key: cord-1036968-poh8882l
authors: Shi, H.; Gandhi, A. A.; Smith, S. A.; Chiang, D.; Yalavarthi, S.; Ali, R. A.; Liu, C.; Sule, G.; Tsou, P.-S.; Zuo, Y.; Kanthi, Y.; Morrissey, J. H.; Knight, J. S.
title: Endothelium-protective, histone-neutralizing properties of the polyanionic agent defibrotide
date: 2021-02-23
journal: medRxiv : the preprint server for health sciences
DOI: 10.1101/2021.02.21.21252160
sha: 863348dd9e7c38c8a20b18f6a7d1173dc7eff23c
doc_id: 1036968
cord_uid: poh8882l

Neutrophil-mediated activation and injury of the endothelium play a role in the pathogenesis of diverse disease states ranging from autoimmunity to cancer to COVID-19. Neutralization of cationic proteins (such as neutrophil extracellular trap/NET-derived histones) with polyanionic compounds has been suggested as a potential strategy for protecting the endothelium from such insults. Here, we report that the FDA-approved polyanionic agent defibrotide (a pleotropic mixture of oligonucleotides) directly engages histones and thereby blocks their pathological effects on endothelium. In vitro, defibrotide counteracted endothelial cell activation and cell death, whether triggered by purified NETs, COVID-19 serum containing high levels of NETs, or recombinant histone H4. In vivo, defibrotide stabilized the endothelium and protected against histone-accelerated inferior vena cava thrombosis in mice. Mechanistically, defibrotide demonstrated direct and tight binding to histone H4 as detected by both electrophoretic mobility shift assay and surface plasmon resonance. Taken together, these data provide insights into the potential role of polyanionic compounds in protecting the endothelium from thromboinflammation with potential implications for myriad NET- and histone-accelerated disease states.

with polyanions has been suggested as an approach to combatting various NET-and histone-associated diseases 29, 30 .

Defibrotide is a pleotropic mixture of oligonucleotides (90% single-stranded phosphodiester oligonucleotides and 10% double-stranded) that is derived from porcine intestinal mucosal DNA and which has antithrombotic, fibrinolytic, and antiinflammatory activities 31, 32 . Defibrotide was initially approved for the treatment of thrombophlebitis and as prophylaxis for deep vein thrombosis in Italy 33,34 (although these approvals are no longer active). Subsequently, it was granted an orphan drug designation by European and American regulatory agencies for the treatment of serious hepatic veno-occlusive disease (VOD) after hematopoietic cell transplantation (Europe) or VOD with renal and/or pulmonary dysfunction posttransplant (United States) 34 . Although defibrotide's mechanisms of action remain incompletely understood, there is evidence that it protects endothelium, modulates platelet activation, potentiates fibrinolysis, decreases thrombin generation and activity, and reduces circulating levels of plasminogen activator inhibitor type 1 [35] [36] [37] [38] [39] .

Defibrotide has also been demonstrated to associate with cationic proteins, for example collagen I 40 .

Here, we hypothesized that the polyanionic properties of defibrotide might mitigate damage of the endothelium by NETs and especially NET-derived cationic proteins. In pursuit of this possibility, we characterized defibrotide's endothelium-protective properties both in vitro and in a mouse model of venous thrombosis. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in In-cell ELISA. In the Biosafety Level-3 facility, confluent monolayers of HUVECs in 96-well microplates were incubated with 2.5% serum or NETs for 6 hours. Some cultures were additionally supplemented with defibrotide. Cells were fixed by adding an equal volume of 8% paraformaldehyde for 30 minutes. Blocking was with 2x blocking solution (ab111541, Abcam) at room temperature for 2 hours. After washing with PBS, cells were incubated with 5 µg/ml primary mouse anti-human antibodies against E-selectin (BBA26, R&D), VCAM-1 (BBA5, R&D), or ICAM-1 (ab2213, Abcam) at 4°C overnight. Next, 100 µl of diluted horseradish peroxidase conjugated rabbit anti-mouse IgG (1:2000, Jackson ImmunoResearch) in 1x blocking solution was added and incubated at room temperature for 1 hour. After washing thoroughly with PBS, 100 µl of TMB substrate was added, and blue color development was measured at OD 650 nm with a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek).

