Stoll, Guido T1 - Platelets and lymphocytes drive progressive penumbral tissue loss during middle cerebral artery occlusion in mice JF - Journal of Neuroinflammation N2 - Background In acute ischemic stroke, cessation of blood flow causes immediate tissue necrosis within the center of the ischemic brain region accompanied by functional failure in the surrounding brain tissue designated the penumbra. The penumbra can be salvaged by timely thrombolysis/thrombectomy, the only available acute stroke treatment to date, but is progressively destroyed by the expansion of infarction. The underlying mechanisms of progressive infarction are not fully understood. Methods To address mechanisms, mice underwent filament occlusion of the middle cerebral artery (MCAO) for up to 4 h. Infarct development was compared between mice treated with antigen-binding fragments (Fab) against the platelet surface molecules GPIb (p0p/B Fab) or rat immunoglobulin G (IgG) Fab as control treatment. Moreover, Rag1\(^{−/−}\) mice lacking T-cells underwent the same procedures. Infarct volumes as well as the local inflammatory response were determined during vessel occlusion. Results We show that blocking of the platelet adhesion receptor, glycoprotein (GP) Ibα in mice, delays cerebral infarct progression already during occlusion and thus before recanalization/reperfusion. This therapeutic effect was accompanied by decreased T-cell infiltration, particularly at the infarct border zone, which during occlusion is supplied by collateral blood flow. Accordingly, mice lacking T-cells were likewise protected from infarct progression under occlusion. Conclusions Progressive brain infarction can be delayed by blocking detrimental lymphocyte/platelet responses already during occlusion paving the way for ultra-early treatment strategies in hyper-acute stroke before recanalization. KW - ischemic penumbra KW - glycoprotein receptor Ib KW - T-cells KW - ischemic stroke KW - thrombo-inflammation KW - middle cerebral artery occlusion Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259172 VL - 18 IS - 1 ER - TY - JOUR A1 - Stegner, David A1 - Klaus, Vanessa A1 - Nieswandt, Bernhard T1 - Platelets as modulators of cerebral ischemia/reperfusion injury JF - Frontiers in Immunology N2 - Ischemic stroke is among the leading causes of disability and death worldwide. In acute ischemic stroke, the rapid recanalization of occluded cranial vessels is the primary therapeutic aim. However, experimental data (obtained using mostly the transient middle cerebral artery occlusion model) indicates that progressive stroke can still develop despite successful recanalization, a process termed “reperfusion injury.” Mounting experimental evidence suggests that platelets and T cells contribute to cerebral ischemia/reperfusion injury, and ischemic stroke is increasingly considered a thrombo-inflammatory disease. The interaction of von Willebrand factor and its receptor on the platelet surface, glycoprotein Ib, as well as many activatory platelet receptors and platelet degranulation contribute to secondary infarct growth in this setting. In contrast, interference with GPIIb/IIIa-dependent platelet aggregation and thrombus formation does not improve the outcome of acute brain ischemia but dramatically increases the susceptibility to intracranial hemorrhage. Here, we summarize the current understanding of the mechanisms and the potential translational impact of platelet contributions to cerebral ischemia/reperfusion injury. KW - thrombo-inflammation KW - ischemic stroke KW - platelet KW - glycoprotein Ibα KW - platelet degranulation Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-195748 SN - 1664-3224 VL - 10 IS - 2505 ER - TY - JOUR A1 - Ammar, Mohamed Raafet A1 - Thahouly, Tamou A1 - Hanauer, André A1 - Stegner, David A1 - Nieswandt, Bernhard A1 - Vitale, Nicolas T1 - PLD1 participates in BDNF-induced signalling in cortical neurons JF - Scientific Reports N2 - The brain-derived neurotrophic factor BDNF plays a critical role in neuronal development and the induction of L-LTP at glutamatergic synapses in several brain regions. However, the cellular and molecular mechanisms underlying these BDNF effects have not been firmly