Biology subjects

Nieswandt, B.

Publications and source records attributed to Nieswandt, B..

11 recordsLinked to original sources

bioRxiv2026

Sub-endothelial platelet activation amplifies neutrophil transmigration within venular walls

Neutrophil recruitment into inflamed tissues requires coordinated transmigration across the multilayered venular wall, yet how this process is regulated beyond the endothelium remains poorly understood. Here, we identify a previously unrecognized platelet-neutrophil circuit that controls this post-endothelial phase. Intravital microscopy of inflamed cremasteric venules showed that pioneer neutrophil transmigration enabled platelet entry into the sub-endothelial compartment of venular walls. There, platelets became activated and released CXCL7 in a GPVI- and GPIb-dependent manner, generating spatially confined chemokine microdomains. These platelet-derived cues directed follower neutrophil migration and promoted their exit across the pericyte layer into the interstitial tissue. Collectively, these findings identify extraluminal platelets as spatial organizers of neutrophil trafficking and uncover a localized intramural feedback mechanism in which neutrophils amplify their own recruitment within venular walls. Graphical summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/739803v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1d22216org.highwire.dtl.DTLVardef@bc0279org.highwire.dtl.DTLVardef@1465d7dorg.highwire.dtl.DTLVardef@a3e755_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Targeting platelet GPVI or GPIIb/IIIa only minimally affect acute inflammation and cardiac repair in experimental permanent myocardial infarction

BackgroundBeyond their role in hemostasis, platelets are recognized as key regulators of inflammatory responses in ischemic diseases, including cardiac ischemia/reperfusion (I/R) injury with key roles of platelet membrane glycoproteins (GP)VI and IIb/IIIa. However, whether platelet-driven thrombo-inflammatory pathways affect acute inflammation and cardiac repair processes in permanent, non-reperfused myocardial infarction (MI) is unknown. MethodsWe targeted GPVI and GPIIb/IIIa in experimental permanent MI in mice. Cardiac, bone marrow, and blood innate immune responses were evaluated by flow cytometry and single-cell RNA-sequencing. Survival and cardiac repair were assessed over the inflammatory and scar formation phase, until day 10 after permanent MI. ResultsPlatelet GPVI immunodepletion by injection of the anti-GPVI antibody JAQ1 did not affect levels of neutrophil or monocyte subsets (Ly6Chi and Ly6Clow) in the bone marrow and blood, and did not alter accumulation of monocytes, macrophage subsets (defined by expression of MHCII and TIM4), or neutrophil subsets (SiglecFhi/low) in the infarcted heart on day 4. GPVI depletion only had a minimal effect on cardiac repair, slightly decreasing interstitial fibrosis in the infarct border zone on day 10. Four days after MI, GPIIb/IIIa inhibition by JON/A-F(ab')2 had no effect on cardiac or systemic innate immune cell levels as measured by flow cytometry and did not affect composition and transcriptomic profile of the cardiac immune infiltrate as revealed by single-cell RNA-sequencing. GPIIb/IIIa inhibition did not improve cardiac remodeling, and was even associated with an increased mortality rate over 10 days post-MI. ConclusionTargeting the GPVI-GPIIb/IIIa axis only had minor effects on post-MI inflammatory responses and cardiac wound healing in permanent myocardial ischemia. Our findings demonstrate that the therapeutic benefits of inhibiting platelet-driven thrombo-inflammation are particularly relevant in the subacute reperfusion phase.

physiology↗

bioRxiv2025

Resting calcium ion fluxes protect cells from fast mitochondrial fragmentation, cell stress responses, and immediate transcriptional reprogramming

Homeostatic calcium ion (Ca2+) fluxes between the endoplasmic reticulum, cytosol, and extracellular space occur not only in response to cell stimulation but also in unstimulated cells. Using murine astrocytes as a model, we asked whether there is a signaling function of these resting Ca2+-fluxes. The data showed that endoplasmic reticulum (ER) Ca{superscript 2} depletion, induced by sarcoplasmic/endoplasmic reticulum Ca{superscript 2}-ATPase (SERCA) inhibition, resulted to prolonged Ca{superscript 2} influx and mitochondrial fragmentation within 10 to 30 minutes. This mitochondrial fragmentation could be prevented in Ca2+- free medium or by inhibiting store-operated Ca2+ entry (SOCE). Similarly, attenuation of STIM proteins, which are vital ER Ca2+ sensors, protected mitochondrial morphology. On the molecular level, ER Ca2+ depletion, achieved either by removing extracellular Ca2+ or through acute SERCA inhibition, led to changes in gene expression of about 13% and 41% of the transcriptome within an hour, respectively. Transcriptome changes were associated with universal biological processes such as transcription, differentiation, or cell stress. Strong increase in expression was observed for the transcription factor ATF4, which is under control of the kinase PERK (EIF2AK3), a key protein involved in ER stress. Corroborating these findings, PERK was rapidly phosphorylated in Ca2+-free medium or after acute pharmacological inhibition of SOCE. In summary, resting, homeostatic Ca2+ fluxes prevent immediate- early cell stress and transcriptional reprogramming.