The signals were corrected by subtracting the mean signal of wells incubated in the absence of the primary antibody.

performed by the UM Advanced Genomics Core, with libraries constructed and subsequently subjected to 150 paired-end cycles on the NovaSeq-6000 platform (Illumina). FastQC (v0.11.8) was used to ensure the quality of data, and sequence alignment were trimmed adapters using Cutadapt (v2.3). Reads were mapped to the reference genome GRCh38 (ENSEMBL) using STAR (v2.6.1b) and assigned count estimates to genes with RSEM (v1.3.1). Alignment options followed ENCODE standards for RNA-seq. FastQC was used in an additional post-alignment step to ensure that only high-quality data were used for expression quantitation and differential expression. Differential expression data were pre-filtered to remove genes with 0 counts in all samples. Differential gene expression analysis was performed using DESeq2, using a negative binomial generalized linear model (thresholds: linear fold change >1.5 or <-1.5, Benjamini-Hochberg FDR (Padj) <0.05). Plots were generated using variations of DESeq2 plotting functions and other packages with R version 3.3.3. Functional analysis, including candidate pathways activated or inhibited in comparison(s) and GO-term enrichments, was performed using iPathway Guide (Advaita).

Cytokines were quantified in supernatants using human IL-1β DuoSet ELISA kit (DY201, R&D systems), human IL-18 ELISA Kit (7620, MBL International), human IL-8 DuoSet ELISA kit (DY208, R&D systems), and human MCP-1 DuoSet ELISA kit (DY279, R&D systems), according to the manufacturers' instructions.

Electrophoretic mobility shift assay (EMSA). 10 µg of defibrotide was incubated with various concentrations (40 µM, 80 µM, 120 µM) of histone H4 in serum-free RPMI for 1 hour at 37°C to form complexes. 120 µM bovine serum albumin was used as a negative protein control. Complexes were then run on a 0.5% agarose gel stained with SYBR safe (Invitrogen) for 30 minutes. The gel was imaged on a Typhoon FLA 7000 biomolecular imager (GE Healthcare). All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in Essen Bioscience, final dilution of 1:200) was added together with histone H4 with or without defibrotide on the following day. Annexin V staining was monitored with the IncuCyte ® S3 microscopy system every 1 hour for 30 hours. Excitation and emission wavelengths were 490 nm and 515 nm, respectively. Images were collected by a Nikon 20x objective. IncuCyte ® S3 integrated software (Essen Bioscience) was used to minimize background fluorescence and quantify fluorescent objects.

For immunofluorescence microscopy, 1x10 5 /well HUVECs were seeded onto coverslips coated with 0.2% gelatin the day before experiments.

The HUVECs were treated with 100 μg/ml histone H4 in the presence or absence of defibrotide for 24 hours. Cells were fixed with 1% paraformaldehyde for 10 minutes, permeabilized with 0.5% TritonX-100 for 10 minutes, and blocked with 5% FBS for 30 minutes. Then the cells were intracellularly stained with 5 µg/ml anti-HMGB1 Alexa All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in Mouse models of venous thrombosis. All experiments were approved by the University of Michigan IACUC. Male C57BL/6 mice were purchased from The Jackson Laboratory (000664), and used at approximately 10 weeks of age. To model large-vein thrombosis was modeled as we have described previously 6 . Mice were injected with either histone (10 mg/kg) or saline 1 hour prior to surgery via tail vein.

Defibrotide (150 mg/kg) or an equal volume of saline were administered intravenously via retro-orbital injection. The first dosage of defibrotide was given 24 hours prior to surgery, and the second dose was delivered just after closure of the abdomen. Thrombus was determined 24 hours later.

and P-selectin were quantified in mice sera using the mouse E-selectin Duoset ELISA (DY575, R&D system) and mouse P-selectin Duoset ELISA (DY737, R&D system) according to the manufacturer's instructions.

