Refine
Has Fulltext
- yes (38)
Is part of the Bibliography
- yes (38) (remove)
Year of publication
Document Type
- Journal article (38)
Language
- English (38) (remove)
Keywords
- ischemic stroke (9)
- platelets (7)
- glycoprotein VI (5)
- platelet activation (5)
- blood (4)
- platelet aggregation (4)
- thrombo-inflammation (4)
- Mice (3)
- integrins (3)
- platelet (3)
- thrombin (3)
- Inflammation (2)
- blood coagulation (2)
- collagens (2)
- factor XII (2)
- flow cytometry (2)
- glycoprotein receptor Ib (2)
- ischemic penumbra (2)
- megakaryocytes (2)
- middle cerebral artery occlusion (2)
- platelet receptors (2)
- thrombopoiesis (2)
- thrombosis (2)
- 2-photon microscopy (1)
- 3D image analysis (1)
- ADAM10 (1)
- Activation (1)
- Adipokine (1)
- Arterial water (1)
- Brain (1)
- CD coreceptors (1)
- CLEC-2 ITAM (1)
- CXCL4 (1)
- CXCL7 (1)
- Cells (1)
- Cerebral-ischemia (1)
- Chemerin (1)
- Chemerin processing (1)
- Coefficient (1)
- Cranial window (1)
- Cytoskeleton (1)
- EHT1864 (1)
- ERK map kinease (1)
- ERK1/2 (1)
- Experimental stroke (1)
- Fluorescence (1)
- GPVI (1)
- GPVI shedding (1)
- Head-injury (1)
- Heart (1)
- Hemodynamic depression (1)
- Identification (1)
- In vivo imaging (1)
- Intravascular coagulation (1)
- JAQ1 (1)
- Magnetic-resonance (1)
- Mast cells (1)
- Metabolism (1)
- Model (1)
- Molecular-weight heparin (1)
- Mouse model (1)
- NAP-2 (1)
- NFκB-activation (1)
- Neurons (1)
- Orai2 (1)
- PF4 (1)
- Peptidase inhibitor 16 (PI16) (1)
- Perfusion (1)
- Protease inhibition (1)
- Protein (1)
- Purification (1)
- RARRES2 (1)
- RKIP (1)
- Rac1 (1)
- Rats (1)
- Src family (1)
- T-cells (1)
- TIG2 (1)
- Thrombus formation (1)
- TspanC8 (1)
- Von-Willebrand-factor (1)
- accelerated atherosclerosis (1)
- acetylsalicylic acid (1)
- actins (1)
- adaptor protein Swiprosin-1/EFhd2 (1)
- adipose tissue (1)
- animal models (1)
- antioxidant function (1)
- apolipoprotein E (1)
- bioenergetics (1)
- biomarkers (1)
- blood flow (1)
- blood platelets (1)
- body weight (1)
- bone marrow (1)
- brain (1)
- cancer (1)
- cascade (1)
- cell-cycle arrest (1)
- cerebellum (1)
- cerebrovascular diseases (1)
- cerebrovascular disorders (1)
- chemokines (1)
- coagulation (1)
- coffin-lowry-syndrome (1)
- cytoskeleton (1)
- cytotoxic T cells (1)
- damage responses (1)
- deficient mice (1)
- demography (1)
- dendric cells (1)
- dendritic growth (1)
- dentate gyrus (1)
- double knockout mice (1)
- embryos (1)
- endothelial cell (1)
- endothelial cell interactions (1)
- enzyme regulation (1)
- experimental autoimmune encephalomyelitis (1)
- fibrin (1)
- fibrinogen (1)
- glycoprotein Ib (1)
- glycoprotein Ibα (1)
- glycoprotein receptor Ibα (1)
- granule cells (1)
- hemostasis, (1)
- hippocampus (1)
- homeostasisIon channels (1)
- human (1)
- hypothalamus (1)
- in vivo (1)
- in-vivo (1)
- insulin (1)
- insulin resistance (1)
- integrin α2 (1)
- interactome (1)
- interspecies comparison (1)
- intracranial bleeding (1)
- kinase Syk (1)
- lesion formation (1)
- leukocyte adhesion (1)
- light sheet fluorescence microscopy (1)
- lipid bilayer (1)
- long-term potentation (1)
- mast cells (1)
