Institut für Experimentelle Biomedizin
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Sonstige beteiligte Institutionen
The implementation of high‐throughput sequencing (HTS) technologies in research and diagnostic laboratories has linked many new genes to rare bleeding, thrombotic, and platelet disorders (BTPD), and revealed multiple genetic variants linked to those disorders, many of them being of uncertain pathogenicity when considering the accepted evidence (variant consequence, frequency in control datasets, number of reported patients, prediction models, and functional assays). The sequencing effort has also resulted in resources for gathering disease‐causing variants associated with specific genes, but for BTPD, such well‐curated databases exist only for a few genes. On the other hand, submissions by individuals or diagnostic laboratories to the variant database ClinVar are hampered by the lack of a submission process tailored to capture the specific features of hemostatic diseases. As we move toward the implementation of HTS in the diagnosis of BTPD, the Scientific and Standardization Committee for Genetics in Thrombosis and Haemostasis has developed and tested a REDCap‐based interface, aimed at the community, to submit curated genetic variants for diagnostic‐grade BTPD genes. Here, we describe the use of the interface and the initial submission of 821 variants from 30 different centers covering 14 countries. This open‐access variant resource will be shared with the community to improve variant classification and regular bulk data transfer to ClinVar.
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.
Glycine and γ-aminobutyric acid (GABA) are the major determinants of inhibition in the central nervous system (CNS). These neurotransmitters target glycine and GABAA receptors, respectively, which both belong to the Cys-loop superfamily of pentameric ligand-gated ion channels (pLGICs). Interactions of the neurotransmitters with the cognate receptors result in receptor opening and a subsequent influx of chloride ions, which, in turn, leads to hyperpolarization of the membrane potential, thus counteracting excitatory stimuli. The majority of glycine receptors and a significant fraction of GABAA receptors (GABAARs) are recruited and anchored to the post-synaptic membrane by the central scaffolding protein gephyrin. This ∼93 kDa moonlighting protein is structurally organized into an N-terminal G-domain (GephG) connected to a C-terminal E-domain (GephE) via a long unstructured linker. Both inhibitory neurotransmitter receptors interact via a short peptide motif located in the large cytoplasmic loop located in between transmembrane helices 3 and 4 (TM3-TM4) of the receptors with a universal receptor-binding epitope residing in GephE. Gephyrin engages in nearly identical interactions with the receptors at the N-terminal end of the peptide motif, and receptor-specific interaction toward the C-terminal region of the peptide. In addition to its receptor-anchoring function, gephyrin also interacts with a rather large collection of macromolecules including different cytoskeletal elements, thus acting as central scaffold at inhibitory post-synaptic specializations. Dysfunctions in receptor-mediated or gephyrin-mediated neurotransmission have been identified in various severe neurodevelopmental disorders. Although biochemical, cellular and electrophysiological studies have helped to understand the physiological and pharmacological roles of the receptors, recent high resolution structures of the receptors have strengthened our understanding of the receptors and their gating mechanisms. Besides that, multiple crystal structures of GephE in complex with receptor-derived peptides have shed light into receptor clustering by gephyrin at inhibitory post-synapses. This review will highlight recent biochemical and structural insights into gephyrin and the GlyRs as well as GABAA receptors, which provide a deeper understanding of the molecular machinery mediating inhibitory neurotransmission.
Psoriasis is a frequent systemic inflammatory autoimmune disease characterized primarily by skin lesions with massive infiltration of leukocytes, but frequently also presents with cardiovascular comorbidities. Especially polymorphonuclear neutrophils (PMNs) abundantly infiltrate psoriatic skin but the cues that prompt PMNs to home to the skin are not well-defined. To identify PMN surface receptors that may explain PMN skin homing in psoriasis patients, we screened 332 surface antigens on primary human blood PMNs from healthy donors and psoriasis patients. We identified platelet surface antigens as a defining feature of psoriasis PMNs, due to a significantly increased aggregation of neutrophils and platelets in the blood of psoriasis patients. Similarly, in the imiquimod-induced experimental in vivo mouse model of psoriasis, disease induction promoted PMN-platelet aggregate formation. In psoriasis patients, disease incidence directly correlated with blood platelet counts and platelets were detected in direct contact with PMNs in psoriatic but not healthy skin. Importantly, depletion of circulating platelets in mice in vivo ameliorated disease severity significantly, indicating that both PMNs and platelets may be relevant for psoriasis pathology and disease severity.
