Publications by authors named "Kimberly Martinod"

38 Publications

The role of platelets in thrombus fibrosis and vessel wall remodeling after venous thrombosis.

J Thromb Haemost 2021 Feb 29;19(2):387-399. Epub 2020 Nov 29.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.

Purpose: Platelets are known to play an important role in venous thrombogenesis, but their role in thrombus maturation, resolution, and postthrombotic vein wall remodeling is unclear. The purpose of this study was to determine the role that circulating platelets play in the later phases of venous thrombosis.

Methods: We used a murine inferior vena cava (IVC) stenosis model. Baseline studies in untreated mice were performed to determine an optimal postthrombotic time point for tissue harvest that would capture both thrombus maturation/resolution and postthrombotic vein wall remodeling. This time point was found to be postoperative day 10. After undergoing IVC ultrasound on day 2 to confirm venous thrombus formation, mice were treated with a daily injection of platelet-depleting antibody (anti-GP1bα) to maintain thrombocytopenia or with control IgG until postoperative day 10, at which time IVC and thrombi were harvested and thrombus length, volume, fibrosis, neovascularization, and smooth muscle cell invasion analyzed. Vein wall fibrosis and intimal thickening were also determined.

Results: Mice that were made thrombocytopenic after venous thrombogenesis had thrombi that were less fibrotic, with fewer invading smooth muscle cells. Furthermore, thrombocytopenia in the setting of venous thrombosis resulted in less postthrombotic vein wall intimal thickening. Thrombus volume did not differ between thrombocytopenic mice and their control peers.

Conclusions: This work suggests that circulating platelets contribute to venous thrombus maturation, fibrosis, and adverse vein wall remodeling, and that that inhibition of platelet recruitment may decrease thrombus and vein wall fibrosis, thus helping thrombolysis and preventing postthrombotic syndrome.
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http://dx.doi.org/10.1111/jth.15134DOI Listing
February 2021

Immunothrombosis and thromboinflammation in host defense and disease.

Platelets 2020 Sep 8:1-11. Epub 2020 Sep 8.

Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf , Hamburg, Germany.

Platelets are increasingly being recognized for playing roles beyond thrombosis and hemostasis. Today we know that they mediate inflammation by direct interactions with innate immune cells or secretion of cytokines/chemokines. Here we review their interactions with neutrophils and monocytes/macrophages in infection and sepsis, stroke, myocardial infarction and venous thromboembolism. We discuss new roles for platelet surface receptors like GPVI or GPIb and also look at platelet contributions to the formation of neutrophil extracellular traps (NETs) as well as to deep vein thrombosis during infection, e.g. in COVID-19 patients.
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http://dx.doi.org/10.1080/09537104.2020.1817360DOI Listing
September 2020

Resolving Thrombosis: Resting and Polarized Macrophages Differentially Degrade Neutrophil Extracellular Traps.

Arterioscler Thromb Vasc Biol 2020 09 26;40(9):1961-1963. Epub 2020 Aug 26.

Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, KU Leuven, Belgium.

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http://dx.doi.org/10.1161/ATVBAHA.120.315013DOI Listing
September 2020

von Willebrand factor increases in experimental cerebral malaria but is not essential for late-stage pathogenesis in mice.

J Thromb Haemost 2020 09 27;18(9):2377-2390. Epub 2020 Aug 27.

Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium.

Background: Cerebral malaria (CM) is the most severe complication of malaria. Endothelial activation, cytokine release, and vascular obstruction are essential hallmarks of CM. Clinical studies have suggested a link between von Willebrand factor (VWF) and malaria pathology.

Objectives: To investigate the contribution of VWF in the pathogenesis of experimental cerebral malaria (ECM).

Methods: Both Vwf and Vwf mice were infected with Plasmodium berghei ANKA (PbANKA) to induce ECM. Alterations of plasma VWF and ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), platelet count, neurological features, and accumulation of platelets and leukocytes in the brain were examined following infection.

Results: Plasma VWF levels significantly increased upon PbANKA infection in Vwf animals. While ADAMTS13 activity was not affected, high molecular weight VWF multimers disappeared at the end-stage ECM, possibly due to an ongoing hypercoagulability. Although the number of reticulocytes, a preferential target for the parasites, was increased in Vwf mice compared to Vwf mice early after infection, parasitemia levels did not markedly differ between the two groups. Interestingly, Vwf mice manifested overall clinical ECM features similar to those observed in Vwf animals. At day 8.5 post-infection, however, clinical ECM features in Vwf mice were slightly more beneficial than in Vwf animals. Despite these minor differences, overall survival was not different between Vwf and Vwf mice. Similarly, PbANKA-induced thrombocytopenia, leukocyte, and platelet accumulations in the brain were not altered by the absence of VWF.