established. Using in vitro cultures of cortical neurons from knockout mice for Pld1 and Rsk2, BDNF was observed to induce a rapid RSK2-dependent activation of PLD and to stimulate BDNF ERK1/2-CREB and mTor-S6K signalling pathways, but these effects were greatly reduced in Pld1\(^{-/-}\) neurons. Furthermore, phospho-CREB did not accumulate in the nucleus, whereas overexpression of PLD1 amplified the BDNF-dependent nuclear recruitment of phospho-ERK1/2 and phospho-CREB. This BDNF retrograde signalling was prevented in cells silenced for the scaffolding protein PEA15, a protein which complexes with PLD1, ERK1/2, and RSK2 after BDNF treatment. Finally PLD1, ERK1/2, and RSK2 partially colocalized on endosomal structures, suggesting that these proteins are part of the molecular module responsible for BDNF signalling in cortical neurons. KW - phospholipase D KW - ERK map kinease KW - long-term potentation KW - brain KW - protein RSK2 KW - dendritic growth KW - neurite outgrowth KW - neurotrophic factor KW - coffin-lowry-syndrome KW - phosphatidic acid Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-139962 VL - 5 IS - 14778 ER - TY - JOUR A1 - Navarro, Stefano A1 - Starke, Andreas A1 - Heemskerk, Johan W. M. A1 - Kuijpers, Marijke J. E. A1 - Stegner, David A1 - Nieswandt, Bernhard T1 - Targeting of a conserved epitope in mouse and human GPVI differently affects receptor function JF - International Journal of Molecular Sciences N2 - Glycoprotein (GP) VI is the major platelet collagen receptor and a promising anti-thrombotic target. This was first demonstrated in mice using the rat monoclonal antibody JAQ1, which completely blocks the Collagen-Related Peptide (CRP)-binding site on mouse GPVI and efficiently inhibits mouse platelet adhesion, activation and aggregation on collagen. Here, we show for the first time that JAQ1 cross-reacts with human GPVI (huGPVI), but not with GPVI in other tested species, including rat, rabbit, guinea pig, swine, and dog. We further demonstrate that JAQ1 differently modulates mouse and human GPVI function. Similar to its effects on mouse GPVI (mGPVI), JAQ1 inhibits CRP-induced activation in human platelets, whereas, in stark contrast to mouse GPVI, it does not inhibit the adhesion, activation or aggregate formation of human platelets on collagen, but causes instead an increased response. This effect was also seen with platelets from newly generated human GPVI knockin mice (hGP6\(^{tg/tg\)). These results indicate that the binding of JAQ1 to a structurally conserved epitope in GPVI differently affects its function in human and mouse platelets. KW - glycoprotein VI KW - JAQ1 KW - platelet receptors KW - platelet activation KW - platelet inhibition Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-286227 SN - 1422-0067 VL - 23 IS - 15 ER - TY - JOUR A1 - Schuhmann, Michael K. A1 - Kraft, Peter A1 - Bieber, Michael A1 - Kollikowski, Alexander M. A1 - Schulze, Harald A1 - Nieswandt, Bernhard A1 - Pham, Mirko A1 - Stegner, David A1 - Stoll, Guido T1 - Targeting platelet GPVI plus rt-PA administration but not α2β1-mediated collagen binding protects against ischemic brain damage in mice JF - International Journal of Molecular Science N2 - Platelet collagen interactions at sites of vascular injuries predominantly involve glycoprotein VI (GPVI) and the integrin α2β1. Both proteins are primarily expressed on platelets and megakaryocytes whereas GPVI expression is also shown on endothelial and integrin α2β1 expression on epithelial cells. We recently showed that depletion of GPVI improves stroke outcome without increasing the risk of cerebral hemorrhage. Genetic variants associated with higher platelet surface integrin α2 (ITGA2) receptor levels have frequently been found to correlate with an increased risk of ischemic stroke in patients. However until now, no preclinical stroke study has addressed whether platelet integrin α2β1 contributes to the pathophysiology of ischemia/reperfusion (I/R) injury. Focal cerebral ischemia was induced in C57BL/6 and Itga2\(^{−/−}\) mice by a 60 min transient middle cerebral artery occlusion (tMCAO). Additionally, wild-type animals were pretreated with anti-GPVI antibody (JAQ1) or Fab fragments of a function