Statistical analysis. Data analysis was performed with GraphPad Prism software version 8. For continuous variables, group means were compared by either t-test (two groups) or one-way ANOVA (more than two groups); correction for multiple comparisons was by Dunnett's, Sidak's, or Tukey's method. For two independent variables, group means were compared by two-way ANOVA (more than two groups); correction for multiple comparisons was by Dunnett's method. Correlations were tested by Pearson's correlation coefficient. Statistical significance was defined as p<0.05. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in 

Human umbilical vein endothelial cells (HUVECs) were cultured with human neutrophilderived NETs in the presence or absence of defibrotide. To gauge the adhesive potential of the cultured HUVECs, gene transcripts associated with the expression of E-selectin, ICAM-1 and VCAM-1 were quantified. In all cases, expression was markedly increased by NETs (Figure 1A-C) , with expression subsequently mitigated by defibrotide (Figure 1A-C) . We reasoned that if these gene expression differences were functionally meaningful, then adhesion of neutrophils to the HUVEC monolayer should track in similar fashion (increased by NETs and decreased by defibrotide). As predicted, calcein-AM-labeled human neutrophils adhered more strongly to NETactivated HUVECs, an effect that was reduced in the presence of defibrotide ( Figure   1D ). Beyond surface adhesion molecules, previous work has also suggested that NETs upregulate expression of tissue factor (TF) by endothelial cells, thereby contributing to the prothrombotic state 43 . Here we found that TF was upregulated by NETs whether measured by gene expression (Figure 1E ) or enzymatic activity ( Figure 1F ); in both contexts, NET-mediated increases were significantly reduced by defibrotide ( Figure 1E-F) . Taken together, these data support the basic premise of the study, namely that defibrotide can neutralize the activation of endothelial cells by NETs. We therefore reasoned that defibrotide might be able to reduce activation of endothelial cells by COVID-19 serum. To address this, we developed an in-cell ELISA platform compatible with our Biosafety Level-3 facility (Figure 2A) . We first confirmed that purified NETs would increase surface expression of E-selectin, ICAM-1, and VCAM-1 in this assay, and that these increases could be mitigated by All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted February 23, 2021. defibrotide ( Figure 2B-D) . Consistent with our hypothesis, we found a similar upregulation of surface E-selectin ( Figure 2E ), ICAM-1 ( Figure 2F ) and VCAM-1 ( Figure 2G ) when HUVECs were cultured with pooled COVID-19 serum, with that upregulation partially counteracted by defibrotide. In summary, these data support the idea that defibrotide can reduce activation of endothelial cells by NETs, and hint at potential clinical utility in diseases such as COVID-19.

Transcriptome profiling confirms a NET-induced proinflammatory signature in endothelial cells, which can be mitigated by defibrotide. The above data demonstrate activation of endothelial cells by NETs in the context of selected genes associated with cell-cell interactions and coagulation. To more broadly understand the pathways associated with endothelial cell activation, we performed RNA sequencing of HUVECs exposed to vehicle, NETs, or NETs + defibrotide. We identified 440 differentially expressed genes (300 upregulated) in HUVECs upon NET stimulation as compared with vehicle. Conversely, there were 229 differentially expressed genes (192 downregulated) when the NETs + defibrotide group was compared to NETs alone. The top regulated genes are displayed in Figure 3A .

Functional gene network analysis of upregulated genes in NET-stimulated HUVECs revealed an inflammatory signature highlighted by meta groups such as the TNF signaling pathway, NF-ĸB signaling pathway, and MAPK signaling pathway ( Figure   3B ). Notably, the same pathways that were upregulated by NETs were likely to be downregulated by defibrotide ( Figure 3C ). Genes downregulated by NETs were also assessed by functional gene network analysis. Important pathways related to cell growth and anti-inflammation were downregulated by NETs, including the Hippo signaling pathway, cAMP signaling pathway, and TGF-β signaling pathway ( Figure   3D ). These same pathways were then upregulated when defibrotide was added to NETs ( Figure 3E ). Taken together, these data confirm the ability of NETs to activate endothelial cells, and demonstrate the ability of defibrotide to reverse those effects. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted February 23, 2021. ; https://doi.org/10.1101/2021.02.21.21252160 doi: medRxiv preprint Blocking histone H4 counteracts HUVEC activation by NETs. As discussed above, part of the original hypothesis was that the polyanionic nature of defibrotide might make it especially effective at neutralizing NET-derived cationic proteins such as histones 2 , which are important mediators of inflammation, tissue injury, and organ dysfunction in the extracellular space 45, 46 . To begin to address this, we asked whether a histone-neutralizing antibody might be effective in our system. Indeed, an anti-histone H4 antibody counteracted the upregulation of HUVEC E-selectin ( Figure   4A ), ICAM-1 (Figure 4B) , VCAM-1 (Figure 4C) , and TF ( Figure 4D ) by NETs. Figure 1) . Similar patterns were also observed for TF gene expression ( Figure 5D ) and enzymatic activity ( Figure 5E ). Given these findings, along with the RNA sequencing data, we investigated whether defibrotide might be working to counterbalance intracellular signaling pathways associated with TLRs or TNF signaling. However, in our hands, defibrotide's protective properties did not extend to HUVEC activation by either LPS or TNF-α (Supplementary Figure 3) . We next tested whether defibrotide might work through direct engagement with histone H4 in the extracellular space. Indeed, using an electrophoretic mobility shift assay (EMSA) 48 , we found evidence of a direct interaction between histone H4 and defibrotide in that histone H4 could retard the migration of defibrotide (a mixture of oligonucleotides) through an agarose gel ( Figure 5F ). In contrast, histone H4 had no impact on the migration of bovine serum All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted February 23, 2021. ; https://doi.org/10.1101/2021.02.21.21252160 doi: medRxiv preprint albumin. A strong interaction between histone H4 and defibrotide was also confirmed by surface plasmon resonance. Assuming an average molecular weight of defibrotide as 16.5 kilodaltons, the equilibrium dissociation constant (KD) between defibrotide and histone H4 was calculated as 53.5 nM (Figure 5G ).