Conclusions: Our study suggests that increased VWF concentration is a hallmark of ECM. However, VWF does not have a major influence in modulating late-stage ECM pathogenesis.
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http://dx.doi.org/10.1111/jth.14932DOI Listing
September 2020

The von Willebrand Factor A1 domain mediates thromboinflammation, aggravating ischemic stroke outcome in mice.

Haematologica 2021 Mar 1;106(3):819-828. Epub 2021 Mar 1.

Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium.

von Willebrand factor (VWF) plays an important role in ischemic stroke. However, the exact mechanism by which VWF mediates progression of ischemic stroke brain damage is not completely understood. Using flow cytometric analysis of single cell suspensions prepared from brain tissue and immunohistochemistry, we investigated the potential inflammatory mechanisms by which VWF contributes to ischemic stroke brain damage in a mouse model of cerebral ischemia/reperfusion injury. Twenty-four hours after stroke, flow cytometric analysis of brain tissue revealed that overall white blood cell recruitment in the ipsilesional brain hemisphere of VWF KO mice was 2 times lower than WT mice. More detailed analysis showed a specific reduction of proinflammatory monocytes, neutrophils and T-cells in the ischemic brain of VWF KO mice compared to WT mice. Interestingly, histological analysis revealed a substantial number of neutrophils and T-cells still within the microcirculation of the stroke brain, potentially contributing to the no-reflow phenomenon. Specific therapeutic targeting of the VWF A1 domain in WT mice resulted in reduced immune cell numbers in the affected brain and protected mice from ischemic stroke brain damage. More specifically, recruitment of proinflammatory monocytes was reduced two-fold, neutrophil recruitment was reduced five-fold and T-cell recruitment was reduced two-fold in mice treated with a VWF A1-targeting nanobody compared to mice receiving a control nanobody. In conclusion, our data identify a potential role for VWF in the recruitment of proinflammatory monocytes, neutrophils and T-cells to the ischemic brain via a mechanism that is mediated by its A1 domain.
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http://dx.doi.org/10.3324/haematol.2019.241042DOI Listing
March 2021

Von Willebrand factor and ADAMTS13 impact on the outcome of Staphylococcus aureus sepsis.

J Thromb Haemost 2020 03 22;18(3):722-731. Epub 2019 Dec 22.

Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.

Background: Previous clinical evidence correlates levels of von Willebrand factor (VWF) and its cleaving protease ADAMTS13 with outcome in septic patients. No previous studies addressed if VWF and ADAMTS13 affected the outcome of Staphylococcus aureus sepsis.

Objectives: We studied the role of VWF and ADAMTS13 in S. aureus sepsis both in patients and in mice.

Methods: VWF levels and ADAMTS13 activity levels were measured in plasma samples from 89 S. aureus bacteremia patients by chemiluminescent assays and were correlated with clinical sepsis outcome parameters. In wild-type mice and mice deficient in VWF and ADAMTS13, we investigated the outcome of S. aureus sepsis and quantified bacterial clearance and organ microthrombi.

Results: In patients with S. aureus bloodstream infections, high VWF levels and low ADAMTS13 activity levels correlated with disease severity and with parameters of inflammation and disseminated intravascular coagulation. In septic mice, VWF deficiency attenuated mortality, whereas ADAMTS13 deficiency increased mortality. Bacterial clearance was enhanced in VWF-deficient mice. The differences in mortality for the studied genotypes were associated with differential loads of organ microthrombi in both liver and kidneys.

Conclusions: In conclusion, this study reports the consistent relation of VWF, ADAMTS13 and their ratio to disease severity in patients and mice with S. aureus sepsis. Targeting VWF multimers and/or the relative ADAMTS13 deficiency that occurs in sepsis should be explored as a potential new therapeutic target in S. aureus endovascular infections.
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http://dx.doi.org/10.1111/jth.14686DOI Listing
March 2020

Extracellular DNA NET-Works With Dire Consequences for Health.

Circ Res 2019 08 1;125(4):470-488. Epub 2019 Aug 1.

From the Program in Cellular and Molecular Medicine (N.S., D.C., D.D.W.), Boston Children's Hospital, MA.

Neutrophils play a central role in innate immune defense. Advances in neutrophil biology have brought to light the capacity of neutrophils to release their decondensed chromatin and form large extracellular DNA networks called neutrophil extracellular traps (NETs). NETs are produced in response to many infectious and noninfectious stimuli and, together with fibrin, block the invasion of pathogens. However, their formation in inflamed blood vessels produces a scaffold that supports thrombosis, generates neo-antigens favoring autoimmunity, and aggravates damage in ischemia/reperfusion injury. NET formation can also be induced by cancer and promotes tumor progression. Formation of NETs within organs can be immediately detrimental, such as in lung alveoli, where they affect respiration, or they can be harmful over longer periods of time. For example, NETs initiate excessive deposition of collagen, resulting in fibrosis, thus likely contributing to heart failure. Here, we summarize the latest knowledge on NET generation and discuss how excessive NET formation mediates propagation of thrombosis and inflammation and, thereby, contributes to various diseases. There are many ways in which NET formation could be averted or NETs neutralized to prevent their detrimental consequences, and we will provide an overview of these possibilities.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.314581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746252PMC
August 2019

Plasma Peptidylarginine Deiminase IV Promotes VWF-Platelet String Formation and Accelerates Thrombosis After Vessel Injury.