physiology↗

bioRxiv2024

Platelets are Protective in Early Abdominal Aortic Aneurysm Formation

BackgroundAbdominal aortic aneurysm (AAA) is a disease associated with the pathophysiologic degradation of the tunica media resulting in aortic dilatation, systemic inflammation, and dysregulated hemostasis. Beyond role its role in initiating primary hemostasis, platelets are a source of ROS, inflammatory cytokines and growth factors necessary for angiogenesis and vascular remodeling. Although platelets contribute to the progression of established aneurysms, their role in the initiation of AAA remains undefined. MethodsLow density lipoprotein receptor deficient (Ldlr-/-) mice were examined for platelet accumulation in the angiotensin II (AngII) model of AAA utilizing in vivo labeling techniques. Two platelet antagonists (clopidogrel and aspirin), a thrombin inhibitor (dabigatran) or genetic deficiencies (protease-activated receptor 4, P2Y12, Lnk) were administered to AngII-infused mice to determine the role of platelets in initiation of AAA. The effect of platelet depletion was examined in multiple mouse strains of AngII-induced AAA and two additional aneurysm models. PheWAS and meta-analysis was analyzed in humans for platelet gene SNPs associated with AAA. ResultsWe show that platelets are recruited rapidly to the aorta after the initiation of AngII infusion. Genetic deficiency of platelet receptors had no effect on abdominal aortic diameter, but augmented rupture-induced death in littermate versus placebo controls during AngII-induced AAA. Moreover, Ldlr-/- mice receiving anti-platelet inhibitors or a thrombin inhibitor also had augmented rupture-induced death. Platelet depletion preceding aneurysm formation resulted in pervasive rupture-induced death in several mouse strains and with three different mouse models of AAA. ConclusionsInhibition of platelet function is detrimental in an early expanding aortic lumen resulting in catastrophic rupture and hemodynamic failure in murine AAA models.

pathology↗

bioRxiv2021

Loss of zinc transporters ZIP1 and ZIP3 augments platelet reactivity in response to G protein-coupled receptor agonists and accelerates thrombus formation in vivo

Zinc (Zn2+) is considered as an important mediator for 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 well-known 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 delayed and less efficient Zn2+ release upon thrombin-stimulated platelet activation. This resulted in a hyperactive platelet response not only in response to thrombin, but also towards other G protein-coupled receptor (GPCR) agonists. Immunoreceptor tyrosine-based activation (ITAM)-coupled receptor agonist signalling, however, was unaffected. Augmented GPCR responses were accompanied by enhanced Ca2+ signalling and PKC activation. Further functional analysis of ZIP1/3 double deficient mice revealed enhanced platelet aggregation, bigger thrombus volume under flow ex vivo and faster in vivo thrombus formation. The current study thereby identifies ZIP1 and ZIP3 as important regulators for the maintenance of platelet Zn2+ homeostasis and function.

biochemistry↗

bioRxiv2021

G6b-B regulates an essential step in megakaryocyte maturation

G6b-B is a megakaryocyte lineage-specific immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor, essential for platelet homeostasis. Mice with a genomic deletion of the entire Mpig6b locus develop severe macrothrombocytopenia and myelofibrosis, which is reflected in humans with null-mutations in MPIG6B. The current model proposes that megakaryocytes lacking G6b-B develop normally, while proplatelet release is hampered, but the underlying molecular mechanism remains unclear. Here, we report on a spontaneous recessive single nucleotide mutation in C57BL/6 mice, localized within the intronic region of the Mpig6b locus that abolishes G6b-B expression and reproduces macrothrombocytopenia, myelofibrosis and osteosclerosis. As the mutation is based on a single nucleotide exchange, Mpig6bmut mice represent an ideal model to study the role of G6b-B. Megakaryocytes from these mice were smaller in size, displayed a less developed demarcation membrane system and reduced expression of receptors. RNA sequencing revealed a striking global reduction in the level of megakaryocyte-specific transcripts, in conjunction with decreased protein levels of the transcription factor GATA-1, and impaired thrombopoietin signaling. The reduced number of mature MKs in the bone marrow was corroborated on a newly developed Mpig6b null mouse strain. Our findings highlight an unexpected essential role of G6b-B in the early differentiation within the megakaryocytic lineage.