Defibrotide strongly protects endothelial cells against histone H4-induced cell death. The above assays were focused on relatively short cell culture times, typically 6 hours. However, we questioned whether the impact of defibrotide on histone H4mediated HUVEC activation would persist over longer periods of time. As reported previously, histones go beyond endothelial cell activation and become cytotoxic upon prolonged exposure in culture 22, 49 . Indeed, we found remarkable protection of cell viability by defibrotide over a 24-hour period ( Figure 6A ). To further confirm these findings, we varied the experiment by introducing kinetic monitoring of surface phosphatidylserine exposure as measured by Annexin V binding. We found a dosedependent relationship between histone H4 and Annexin V binding ( Supplementary   Figure 4) , and found strong and stepwise protection when HUVECs were also cultured with defibrotide concentrations ranging from 10-40 μg/ml ( Figure 6B ). We also found increased levels of both IL-1β and IL-18 in culture supernatants, both of which were reduced by defibrotide ( Figure 6C-D) , supporting the idea that histone H4-mediated cell death may be on the spectrum of pyroptosis. To further substantiate these data, we also assessed translocation and subsequent release of the alarmin HMGB1, which is known to track with inflammatory forms of cell death including pyroptosis 50 . By microscopy, we observed the translocation of HMGB1 from nucleus to cytoplasm upon exposure of HUVECs to histone H4, with reversal of this effect by defibrotide (Figure 6E) . Measurement of HMGB1 in culture supernatants mirrored these findings, with histone H4 triggering HMGB1 release and defibrotide neutralizing that effect (Figure 6F) . Taken together, these data demonstrate that longer-term exposure of HUVECs to histone H4 triggers cell death (which may be of All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in (Figure 7A) 6, 51 . First, we asked whether thrombus accretion was impacted by injection of calf thymus histones, and indeed found this to be the case (Figure 7B-C) . As part of these experiments, we also administered defibrotide intravenously shortly after injection of the histones. With this approach, both thrombus accretion (Figure 7B -C) and thrombus length (Supplementary Figure 5) were reduced essentially to the levels seen in control mice. In support of endothelial cell activation contributing to the histone-accentuated thrombosis phenotype, both soluble E-selectin and soluble P-selectin tracked closely with thrombus accretion ( Figure 7D-E) , as did infiltration of leukocytes, whether scored as Ly6G+

(neutrophils) or CD45+ (most leukocytes) (Figure 7F-H) . In support of this concept, there was a strong correlation between either soluble E-selectin or soluble P-selectin and thrombus size (Supplementary Figure 6) . Taken together, these data confirm the proinflammatory and prothrombotic impact of histones in vivo and demonstrate that defibrotide has the potential to neutralize these properties.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in 

As evidence continues to implicate NETs and NET-derived histones in the pathophysiology of disease states ranging from infection (including COVID-19) to autoimmunity to cancer 52 , the search for NET-targeting therapeutics takes on additional importance. Here, we explored the extent to which an FDA-approved drug defibrotide might protect endothelial cells from NETs and extracellular histones. We found defibrotide to counteract endothelial cell activation and hypercoagulability triggered by NETs, COVID-19 serum with high NETs, and histone H4.