Circ Res 2019 08 28;125(5):507-519. Epub 2019 Jun 28.

From the Program in Cellular and Molecular Medicine (N.S., D.M.M., K.M., D.C., C.S., D.D.W.), Boston Children's Hospital, MA.

Rationale: PAD4 (peptidylarginine deiminase type IV), an enzyme essential for neutrophil extracellular trap formation (NETosis), is released together with neutrophil extracellular traps into the extracellular milieu. It citrullinates histones and holds the potential to citrullinate other protein targets. While NETosis is implicated in thrombosis, the impact of the released PAD4 is unknown.

Objective: This study tests the hypothesis that extracellular PAD4, released during inflammatory responses, citrullinates plasma proteins, thus affecting thrombus formation.

Methods And Results: Here, we show that injection of r-huPAD4 in vivo induces the formation of VWF (von Willebrand factor)-platelet strings in mesenteric venules and that this is dependent on PAD4 enzymatic activity. VWF-platelet strings are naturally cleaved by ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type-1 motif-13). We detected a reduction of endogenous ADAMTS13 activity in the plasma of wild-type mice injected with r-huPAD4. Using mass spectrometry and in vitro studies, we found that r-huPAD4 citrullinates ADAMTS13 on specific arginine residues and that this modification dramatically inhibits ADAMTS13 enzymatic activity. Elevated citrullination of ADAMTS13 was observed in plasma samples of patients with sepsis or noninfected patients who were elderly (eg, age >65 years) and had underlying comorbidities (eg, diabetes mellitus and hypertension) as compared with healthy donors. This shows that ADAMTS13 is citrullinated in vivo. VWF-platelet strings that form on venules of Adamts13 mice were immediately cleared after injection of r-huADAMTS13, while they persisted in vessels of mice injected with citrullinated r-huADAMTS13. Next, we assessed the effect of extracellular PAD4 on platelet-plug formation after ferric chloride-induced injury of mesenteric venules. Administration of r-huPAD4 decreased time to vessel occlusion and significantly reduced thrombus embolization.

Conclusions: Our data indicate that PAD4 in circulation reduces VWF-platelet string clearance and accelerates the formation of a stable platelet plug after vessel injury. We propose that this effect is, at least in part, due to ADAMTS13 inhibition.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.314571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697196PMC
August 2019

von Willebrand factor in experimental malaria-associated acute respiratory distress syndrome.

J Thromb Haemost 2019 08 12;17(8):1372-1383. Epub 2019 Jun 12.

Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium.

Background: Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a lethal complication of severe malaria, characterized by marked pulmonary inflammation. Patient studies have suggested a link between von Willebrand factor (VWF) and malaria severity.

Objectives: To investigate the role of VWF in the pathogenesis of experimental MA-ARDS.

Methods: Plasmodium berghei NK65-E (PbNK65) parasites were injected in Vwf and Vwf mice. Pathological parameters were assessed following infection.

Results: In accordance with patients with severe malaria, plasma VWF levels were increased and ADAMTS13 activity levels were reduced in experimental MA-ARDS. ADAMTS13- and plasmin-independent reductions of high molecular weight VWF multimers were observed at the end stage of disease. Thrombocytopenia was VWF-independent because it was observed in both Vwf and Vwf mice. Interestingly, Vwf mice had a shorter survival time compared with Vwf controls following PbNK65 infection. Lung edema could not explain this shortened survival because alveolar protein levels in Vwf mice were approximately two times lower than in Vwf controls. Parasite load, on the other hand, was significantly increased in Vwf mice compared with Vwf mice in both peripheral blood and lung tissue. In addition, anemia was only observed in PbNK65-infected Vwf mice. Of note, Vwf mice presented with two times more reticulocytes, a preferential target of the parasites.

Conclusions: This study suggests that parasite load together with malarial anemia, rather than alveolar leakage, might contribute to shortened survival in PbNK65-infected Vwf mice. VWF deficiency is associated with early reticulocytosis following PbNK65 infection, which potentially explains the increase in parasite load.
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http://dx.doi.org/10.1111/jth.14485DOI Listing
August 2019

Neutrophil Extracellular Traps in Arterial and Venous Thrombosis.