cell biology↗

bioRxiv2021

Microthrombocytopenia caused by impaired microtubule stability in RhoB-deficient mice

Megakaryocytes are large cells in the bone marrow, which give rise to blood platelets. Platelet biogenesis involves megakaryocyte maturation, the localization of mature cells in close proximity to bone marrow sinusoids and the formation of protrusions, which are shed into the circulation. Rho GTPases play important roles in platelet biogenesis and function. RhoA-deficient mice display macrothrombocytopenia and a striking mislocalization of megakaryocytes into bone marrow sinusoids and a specific defect in G-protein signaling in platelets. However, the role of the closely related protein RhoB in megakaryocytes or platelets remains unknown. In this study, we show that, in contrast to RhoA deficiency, genetic ablation of RhoB in mice results in microthrombocytopenia (decreased platelet count and size). RhoB-deficient platelets displayed mild functional defects predominantly upon induction of the collagen/glycoprotein VI pathway. Megakaryocyte maturation and localization within the bone marrow, as well as actin dynamics were not affected in the absence of RhoB. However, in vitro generated proplatelets revealed pronouncedly impaired microtubule organization. Furthermore, RhoB-deficient platelets and megakaryocytes displayed selective defects in microtubule dynamics/stability, correlating with pronouncedly reduced levels of acetylated -tubulin. Our findings imply that absence of this tubulin posttranslational modification results in decreased microtubule stability leading to microthrombocytopenia in RhoB-deficient mice. Our data thus points to specifically impaired microtubule - but not actin - dynamics as a general mechanism underlying the manifestation of microthrombocytopenia in vivo. We furthermore demonstrate that RhoA and RhoB have specific, non-redundant functions in the megakaryocyte lineage. KEY POINTSO_LIRhoB-deficient mice display microthrombocytopenia C_LIO_LIRhoB has different functions in the megakaryocyte lineage than RhoA and regulates microtubule dynamics C_LI

cell biology↗

bioRxiv2021

Antibody-mediated depletion of human CLEC-2 in a novel humanised mouse model

Platelet C-type lectin-like receptor 2 (CLEC-2) has been proposed as a potential anti-thrombotic target as genetic or antibody-mediated receptor deficiency prevents occlusive thrombus formation in mice. This occurs through interaction with an unknown ligand as the endogenous ligand podoplanin is not present in the vasculature. However, the CLEC-2-podoplanin interaction does have an important role in tumour metastasis. There are currently no methods to test potential human therapeutics targeting CLEC-2, such as antibodies, in vivo. We have therefore generated and characterised a humanised CLEC-2 mouse (hCLEC-2KI) and developed a novel monoclonal anti-human CLEC-2 antibody, HEL1, for in vivo testing. hCLEC-2KI mice were phenotypically normal and had comparable platelet glycoprotein receptor expression, activation and aggregation to wildtype platelets. hCLEC-2KI mice had both comparable bleeding and vessel occlusion times to WT mice. Challenging hCLEC-2KI mice with HEL1 or a second monoclonal anti-hCLEC-2 antibody, AYP1, resulted in transient thrombocytopenia as well as CLEC-2 depletion for more than 2 weeks but had no effect on haemostasis. This illustrates the power of the humanised CLEC-2 mouse model in evaluating novel therapeutics in vivo, including antibodies that target CLEC-2, as well as the limited effect on haemostasis when targeting CLEC-2.

physiology↗

bioRxiv2021

Mapping densely distributed membrane receptors in blood platelets with expansion microscopy