blocking antibody against integrin α2β1 (LEN/B). In anti-GPVI treated animals, intravenous (IV) recombinant tissue plasminogen activator (rt-PA) treatment was applied immediately prior to reperfusion. Stroke outcome, including infarct size and neurological scoring was determined on day 1 after tMCAO. We demonstrate that targeting the integrin α2β1 (pharmacologic; genetic) did neither reduce stroke size nor improve functional outcome on day 1 after tMCAO. In contrast, depletion of platelet GPVI prior to stroke was safe and effective, even when combined with rt-PA treatment. Our results underscore that GPVI, but not ITGA2, is a promising and safe target in the setting of ischemic stroke. KW - ischemic stroke KW - integrin α2 KW - glycoprotein VI KW - recombinant tissue-type plasminogen activator KW - intracranial bleeding KW - transient middle cerebral artery occlusion Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201700 SN - 1422-0067 VL - 20 IS - 8 ER - TY - JOUR A1 - Navarro, Stefano A1 - Stegner, David A1 - Nieswandt, Bernhard A1 - Heemskerk, Johan W. M. A1 - Kuijpers, Marijke J. E. T1 - Temporal roles of platelet and coagulation pathways in collagen- and tissue factor-induced thrombus formation JF - International Journal of Molecular Sciences N2 - In hemostasis and thrombosis, the complex process of thrombus formation involves different molecular pathways of platelet and coagulation activation. These pathways are considered as operating together at the same time, but this has not been investigated. The objective of our study was to elucidate the time-dependency of key pathways of thrombus and clot formation, initiated by collagen and tissue factor surfaces, where coagulation is triggered via the extrinsic route. Therefore, we adapted a microfluidics whole-blood assay with the Maastricht flow chamber to acutely block molecular pathways by pharmacological intervention at desired time points. Application of the technique revealed crucial roles of glycoprotein VI (GPVI)-induced platelet signaling via Syk kinase as well as factor VIIa-induced thrombin generation, which were confined to the first minutes of thrombus buildup. A novel anti-GPVI Fab EMF-1 was used for this purpose. In addition, platelet activation with the protease-activating receptors 1/4 (PAR1/4) and integrin αIIbβ3 appeared to be prolongedly active and extended to later stages of thrombus and clot formation. This work thereby revealed a more persistent contribution of thrombin receptor-induced platelet activation than of collagen receptor-induced platelet activation to the thrombotic process. KW - coagulation KW - fibrin KW - glycoprotein VI KW - platelet receptors KW - spatiotemporal thrombus KW - thrombin Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284219 SN - 1422-0067 VL - 23 IS - 1 ER - TY - JOUR A1 - Nieswandt, Bernhard A1 - Morowski, Martina A1 - Brachs, Sebastian A1 - Mielenz, Dirk A1 - Dütting, Sebastian T1 - The Adaptor Protein Swiprosin-1/EFhd2 Is Dispensable for Platelet Function in Mice N2 - Background Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity, but may also cause pathological vessel occlusion. Reorganizations of the platelet cytoskeleton and agonist-induced intracellular Ca2+-mobilization are crucial for platelet hemostatic function. EF-hand domain containing 2 (EFhd2, Swiprosin-1) is a Ca2+-binding cytoskeletal adaptor protein involved in actin remodeling in different cell types, but its function in platelets is unknown. Objective Based on the described functions of EFhd2 in immune cells, we tested the hypothesis that EFhd2 is a crucial adaptor protein for platelet function acting as a regulator of Ca2+-mobilization and cytoskeletal rearrangements. Methods and Results We generated EFhd2-deficient mice and analyzed their platelets in vitro and in vivo. Efhd2-/- mice displayed normal platelet count and size, exhibited an unaltered in vivo life span and showed normal Ca2+-mobilization and activation/aggregation responses to classic agonists. Interestingly, upon stimulation of the immunoreceptor tyrosine-based activation motif-coupled receptor glycoprotein (GP) VI, Efhd2-/- platelets showed a slightly increased coagulant activity. Furthermore, absence