- mechanisms (1)
- metalloproteinase (1)
- mice (1)
- mitogenic signaling (1)
- mouse (1)
- mouse models (1)
- multiple sclerosis (1)
- mutant p53 (1)
- network (1)
- neurite outgrowth (1)
- neuroinflammation (1)
- neurology (1)
- neuronal dendrites (1)
- neuronal differentiation (1)
- neurons (1)
- neurotrophic factor (1)
- nitric oxide synthase (1)
- occlusion (1)
- optical clearing (1)
- p53-dependent apoptosis (1)
- p53-inducible regulator (1)
- phosphatidic acid (1)
- phospholipase C gamma 2 (1)
- phospholipase D (1)
- platelet adhesion (1)
- platelet degranulation (1)
- platelet factor 4 (1)
- platelet inhibition (1)
- platelet receptor (1)
- podoplanin (1)
- protein RSK2 (1)
- proteome (1)
- recombinant tissue-type plasminogen activator (1)
- regulatory circuit downstream (1)
- restoration (1)
- senescence (1)
- serotonin (1)
- sex addiction (1)
- shedding (1)
- signaling (1)
- spatiotemporal thrombus (1)
- stroke (1)
- subthreshold IKK activation (1)
- tMCAO (1)
- tetraspanin (1)
- thromboxane (1)
- transcriptome (1)
- transient middle cerebral artery (1)
- transient middle cerebral artery occlusion (1)
- tumor microenvironment (1)
- tumorigenesis (1)
- tyrosine-protein kinase (1)
- vascular structure (1)
Institute
- Rudolf-Virchow-Zentrum (35)
- Institut für Experimentelle Biomedizin (23)
- Neurologische Klinik und Poliklinik (13)
- Institut für diagnostische und interventionelle Neuroradiologie (ehem. Abteilung für Neuroradiologie) (5)
- Medizinische Klinik und Poliklinik I (3)
- Theodor-Boveri-Institut für Biowissenschaften (3)
- Institut für Anatomie und Zellbiologie (2)
- Medizinische Klinik und Poliklinik II (2)
- Neurochirurgische Klinik und Poliklinik (2)
- Deutsches Zentrum für Herzinsuffizienz (DZHI) (1)
- Graduate School of Life Sciences (1)
- Institut für Klinische Epidemiologie und Biometrie (1)
- Institut für Klinische Neurobiologie (1)
- Institut für Medizinische Strahlenkunde und Zellforschung (1)
- Institut für Pharmakologie und Toxikologie (1)
- Klinik und Poliklinik für Psychiatrie, Psychosomatik und Psychotherapie (1)
- Lehrstuhl für Molekulare Psychiatrie (1)
- Pathologisches Institut (1)
- Physikalisches Institut (1)
- Physiologisches Institut (1)
Background
Effective inhibition of thrombosis without generating bleeding risks is a major challenge in medicine. Accumulating evidence suggests that this can be achieved by inhibition of coagulation factor XII (FXII), as either its knock-out or inhibition in animal models efficiently reduced thrombosis without affecting normal hemostasis. Based on these findings, highly specific inhibitors for human FXII(a) are under development. However, currently, in vivo studies on their efficacy and safety are impeded by the lack of an optimized animal model expressing the specific target, that is, human FXII.
Objective
The primary objective of this study is to develop and functionally characterize a humanized FXII mouse model.
Methods
A humanized FXII mouse model was generated by replacing the murine with the human F12 gene (genetic knock-in) and tested it in in vitro coagulation assays and in in vivo thrombosis models.
