Biology subjects

Watson, S. P.

Publications and source records attributed to Watson, S. P..

4 recordsLinked to original sources

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

Analysis of Preplatelets and Their Barbell Platelet Derivatives by Imaging Flow Cytometry

Circulating large "preplatelets" undergo fission via barbell platelet intermediates into two smaller, mature platelets. In this study, we determine whether preplatelets and/or barbells are equivalent to reticulated/immature platelets by using ImageStream flow cytometry (ISFC) and super-resolution microscopy. Immature platelets, preplatelets and barbells were quantified in healthy and thrombocytopenic mice, healthy human volunteers, and patients with immune thrombocytopenia (ITP) or undergoing chemotherapy. Preplatelets and barbells were 1.9%+0.18/1.7%+0.48 (n=6) and 3.3%+1.6/0.5%+0.27 (n=12) of total platelet counts in murine and human whole blood, respectively. Both preplatelets and barbells exhibited high expression of HLA-I with high thiazole orange and mitotracker fluorescence. Tracking dye experiments confirmed that preplatelets transform into barbells and undergo fission ex vivo to increase platelet counts, with dependence upon the cytoskeleton and normal mitochondrial respiration. Samples from antibody-induced thrombocytopenia in mice and patients with ITP had increased levels of both preplatelets and barbells correlating with immature platelet levels. Furthermore, barbells were absent post-chemotherapy in patients. In mice, in vivo biotinylation confirmed that barbells, but not all large platelets, were immature. This study demonstrates that a subpopulation of large platelets are immature preplatelets that can transform into barbells and undergo fission during maturation.

cell biology↗

bioRxiv2020

CLEC-2 promotes inflammatory peritoneal macrophage emigration to draining lymph nodes during endotoxemia

Macrophage recruitment during sterile inflammation and infection is essential to clear pathogens, apoptotic cells and debris. However, persistent macrophage accumulation leads to chronic inflammation. Platelets are emerging as key modulators of the inflammatory response. Here, we identify that platelet C-type-lectin-like receptor-2 (CLEC-2) is a crucial immunomodulatory receptor through the interaction with podoplanin, upregulated on inflammatory macrophages. Mechanistically, platelet CLEC-2 upregulates the expression of podoplanin and its co-ligands CD44 and ERM proteins, leading to actin rearrangement and promotion of cell migration; this is mimicked by recombinant CLEC-2-Fc (rCLEC-2-Fc). Treatment of LPS-challenged mice with rCLEC-2-Fc induces a rapid emigration of peritoneal macrophages to mesenteric lymph nodes, through a gradient generated by the podoplanin ligand, CCL21, to prime T cells. We propose that crosslinking podoplanin using rCLEC-2-Fc is a novel, cell-specific strategy to accelerate macrophage removal from the site of inflammation, and hence promote the resolution of the inflammatory response. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/423770v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@e37acborg.highwire.dtl.DTLVardef@92a5e1org.highwire.dtl.DTLVardef@1c8832eorg.highwire.dtl.DTLVardef@11c5c97_HPS_FORMAT_FIGEXP M_FIG C_FIG SummaryPersistent macrophage accumulation in inflamed tissue leads to chronic inflammation and organ damage. Bourne et al. identify recombinant CLEC-2-Fc crosslinking podoplanin on inflammatory macrophages, as a cell-specific strategy to accelerate their emigration to draining lymph nodes, and reduce local inflammation.

immunology↗

bioRxiv2020

Immobilised collagen prevents shedding and induces sustained GPVI clustering and signalling in platelets.

Collagen, the most thrombogenic constituent of blood vessel walls, activates platelets through glycoprotein VI (GPVI). In suspension, following platelet activation by collagen, GPVI is cleaved by A Disintegrin And Metalloproteinase (ADAM)10 and ADAM17. In this study, we use single-molecule localization microscopy and a 2-level DBSCAN-based clustering tool to show that GPVI remains clustered along immobilised collagen fibres for at least 3 hours in the absence of significant shedding. Tyrosine phosphorylation of spleen tyrosine kinase (Syk) and Linker of Activated T cells (LAT), and elevation of intracellular Ca2+, are sustained over this period. Syk, but not Src kinase-dependent signalling is required to maintain clustering of the collagen integrin α2β1, whilst neither is required for GPVI. We propose that clustering of GPVI on immobilised collagen protects GPVI from shedding in order to maintain sustained Src and Syk-kinases dependent signalling, activation of integrin α2β1 and continued adhesion.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