Mechanistically, our evidence points to a direct interaction between defibrotide and cationic proteins such as histone H4 as an important aspect of these protective effects.

Polyanionic substances naturally exist in the extracellular environment where they play a variety of biological roles 53 Histones bind DNA tightly mainly due to charge-charge interactions, with a possible role for specific DNA sequence motifs 60 . Our results found a strong interaction between histone H4 and defibrotide, which was very resistant to dissociation.

Considering defibrotide is a natural product (i.e., not produced by a DNA synthesizer), the possibility of any specific sequence dominating its effect is low. We therefore speculate that the main binding force between histone H4 and defibrotide also comes from charge-charge interactions. An interesting unknown is the extent to All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted February 23, 2021. ; https://doi.org/10.1101/2021.02.21.21252160 doi: medRxiv preprint which the degradation of defibrotide in vivo may be delayed upon binding histone H4.

Typically, phosphodiester oligonucleotides would be rapidly degraded in plasma; however, we found a protective role in an animal model over 24 hours without the need for redosing. A deeper understanding of these in vivo properties should be a priority for future research.

As a major component of NETs, histones are responsible for microvascular dysfunction during sepsis where they trigger neutrophil migration, endothelial injury, hemorrhage, and thrombosis 17 . Compared with other histones, histone H4 has the strongest impact on platelets, enhancing thrombin generation and accelerating thrombosis 61 . Histone H4 has also been reported as the major histone mediator of membrane lysis of smooth muscle cells and arterial tissue damage and inflammation in atherosclerosis 62 . Our study has now also revealed that neutralizing histone H4 significantly mitigates NET-mediated activation of HUVECs. Whether defibrotide preferentially neutralizes histone H4 as compared with other histones is an area deserving of further research.

Multiple organ dysfunction syndrome (MODS) is widely considered to be the leading cause of morbidity and mortality for patients admitted to an intensive care unit, where it encompasses heterogeneous disease states such as sepsis, shock, trauma, severe burn, and pancreatitis [63] [64] [65] [66] . Systemic inflammation and vascular coagulopathy account for the main pathological processes of MODS 67 , and of course also characterize aspects of COVID-19 [68] [69] [70] [71] and the closely related catastrophic antiphospholipid syndrome 72 . In MODS, endothelial cell activation is considered a precursor to tissue damage and end-organ dysfunction with upregulation of adhesion molecules triggered by cytokines, microbial proteins, and various cationic proteins from necrotic cells 73 . One recent study evaluated circulating histones in a cohort of 420 ICU patients with sepsis, severe trauma, or severe pancreatitis and identified circulating histones as major mediators of MODS in these patients 74 . One may All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted February 23, 2021. ; https://doi.org/10.1101/2021.02.21.21252160 doi: medRxiv preprint consider whether administration of defibrotide in an early phase of MODS might neutralize cationic proteins such as histones to stabilize the endothelium and break the vicious thromboinflammatory cycle. We are hopeful that further preclinical (and soon clinical) work may have the opportunity to explore the potential of defibrotide as a NET-and histone-neutralizing drug.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted February 23, 2021. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted February 23, 2021. presence of Annexin V red agent. The plate was imaged every hour using the IncuCyte ® S3 timelapse microscope for 30 hours. Mean ± standard deviation for three independent experiments is presented; ****p<0.0001by two-way ANOVA corrected by Dunnett's test.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted February 23, 2021. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted February 23, 2021. ; https://doi.org/10.1101/2021.02.21.21252160 doi: medRxiv preprint

Neutrophils and immunity: challenges and opportunities

Neutrophil extracellular traps kill bacteria

Neutrophils scan for activated platelets to initiate inflammation

Histones induce phosphatidylserine exposure and a procoagulant phenotype in human red blood cells

Extracellular DNA traps promote thrombosis

In Vivo Role of Neutrophil Extracellular Traps in Antiphospholipid Antibody-Mediated Venous Thrombosis

Neutrophil extracellular traps promote deep vein thrombosis in mice

VWF-mediated leukocyte recruitment with chromatin decondensation by PAD4 increases myocardial ischemia/reperfusion injury in mice