Results
These hF12\(^{KI}\) mice were indistinguishable from wild-type mice in all tested assays of coagulation and platelet function in vitro and in vivo, except for reduced expression levels of hFXII compared to human plasma. Targeting FXII by the anti-human FXIIa antibody 3F7 increased activated partial thromboplastin time dose-dependently and protected hF12\(^{KI}\) mice in an arterial thrombosis model without affecting bleeding times.
Conclusion
These data establish the newly generated hF12\(^{KI}\) mouse as a powerful and unique model system for in vivo studies on anti-FXII(a) biologics, supporting the development of efficient and safe human FXII(a) inhibitors.
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.
Comparison of the central human and mouse platelet signaling cascade by systems biological analysis
(2020)
Background
Understanding the molecular mechanisms of platelet activation and aggregation is of high interest for basic and clinical hemostasis and thrombosis research. The central platelet protein interaction network is involved in major responses to exogenous factors. This is defined by systemsbiological pathway analysis as the central regulating signaling cascade of platelets (CC).
Results
The CC is systematically compared here between mouse and human and major differences were found. Genetic differences were analysed comparing orthologous human and mouse genes. We next analyzed different expression levels of mRNAs. Considering 4 mouse and 7 human high-quality proteome data sets, we identified then those major mRNA expression differences (81%) which were supported by proteome data. CC is conserved regarding genetic completeness, but we observed major differences in mRNA and protein levels between both species. Looking at central interactors, human PLCB2, MMP9, BDNF, ITPR3 and SLC25A6 (always Entrez notation) show absence in all murine datasets. CC interactors GNG12, PRKCE and ADCY9 occur only in mice. Looking at the common proteins, TLN1, CALM3, PRKCB, APP, SOD2 and TIMP1 are higher abundant in human, whereas RASGRP2, ITGB2, MYL9, EIF4EBP1, ADAM17, ARRB2, CD9 and ZYX are higher abundant in mouse. Pivotal kinase SRC shows different regulation on mRNA and protein level as well as ADP receptor P2RY12.
Conclusions
Our results highlight species-specific differences in platelet signaling and points of specific fine-tuning in human platelets as well as murine-specific signaling differences.
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
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.
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.
Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard–Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V.
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.
Background:
Ischemic stroke causes a strong inflammatory response that includes T cells, monocytes/macrophages, and neutrophils. Interaction of these immune cells with platelets and endothelial cells facilitates microvascular dysfunction and leads to secondary infarct growth. We recently showed that blocking of platelet glycoprotein (GP) receptor Ib improves stroke outcome without increasing the risk of intracerebral hemorrhage. Until now, it has been unclear whether GPIb only mediates thrombus formation or also contributes to the pathophysiology of local inflammation.
Methods:
Focal cerebral ischemia was induced in C57BL/6 mice by a 60-min transient middle cerebral artery occlusion (tMCAO). Animals were treated with antigen-binding fragments (Fab) against the platelet surface molecules GPIb (p0p/B Fab). Rat immunoglobulin G (IgG) Fab was used as control treatment. Stroke outcome, including infarct size and functional deficits as well as the local inflammatory response, was assessed on day 1 after tMCAO.
Results:
Blocking of GPIb reduced stroke size and improved functional outcome on day 1 after tMCAO without increasing the risk of intracerebral hemorrhage. As expected, disruption of GPIb-mediated pathways in platelets significantly reduced thrombus burden in the cerebral microvasculature. In addition, inhibition of GPIb limited the local inflammatory response in the ischemic brain as indicated by lower numbers of infiltrating T cells and macrophages and lower expression levels of inflammatory cytokines compared with rat IgG Fab-treated controls.
Conclusion:
In acute ischemic stroke, thrombus formation and inflammation are closely intertwined (“thrombo-inflammation”). Blocking of platelet GPIb can ameliorate thrombo-inflammation.
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.