Interrogating small platelets and their densely packed, highly abundant receptor landscape is key to understand platelet clotting, a process that can save lives when stopping blood loss after an injury, but also kill when causing heart attack, stroke or pulmonary embolism. The underlying key receptor distributions and interactions, in particular the relevance of integrin clustering, are not fully understood is because of highly abundant and densely distributed GPIIb/IIIa receptors. This makes receptor distributions difficult to assess even by super-resolution fluorescence microscopy. Here, we combine dual-color expansion and confocal microscopy with colocalization analysis to assess platelet receptor organization without the need of a super-resolution microscope. We show that 4x expansion is highly straight-forward for super-resolution microscopy of platelets, while 10x expansion provides higher precision at the price of increased efforts in sample preparation and imaging. Quantifying various receptor colocalization scenarios we demonstrate that expansion microscopy can pinpoint receptor distributions and interactions in resting and activated platelets being superior to conventional methods that fail in such dense 3D scenarios with highly abundant receptors. We reveal the presence of GPIIb/IIIa clusters in resting platelets, which are not affected by platelet activation indicating that they contribute to the rapid platelet response during platelet clotting. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/431449v3_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@c4e38forg.highwire.dtl.DTLVardef@37c1adorg.highwire.dtl.DTLVardef@8c8597org.highwire.dtl.DTLVardef@196bd69_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Loss of Hem1 disrupts macrophage function and impacts on migration, phagocytosis and integrin-mediated adhesion

The hematopoietic-specific protein 1 (Hem1) comprises an essential subunit of the WAVE Regulatory Complex (WRC) in immune cells. WRC has a fundamental role in Arp2/3 complex activation and the protrusion of branched actin networks in motile cells. Hem1 deficiency leads to suppression of the entire WRC in immune cells. Defective WRC function in macrophages results in loss of lamellipodia and migration defects. Moreover, phagocytosis, commonly accompanied by lamellipodium protrusion during cup formation, is altered in Hem1 null cells concerning frequency and efficacy. When analyzing cell spreading, adhesion and podosome formation, we found that Hem1 null cells are capable, in principle, of podosome formation and consequently, do not show any quantitative differences in extracellular matrix degradation. Their adhesive behavior, however, was significantly altered. Specifically, adhesion as well as de-adhesion of Hem1 null cells was strongly compromised, likely contributing to the observed reduced efficiency of phagocytosis. In line with this, phosphorylation of the prominent adhesion component paxillin was diminished. Non-hematopoietic somatic cells disrupted in expression for both Hem1 and its ubiquitous orthologue Nck-associated protein 1 (Nap1) or the essential WRC components Sra-1/PIR121 did not only confirm defective paxillin phosphorylation, but also revealed that paxillin turnover in focal adhesions is accelerated in the absence of WRC. Finally, adhesion assays using platelets lacking functional WRC as model system unmasked radically decreased IIb{beta}3 integrin activation. Our results thus demonstrate that WRC-driven actin networks impact on integrin-dependent processes controlling formation and dismantling of different types of cell-substratum adhesion. One sentence summaryInterference of Hem1 function in mice and cells uncovers a hitherto unrecognized role in integrin-mediated cell adhesion that is crucial for macrophage function and connects to recently discovered immunodeficiencies in patients carrying Hem1 mutations.

cell biology↗

bioRxiv2017

C-type lectin-like receptor 2 (CLEC-2)-dependent DC migration is controlled by tetraspanin CD37

Cell migration is central to evoke a potent immune response. Dendritic cell (DC) migration to lymph nodes is dependent on the interaction of C-type lectin-like receptor 2 (CLEC-2) expressed by DCs, with podoplanin expressed by lymph node stromal cells (LNSCs). However, the underlying molecular mechanisms by which CLEC-2 influences DC migration remain elusive. Here, we show that CLEC-2-dependent DC migration is tightly controlled by tetraspanin CD37, a membrane-organizing protein. Our findings demonstrate a specific molecular interaction between CLEC-2 and CD37. Myeloid cells lacking CD37 (Cd37-/-) expressed less CLEC-2 on their surface compared to wild-type cells, indicating that CD37 is required to stabilize membrane expression of CLEC-2. In addition, CLEC-2-expressing DCs lacking CD37 showed impaired adhesion, migration velocity and displacement on LNSCs. Moreover, Cd37-/- DCs failed to form actin protrusions in a 3D collagen matrix upon podoplanin-induced CLEC-2 stimulation, phenocopying CLEC-2-deficient DCs (CD11c{Delta}CLEC-2). Microcontact printing experiments revealed that CD37 is required for CLEC-2 recruitment in the membrane to its ligand podoplanin. This study demonstrates that tetraspanin CD37 controls CLEC-2 membrane organization and provides new molecular insights underlying CLEC-2-dependent DC migration.

cell biology↗