Biology subjects

O'Donnell, J. S.

Publications and source records attributed to O'Donnell, J. S..

5 recordsLinked to original sources

bioRxiv2026

Type 2B VWD substitutions trigger enhanced macrophage-mediated clearance that is attenuated by the BT200 aptamer

Type 2B von Willebrand disease (VWD) is characterized by missense variants in exon 28 of the von Willebrand factor (VWF) gene that encodes the VWF-A1 domain. These missense mutations result in single amino acid residue substitutions that cause activation of VWF-A1 and promote spontaneous interaction with platelet GPIba;. Importantly, enhanced VWF clearance has been shown to play a key pathogenic role in >90% patients with Type 2B VWD. Although this VWF clearance occurs via mechanisms that are independent of VWF-platelet complex formation, the biological mechanisms responsible for the increased clearance of type 2B VWD variants remain poorly understood. In this study, we investigated a series of different type 2B amino acid substitutions within the VWF-A1 domain (R1306W, R1308C, W1313C and R1379L). We demonstrate that these variants all exhibit significantly increased macrophage binding. In part, the enhanced macrophage interactions are mediated via increased binding of type 2B VWD variants to LRP1 extracellular cluster II and IV. Our findings further demonstrate that the K1405-K1408 lysine cluster in the VWF-A1 domain plays a key role in enabling enhanced LRP1 interactions for type 2B VWD variants. In addition, we show that type 2B VWF variants demonstrate significantly enhanced interaction with the macrophage MGL receptor. Finally, and importantly from a clinical perspective, we demonstrate that the increase in macrophage binding for type 2B variants is significantly attenuated in the presence of BT200. Collectively, our findings have direct translational relevance with respect to the clinical heterogeneity and treatment of type 2B VWD.

biochemistry↗

bioRxiv2024

Trained Immunity Causes Myeloid Cell Hypercoagulability

Venous thromboembolism is common in individuals with chronic inflammatory diseases, but the pathogenic basis for this increased thrombotic risk remains poorly understood. Myeloid cell trained immunity describes persistent innate immune cell memory arising from prior exposure to an inflammatory stimulus, leading to an enhanced immune response to subsequent unrelated stimuli. We identify enhanced myeloid cell prothrombotic activity as a novel maladaptive consequence of trained immunity. LPS stimulation of murine bone marrow-derived macrophages trained previously with either {beta}-glucan or free haem exhibited significantly enhanced procoagulant and antifibrinolytic gene expression and activity compared to macrophages stimulated with LPS alone. The {beta}-glucan training-mediated increase in activated myeloid cell procoagulant activity was mediated by enhanced acid sphingomyelinase-mediated tissue factor (TF) functional decryption. Furthermore, pre-treatment with methyltransferase and acetyltransferase inhibitors to erase epigenetic marks associated with innate immune memory diminished trained macrophage TF gene expression in {beta}-glucan-trained macrophages. Functional analysis of splenic monocytes isolated from {beta}-glucan-trained mice revealed enhanced procoagulant activity up to 4 weeks after {beta}-glucan administration compared to monocytes from control mice over the same time period. Remarkably, monocyte procoagulant activity increased proportionately with time since {beta}-glucan administration, before plateauing at 4 weeks. Furthermore, haematopoietic progenitor cells and bone marrow interstitial fluid isolated from {beta}-glucan-trained mice possessed enhanced procoagulant activity compared to control mice. Trained immunity and associated metabolic perturbations may therefore represent novel therapeutic vulnerabilities in immunothrombotic disease development, opening new avenues for targeted intervention.

immunology↗

bioRxiv2024

Tissue factor-dependent colitogenic CD4+ T cell thrombogenicity is regulated by activated protein C signalling.