of EFhd2 had no significant impact on integrin-mediated clot retraction, actomyosin rearrangements and spreading of activated platelets on fibrinogen. In vivo EFhd2-deficiency resulted in unaltered hemostatic function and unaffected arterial thrombus formation. Conclusion These results show that EFhd2 is not essential for platelet function in mice indicating that other cytoskeletal adaptors may functionally compensate its loss. KW - adaptor protein Swiprosin-1/EFhd2 KW - platelets KW - platelet activation KW - platelet aggregation KW - cytoskeleton KW - thrombin KW - blood KW - actins KW - collagens Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-113316 ER - TY - JOUR A1 - Koo, Chek Ziu A1 - Matthews, Alexandra L. A1 - Harrison, Neale A1 - Szyroka, Justyna A1 - Nieswandt, Bernhard A1 - Gardiner, Elizabeth E. A1 - Poulter, Natalie S. A1 - Tomlinson, Michael G. T1 - The platelet collagen receptor GPVI is cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 molecular scissors JF - International Journal of Molecular Sciences N2 - The platelet-activating collagen receptor GPVI represents the focus of clinical trials as an antiplatelet target for arterial thrombosis, and soluble GPVI is a plasma biomarker for several human diseases. A disintegrin and metalloproteinase 10 (ADAM10) acts as a ‘molecular scissor’ that cleaves the extracellular region from GPVI and many other substrates. ADAM10 interacts with six regulatory tetraspanin membrane proteins, Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33, which are collectively termed the TspanC8s. These are emerging as regulators of ADAM10 substrate specificity. Human platelets express Tspan14, Tspan15 and Tspan33, but which of these regulates GPVI cleavage remains unknown. To address this, CRISPR/Cas9 knockout human cell lines were generated to show that Tspan15 and Tspan33 enact compensatory roles in GPVI cleavage, with Tspan15 bearing the more important role. To investigate this mechanism, a series of Tspan15 and GPVI mutant expression constructs were designed. The Tspan15 extracellular region was found to be critical in promoting GPVI cleavage, and appeared to achieve this by enabling ADAM10 to access the cleavage site at a particular distance above the membrane. These findings bear implications for the regulation of cleavage of other ADAM10 substrates, and provide new insights into post-translational regulation of the clinically relevant GPVI protein. KW - ADAM10 KW - GPVI KW - tetraspanin KW - platelet KW - shedding KW - TspanC8 KW - metalloproteinase Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284468 SN - 1422-0067 VL - 23 IS - 5 ER - TY - JOUR A1 - Stegner, David A1 - van Eeuwijk, Judith M.M. A1 - Angay, Oğuzhan A1 - Gorelashvili, Maximilian G. A1 - Semeniak, Daniela A1 - Pinnecker, Jürgen A1 - Schmithausen, Patrick A1 - Meyer, Imke A1 - Friedrich, Mike A1 - Dütting, Sebastian A1 - Brede, Christian A1 - Beilhack, Andreas A1 - Schulze, Harald A1 - Nieswandt, Bernhard A1 - Heinze, Katrin G. T1 - Thrombopoiesis is spatially regulated by the bone marrow vasculature JF - Nature Communications N2 - In mammals, megakaryocytes (MKs) in the bone marrow (BM) produce blood platelets, required for hemostasis and thrombosis. MKs originate from hematopoietic stem cells and are thought to migrate from an endosteal niche towards the vascular sinusoids during their maturation. Through imaging of MKs in the intact BM, here we show that MKs can be found within the entire BM, without a bias towards bone-distant regions. By combining in vivo two-photon microscopy and in situ light-sheet fluorescence microscopy with computational simulations, we reveal surprisingly slow MK migration, limited intervascular space, and a vessel-biased MK pool. These data challenge the current thrombopoiesis model of MK migration and support a modified model, where MKs at sinusoids are replenished by sinusoidal precursors rather than cells from a distant periostic niche. As MKs do not need to migrate to reach the vessel, therapies to increase MK numbers might be sufficient to raise platelet counts. KW - bone marrow KW - megakaryocytes KW - thrombopoiesis Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170591 VL - 8 IS - 127 ER -