CD8 T cells protect the liver against viral infection, but can also cause severe liver damage that may even lead to organ failure. Given the lack of mechanistic insights and specific treatment options in patients with acute fulminant hepatitis, we develop a mouse model reflecting a severe acute virus-induced CD8 T cell-mediated hepatitis. Here we show that antigen-specific CD8 T cells induce liver damage in a perforin-dependent manner, yet liver failure is not caused by effector responses targeting virus-infected hepatocytes alone. Additionally, CD8 T cell mediated elimination of cross-presenting liver sinusoidal endothelial cells causes endothelial damage that leads to a dramatically impaired sinusoidal perfusion and indirectly to hepatocyte death. With the identification of perforin-mediated killing as a critical pathophysiologic mechanism of liver failure and the protective function of a new class of perforin inhibitor, our study opens new potential therapeutic angles for fulminant viral hepatitis.
In the recent two decades, LIM and SH3 protein 1 (LASP1) has been developed from a simple actin-binding structural protein to a tumor biomarker and subsequently to a complex, nuclear transcriptional regulator. Starting with a brief historical perspective, this review will mainly compare and contrast LASP1 and LASP2 from the angle of the newest data and importantly, examine their role in transcriptional regulation. We will summarize the current knowledge through pictorial models and tables including the roles of different microRNAs in the differential regulation of LASP1 levels and patient outcome rather than specify in detail all tumor entities. Finally, the novel functional roles of LASP1 in secretion of vesicles, expression of matrix metalloproteinases and transcriptional regulation as well as the activation of survival and proliferation pathways in different cancer types are described.
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.
Kisspeptins (KPs, KISS1) and their receptor (KISS1R) play a pivotal role as metastasis suppressor for many cancers. Low or lost KP expression is associated with higher tumor grade, increased metastatic potential, and poor prognosis. Therefore, KP expression has prognostic relevance and correlates with invasiveness in cancers. Furthermore, KISS1R represents a very promising target for molecular imaging and therapy for KISS1R-expressing tumors. The goal of this study was to evaluate the developed KISS1-54 derivative, [\(^{68}\)Ga]KISS1-54, as a PET-imaging probe for KISS1R-expressing tumors. The NODAGA-KISS1-54 peptide was labeled by Gallium-68, and the stability of the resulting [\(^{68}\)Ga]KISS1-54 evaluated in injection solution and human serum, followed by an examination in different KISS1R-expressing tumor cell lines, including HepG2, HeLa, MDA-MB-231, MCF7, LNCap, SK-BR-3, and HCT116. Finally, [\(^{68}\)Ga]KISS1-54 was tested in LNCap- and MDA-MB-231-bearing mice, using µ-PET, assessing its potential as an imaging probe for PET. [\(^{68}\)Ga]KISS1-54 was obtained in a 77 ± 7% radiochemical yield and at a >99% purity. The [\(^{68}\)Ga]KISS1-54 cell uptake amounted to 0.6–4.4% per 100,000 cells. Moreover, the accumulation of [\(^{68}\)Ga]KISS1-54 was effectively inhibited by nonradioactive KISS1-54. In [\(^{68}\)Ga]KISS1-54-PET, KISS1R-positive LNCap-tumors were clearly visualized as compared to MDA-MB-231-tumor implant with predominantly intracellular KISS1R expression. Our first results suggest that [\(^{68}\)Ga]KISS1-54 is a promising candidate for a radiotracer for targeting KISS1R-expressing tumors via PET.
Introduction
Pro-thrombotic events are one of the prevalent causes of intensive care unit (ICU) admissions among COVID-19 patients, although the signaling events in the stimulated platelets are still unclear.
Methods
We conducted a comparative analysis of platelet transcriptome data from healthy donors, ICU, and non-ICU COVID-19 patients to elucidate these mechanisms. To surpass previous analyses, we constructed models of involved networks and control cascades by integrating a global human signaling network with transcriptome data. We investigated the control of platelet hyperactivation and the specific proteins involved.
Results
Our study revealed that control of the platelet network in ICU patients is significantly higher than in non-ICU patients. Non-ICU patients require control over fewer proteins for managing platelet hyperactivity compared to ICU patients. Identification of indispensable proteins highlighted key subnetworks, that are targetable for system control in COVID-19-related platelet hyperactivity. We scrutinized FDA-approved drugs targeting indispensable proteins and identified fostamatinib as a potent candidate for preventing thrombosis in COVID-19 patients.
Discussion
Our findings shed light on how SARS-CoV-2 efficiently affects host platelets by targeting indispensable and critical proteins involved in the control of platelet activity. We evaluated several drugs for specific control of platelet hyperactivity in ICU patients suffering from platelet hyperactivation. The focus of our approach is repurposing existing drugs for optimal control over the signaling network responsible for platelet hyperactivity in COVID-19 patients. Our study offers specific pharmacological recommendations, with drug prioritization tailored to the distinct network states observed in each patient condition. Interactive networks and detailed results can be accessed at https://fostamatinib.bioinfo-wuerz.eu/.
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.