Biology subjects

Di Paola, J.

Publications and source records attributed to Di Paola, J..

4 recordsLinked to original sources

bioRxiv2026

TNFα drives platelet hyperreactivity and thromboinflammation through regulation of hematopoietic stem and progenitor cells.

TNF is the primary age-related cytokine responsible for hyperreactive platelet formation. In mouse models, increased TNF induced abnormal megakaryocyte development and platelet activity. Here, we extend these findings to demonstrate that TNF drives this thrombotic phenotype through expansion of the hematopoietic stem and progenitor cell (HSPCs) compartment. Using HSPC-specific TNF receptor labeling and single cell RNA sequencing, we found that TNF receptors are absent from megakaryocytes and their progenitors (MkPs) indicating that TNF does not directly act on these cells. Chronic TNF exposure expanded HSPCs in the bone marrow and extramedullary tissues - and these expanded HSPCs retained functional repopulation capacity. Using species-specificity TNF receptor activation, we further demonstrate that TNFR1 signaling is sufficient to induce platelet hyperreactivity independent of HSPC expansion. Unlike emergency hematopoiesis, chronic TNF promoted megakaryopoiesis through the canonical hematopoietic hierarchy as demonstrated by lineage-tracing studies. Mechanistically, chronic TNF induced a distinct transcriptional program in single cell RNA sequencing of HSPCs and megakaryocytes. Together, these findings establish that chronic TNF promotes hyperreactive platelet formation not through direct effects on megakaryocytes or MkPs but by expanding and transcriptionally reprogramming HSPCs, thereby imprinting a TNF dependent program that persists through megakaryopoiesis and ultimately produces hyperreactive platelets.

cell biology↗

bioRxiv2024

LRRC8 complexes are adenosine nucleotide release channels regulating platelet activation and arterial thrombosis

Platelet shape and volume changes are early mechanical events contributing to platelet activation and thrombosis. Here, we identify single-nucleotide polymorphisms in Leucine-Rich Repeat Containing 8 (LRRC8) protein subunits that form the Volume-Regulated Anion Channel (VRAC) which are independently associated with altered mean platelet volume. LRRC8A is required for functional VRAC in megakaryocytes (MKs) and regulates platelet volume, adhesion, and agonist-stimulated activation, aggregation, ATP secretion and calcium mobilization. MK-specific LRRC8A cKO mice have reduced arteriolar thrombus formation and prolonged arterial thrombosis without affecting bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced ATP/ADP release to amplify agonist-stimulated calcium and PI3K-AKT signaling via P2X1, P2Y1 and P2Y12 receptors. Small-molecule LRRC8 channel inhibitors recapitulate defects observed in LRRC8A-null platelets in vitro and in vivo. These studies identify the mechanoresponsive LRRC8 channel complex as an ATP/ADP release channel in platelets which regulates platelet function and thrombosis, providing a proof-of-concept for a novel anti-thrombotic drug target.

cell biology↗

bioRxiv2022

Multiomic Profiling Reveals Metabolic Alterations Mediating Aberrant Platelet Activity and Inflammation in Myeloproliferative Neoplasms

Platelets from patients with myeloproliferative neoplasms (MPNs) exhibit a hyperreactive phenotype. Here, we found elevated P-selectin exposure and platelet-leukocyte aggregates indicating activation of platelets from essential thrombocythemia (ET) patients. Single cell RNA-seq analysis of primary samples revealed significant enrichment of transcripts related to platelet activation, mTOR and oxidative phosphorylation (OXPHOS) in ET patient platelets. These observations were validated via proteomic profiling. Platelet metabolomics revealed distinct metabolic phenotypes consisting of elevated ATP generation, accompanied by increases in the levels of multiple intermediates of the tricarboxylic acid (TCA) cycle, but lower alpha-ketoglutarate (-KG) in MPN patients. Inhibition of PI3K/AKT/mTOR signaling significantly reduced metabolic responses and hyperreactivity in MPN patient platelets, while -KG supplementation markedly reduced oxygen consumption and ATP generation. Ex vivo incubation of platelets from both MPN patients and Jak2 V617F mice with -KG significantly reduced platelet activation responses. Oral -KG supplementation of Jak2 V617F mice decreased splenomegaly and reduced hematocrit, monocyte and platelet counts. Finally, -KG incubation significantly decreased proinflammatory cytokine secretion from MPN CD14+ monocytes. Our results reveal a previously unrecognized metabolic disorder in conjunction with aberrant PI3K/AKT/mTOR signaling, contributing to platelet hyperreactivity in MPN patients.

cancer biology↗

bioRxiv2018

Recovery and analysis of transcriptome subsets from pooled single-cell RNA-seq libraries

Single-cell RNA sequencing (scRNA-seq) methods generate sparse gene expression profiles for thousands of single cells in a single experiment. The information in these profiles is sufficient to classify cell types by distinct expression patterns but the high complexity of scRNA-seq libraries often prevents full characterization of transcriptomes from individual cells. To extract more focused gene expression information from scRNA-seq libraries, we developed a strategy to physically recover the DNA molecules comprising transcriptome subsets, enabling deeper interrogation of the isolated molecules by another round of DNA sequencing. We applied the method in cell-centric and gene-centric modes to isolate cDNA fragments from scRNA-seq libraries. First, we resampled the transcriptomes of rare, single megakaryocytes from a complex mixture of lymphocytes and analyzed them in a second round of DNA sequencing, yielding up to 20-fold greater sequencing depth per cell and increasing the number of genes detected per cell from a median of 1,313 to 2,002. We similarly isolated mRNAs from targeted T cells to improve the reconstruction of their VDJ-rearranged immune receptor mRNAs. Second, we isolated CD3D mRNA fragments expressed across cells in a scRNA-seq library prepared from a clonal T cell line, increasing the number of cells with detected CD3D expression from 59.7% to 100%. Transcriptome resampling is a general approach to recover targeted gene expression information from single-cell RNA sequencing libraries that enhances the utility of these costly experiments, and may be applicable to the targeted recovery of molecules from other single-cell assays.

genomics↗