Peptidylarginine deiminase inhibition reduces vascular damage and modulates innate immune responses in murine models of atherosclerosis

Neutrophil extracellular traps induce endothelial dysfunction in systemic lupus erythematosus through the activation of matrix metalloproteinase-2

Neutrophil extracellular traps in COVID-19

Vascular occlusion by neutrophil extracellular traps in COVID-19

Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome

SARS-CoV-2-triggered neutrophil extracellular traps mediate COVID-19 pathology

Immunothrombotic Dysregulation in COVID-19 Pneumonia Is Associated With Respiratory Failure and Coagulopathy

Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans

Extracellular histones are major mediators of death in sepsis

HMGB1 and Histones Play a Significant Role in Inducing Systemic Inflammation and Multiple Organ Dysfunctions in Severe Acute Pancreatitis

Extracellular histones are clinically relevant mediators in the pathogenesis of acute respiratory distress syndrome

Circulating histones are mediators of trauma-associated lung injury

Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones

Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Extracellular Histones Are Mediators of Death through TLR2 and TLR4 in Mouse Fatal Liver Injury

Histones from Dying Renal Cells Aggravate Kidney Injury <em>via</em> TLR2 and TLR4

Histones Induce the Procoagulant Phenotype of Endothelial Cells through Tissue Factor Up-Regulation and Thrombomodulin Down-Regulation

Histones trigger sterile inflammation by activating the NLRP3 inflammasome

Extracellular Histone H3 Induces Pyroptosis During Sepsis and May Act Through NOD2 and VSIG4/NLRP3 Pathways

Histones stimulate von Willebrand factor release in vitro and in vivo

Actin and DNA Protect Histones from Degradation by Bacterial Proteases but Inhibit Their Antimicrobial Activity

Neutralizing the pathological effects of extracellular histones with small polyanions

A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in vascular disorders

Defibrotide, a polydisperse mixture of single-stranded phosphodiester oligonucleotides with lifesaving activity in severe hepatic veno-occlusive disease: protein-nucleic acid interactions

Extracellular histones are major mediators of death in sepsis

Indirect regulation of HMGB1 release by gasdermin D

Adenosine receptor agonism protects against NETosis and thrombosis in antiphospholipid syndrome

Neutrophil extracellular traps in immunity and disease

Polyanionic drugs and viral oncogenesis: a novel approach to control infection, tumor-associated inflammation and angiogenesis

More to "heparin" than anticoagulation

Therapeutic applications of glycosaminoglycans

Fibroblast growth factors/fibroblast growth factor receptors as targets for the development of anti-angiogenesis strategies

The basic domain in HIV-1 Tat protein as a target for polysulfonated heparin-mimicking extracellular Tat antagonists

Inhibition of growth factor mitogenicity and growth of tumor cell xenografts by a sulfonated distamycin A derivative

The polyanions heparin and suramin impede binding of free adenine to a DNA glycosylase from C. pseudotuberculosis

The structure of DNA in the nucleosome core

Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4

Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

Multiple organ failure. Pathophysiology and potential future therapy

Multiple organ failure syndrome in the 1990s. Systemic inflammatory response and organ dysfunction

Multiple organ dysfunction score: a reliable descriptor of a complex clinical outcome

Use of the SOFA score to assess the incidence of organ dysfunction/failure in intensive care units: results of a multicenter, prospective study. Working group on "sepsis-related problems" of the European Society of Intensive Care Medicine

Inflammation, coagulopathy, and the pathogenesis of multiple organ dysfunction syndrome

Prevalence of venous thromboembolism in patients with severe novel coronavirus pneumonia

Incidence of thrombotic complications in critically ill ICU patients with COVID-19

Prothrombotic autoantibodies in serum from patients hospitalized with COVID-19

The diagnosis and clinical management of the catastrophic antiphospholipid syndrome: A comprehensive review

The cytokine storm and factors determining the sequence and severity of organ dysfunction in multiple organ dysfunction syndrome

Circulating Histones Are Major Mediators of Multiple Organ Dysfunction Syndrome in Acute Critical Illnesses

All authors participated in writing the manuscript and gave approval before submission.

The authors have no relevant financial conflicts to report. Defibrotide was provided by Jazz Pharmaceuticals. The work was also partially supported by a grant from Jazz