Refine
Has Fulltext
- yes (26)
Is part of the Bibliography
- yes (26)
Year of publication
Document Type
- Journal article (26) (remove)
Language
- English (26)
Keywords
- ischemic stroke (7)
- glycoprotein VI (4)
- platelets (4)
- thrombo-inflammation (4)
- platelet (3)
- platelet activation (3)
- thrombin (3)
- thrombosis (3)
- collagens (2)
- glycoprotein receptor Ib (2)
- ischemic penumbra (2)
- megakaryocytes (2)
- middle cerebral artery occlusion (2)
- platelet aggregation (2)
- platelet receptors (2)
- signaling (2)
- thrombopoiesis (2)
- 3D image analysis (1)
- ADAM10 (1)
- Cytoskeleton (1)
- ERK map kinease (1)
- ERK1/2 (1)
- GPVI (1)
- JAQ1 (1)
- Mast cells (1)
- Orai2 (1)
- RKIP (1)
- T-cells (1)
- TspanC8 (1)
- ZIP (1)
- actins (1)
- adaptor protein Swiprosin-1/EFhd2 (1)
- adipose tissue (1)
- animal models (1)
- arterial thrombus formation (1)
- bioenergetics (1)
- bleeding (1)
- blood (1)
- blood coagulation (1)
- blood flow (1)
- blood platelets (1)
- body weight (1)
- bone marrow (1)
- brain (1)
- cancer (1)
- cascade (1)
- cerebellum (1)
- cerebrovascular disorders (1)
- coagulation (1)
- coagulation system (1)
- coffin-lowry-syndrome (1)
- collagen (1)
- cytoskeleton (1)
- dendritic growth (1)
- dentate gyrus (1)
- embryos (1)
- enzyme regulation (1)
- factor XII (1)
- fibrin (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)
- insulin (1)
- insulin resistance (1)
- integrin α2 (1)
- integrins (1)
- interactome (1)
- interspecies comparison (1)
- intracranial bleeding (1)
- light sheet fluorescence microscopy (1)
- long-term potentation (1)
- metalloproteinase (1)
- metals (1)
- microfluidics (1)
- mouse (1)
- mouse models (1)
- network (1)
- neurite outgrowth (1)
- neurology (1)
- neuronal dendrites (1)
- neuronal differentiation (1)
- neurons (1)
- neurotrophic factor (1)
- occlusion (1)
- optical clearing (1)
- phosphatidic acid (1)
- phospholipase D (1)
- platelet degranulation (1)
- platelet inhibition (1)
- protein RSK2 (1)
- proteome (1)
- recombinant tissue-type plasminogen activator (1)
- regulatory circuit downstream (1)
- serotonin (1)
- shedding (1)
- spatiotemporal thrombus (1)
- tMCAO (1)
- tetraspanin (1)
- transcriptome (1)
- transient middle cerebral artery (1)
- transient middle cerebral artery occlusion (1)
- tumor microenvironment (1)
- vascular structure (1)
- zinc (1)
Institute
- Institut für Experimentelle Biomedizin (26) (remove)
EU-Project number / Contract (GA) number
- 766118 (2)
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.
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:
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
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.
Genetically modified mice are indispensable for establishing the roles of platelets in arterial thrombosis and hemostasis. Microfluidics assays using anticoagulated whole blood are commonly used as integrative proxy tests for platelet function in mice. In the present study, we quantified the changes in collagen-dependent thrombus formation for 38 different strains of (genetically) modified mice, all measured with the same microfluidics chamber. The mice included were deficient in platelet receptors, protein kinases or phosphatases, small GTPases or other signaling or scaffold proteins. By standardized re-analysis of high-resolution microscopic images, detailed information was obtained on altered platelet adhesion, aggregation and/or activation. For a subset of 11 mouse strains, these platelet functions were further evaluated in rhodocytin- and laminin-dependent thrombus formation, thus allowing a comparison of glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2) and integrin α6β1 pathways. High homogeneity was found between wild-type mice datasets concerning adhesion and aggregation parameters. Quantitative comparison for the 38 modified mouse strains resulted in a matrix visualizing the impact of the respective (genetic) deficiency on thrombus formation with detailed insight into the type and extent of altered thrombus signatures. Network analysis revealed strong clusters of genes involved in GPVI signaling and Ca2+ homeostasis. The majority of mice demonstrating an antithrombotic phenotype in vivo displayed with a larger or smaller reduction in multi-parameter analysis of collagen-dependent thrombus formation in vitro. Remarkably, in only approximately half of the mouse strains that displayed reduced arterial thrombosis in vivo, this was accompanied by impaired hemostasis. This was also reflected by comparing in vitro thrombus formation (by microfluidics) with alterations in in vivo bleeding time. In conclusion, the presently developed multi-parameter analysis of thrombus formation using microfluidics can be used to: (i) determine the severity of platelet abnormalities; (ii) distinguish between altered platelet adhesion, aggregation and activation; and (iii) elucidate both collagen and non-collagen dependent alterations of thrombus formation. This approach may thereby aid in the better understanding and better assessment of genetic variation that affect in vivo arterial thrombosis and hemostasis.
Zinc (Zn2+) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn2+ storage and release. To visualize Zn2+ storage in human and mouse platelets, the Zn2+ specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d−/− mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn2+ release upon activation. Platelets from Nbeal2−/− mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn2+ levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn2+ homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2−/− and Unc13d−/− mice, and the impairment could be partially restored by extracellular Zn2+. Altogether, we conclude that the release of ionic Zn2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn2+ in platelet-dependent fibrin formation.