Inflammatory bowel disease (IBD) patients experience up to 6-fold increased risk of venous thromboembolism (VTE) compared to the general population, although the mechanistic basis for this increased risk remains poorly defined. We found that colitogenic CD4+ T cells express tissue factor (TF) and promote rapid TF-dependent plasma thrombin generation in T cell-dependent calibrated automated thrombinography assays. Furthermore, we identified the presence of TF+CD4+CD3+ T cells in the colons of both mice with colitis and paediatric IBD patients during active disease. TF is typically expressed in an encrypted state and requires decryption for optimal procoagulant activity. Notably, flow cytometric analysis demonstrated that activated CD4+ T cells express significantly increased acid sphingomyelinase and protein disulphide isomerase, critical mediators for TF decryption, on their cell membrane compared to naive T cells. The protein C (PC) pathway is an important regulator of TF-mediated thrombin generation. Pertinently, pre-clinical studies suggest an important role for diminished PC pathway activity in IBD pathophysiology. To understand how this process might be regulated, we performed meta-transcriptomic and gene expression analysis of IBD patient gut biopsy tissue, identifying dysregulated expression of genes involved in the regulation of coagulation, including PC (PROC) and its receptor (EPCR; PROCR). Subsequent functional studies revealed that activated protein C (APC) signalling reduced colitogenic T cell generation and activity, potently impaired TF decryption and significantly reduced T cell-mediated thrombin generation and clot formation. These data identify TF-mediated colitogenic T cell thrombogenicity and demonstrate a new role for APC signalling in regulating T cell thrombo-inflammatory activity.

immunology↗

bioRxiv2023

HTLV-1 reverse transcriptase homology model provides structural basis for sensitivity to existing nucleoside/nucleotide reverse transcriptase inhibitors

The human T-lymphotropic virus type 1 (HTLV-1) infects millions of people globally and is endemic to various resource-limited regions. Infections persist for life and are associated with increased susceptibility to opportunistic infections and severe diseases including adult T cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy-tropical spastic paraparesis (HAM-TSP). No HTLV-1-specific anti-retrovirals have been developed and it is unclear whether existing anti-retrovirals developed for treatment of human immunodeficiency virus (HIV) have efficacy against HTLV-1. To understand the structural basis for therapeutic binding, homology modelling and machine learning were used to develop a structural model of the HTLV-1 reverse transcriptase. With this, molecular docking experiments using a panel of FDA-approved inhibitors of viral reverse transcriptases to assess their capacity for binding, and in turn, inhibition. Importantly, nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) but not non-nucleoside reverse transcriptase inhibitors (NNRTIs) were capable of binding the HTLV-1 reverse transcriptase, with similar affinity to HIV-1 reverse transcriptase. By strengthening the rationale for clinical testing of therapies such as tenofovir alafenamide, zidovudine, lamivudine, and azvudine for treatment of HTLV-1, this study has demonstrated the power of in silico structural biology approaches in drug design and therapeutic testing.

pharmacology and toxicology↗

bioRxiv2023

GLYCOLYTIC REPROGRAMMING FUELS MYELOID CELL-DRIVEN HYPERCOAGULABILITY

BackgroundMyeloid cell metabolic reprogramming is a hallmark of inflammatory disease, however, its role in inflammation-induced hypercoagulability is poorly understood. Objective/MethodsUsing novel myeloid cell-based global haemostasis assays and murine models of immunometabolic disease, we evaluated the role of inflammation-associated metabolic reprogramming in regulating blood coagulation. ResultsGlycolysis was essential for enhanced activated myeloid cell tissue factor expression and decryption, driving increased cell-dependent thrombin generation in response to inflammatory challenge. Similarly, inhibition of glycolysis enhanced activated macrophage fibrinolytic activity via reduced plasminogen activator inhibitor 1 (PAI-1)-activity. Macrophage polarisation or activation markedly increased endothelial protein C receptor (EPCR) expression on monocytes and macrophages, leading to increased myeloid cell-dependent protein C activation. Importantly, inflammation-dependent EPCR expression on tissue-resident macrophages was also observed in vivo. Adipose tissue macrophages from obese mice fed a high-fat diet exhibited significantly enhanced EPCR expression and APC generation compared to macrophages isolated from the adipose tissue of healthy mice. Similarly, the induction of colitis in mice prompted infiltration of EPCR+ innate myeloid cells within inflamed colonic tissue that were absent from the intestinal tissue of healthy mice. ConclusionCollectively, this study identifies immunometabolic regulation of myeloid cell hypercoagulability, opening new therapeutic possibilities for targeted mitigation of thrombo-inflammatory disease. ESSENTIALSO_LIInflammation-mediated glycolytic reprogramming enables myeloid cell-induced hypercoagulability and antifibrinolytic activity. C_LIO_LI2-Deoxy-D-glucose (2-DG) inhibits the expression of transcription factors necessary for inflammation-induced procoagulant gene expression. C_LIO_LIMyeloid cell membrane regulation of tissue factor procoagulant activity is glycolysis-dependent. C_LIO_LIActivation of myeloid innate immunity dysregulates activated protein C anticoagulant pathway activity. C_LI

immunology↗