Semin Thromb Hemost 2019 Feb 11;45(1):86-93. Epub 2019 Jan 11.

Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium.

Thrombotic complications are still a major health risk worldwide. Our view on the pathophysiology of thrombosis has significantly changed since the discovery of neutrophil extracellular traps (NETs) and their prothrombotic characteristics. Generated by neutrophils that release their decondensed chromatin as a network of extracellular fibers, NETs promote thrombus formation by serving as a scaffold that activates platelets and coagulation. The thrombogenic involvement of NETs has been described in various settings of thrombosis, including stroke, myocardial infarction, and deep vein thrombosis. The aim of this review is to summarize existing evidence showing the presence of NETs in human thrombus material. Following an introduction on NETs and their role in thrombus formation, the authors address studies showing the presence of NETs in arterial or venous thrombi. In addition, they focus on potential novel therapeutic opportunities to resolve or prevent thrombosis by targeting NETs.
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http://dx.doi.org/10.1055/s-0038-1677040DOI Listing
February 2019

Solid peripheral tumor leads to systemic inflammation, astrocyte activation and signs of behavioral despair in mice.

PLoS One 2018 15;13(11):e0207241. Epub 2018 Nov 15.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, United States of America.

Prevalence of depression is higher in patients with cancer than in the general population. Sustained systemic inflammation has been associated with depressive behavior and it has been reported that depressed patients commonly display alterations in their immune system. We previously showed that cancer in mice induces a systemic environment that promotes neutrophil activation and leukocytosis. We thus hypothesized that the peripheral systemic response to a solid tumor leads to endothelial activation, which may promote inflammatory changes in the brain with behavioral consequences. Using the Lewis lung carcinoma (LLC) model, we show that tumor growth induces a progressive increase in peripheral inflammation as observed by elevated interleukin-6 (IL-6). In behavioral studies, tumor-bearing mice showed no sign of motor, coordination or short term working memory deficits as assessed by rotarod, balance-beam, and novel object recognition tests. However, there was an impairment in the grip strength test and interestingly, an anxious and despair-like phenotype in the elevated plus-maze, and tail suspension tests, respectively. Immunostaining of perfused brains revealed fibrin accumulation in the vasculature with some leakage into the parenchyma, a process known to activate endothelial cells. Taken together, our results suggest that the inflamed and prothrombotic systemic environment created by the growth of a peripherally-located solid tumor induces endothelial activation, accumulation of fibrin in the brain and astrocyte activation, perhaps leading to depressive-like behavior.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0207241PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237350PMC
April 2019

von Willebrand factor deficiency does not influence angiotensin II-induced abdominal aortic aneurysm formation in mice.

Sci Rep 2018 11 9;8(1):16645. Epub 2018 Nov 9.

Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium.

Abdominal aortic aneurysm (AAA) refers to a localized dilation of the abdominal aorta that exceeds the normal diameter by 50%. AAA pathophysiology is characterized by progressive inflammation, vessel wall destabilization and thrombus formation. Our aim was to investigate the potential involvement of von Willebrand factor (VWF), a thrombo-inflammatory plasma protein, in AAA pathophysiology using a dissection-based and angiotensin II infusion-induced AAA mouse model. AAA formation was induced in both wild-type and VWF-deficient mice by subcutaneous implantation of an osmotic pump, continuously releasing 1000 ng/kg/min angiotensin II. Survival was monitored, but no significant difference was observed between both groups. After 28 days, the suprarenal aortic segment of the surviving mice was harvested. Both AAA incidence and severity were similar in wild-type and VWF-deficient mice, indicating that AAA formation was not significantly influenced by the absence of VWF. Although VWF plasma levels increased after the infusion period, these increases were not correlated with AAA progression. Also detailed histological analyses of important AAA hallmarks, including elastic degradation, intramural thrombus formation and leukocyte infiltration, did not reveal differences between both groups. These data suggest that, at least in the angiotensin II infusion-induced AAA mouse model, the role of VWF in AAA pathophysiology is limited.
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http://dx.doi.org/10.1038/s41598-018-35029-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6226453PMC
November 2018

Neutrophil cytoplasts induce T17 differentiation and skew inflammation toward neutrophilia in severe asthma.

Sci Immunol 2018 08;3(26)

Pulmonary and Critical Care Medicine Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.

Severe asthma is a debilitating and treatment refractory disease. As many as half of these patients have complex neutrophil-predominant lung inflammation that is distinct from milder asthma with type 2 eosinophilic inflammation. New insights into severe asthma pathogenesis are needed. Concomitant exposure of mice to an aeroallergen and endotoxin during sensitization resulted in complex neutrophilic immune responses to allergen alone during later airway challenge. Unlike allergen alone, sensitization with allergen and endotoxin led to NETosis. In addition to neutrophil extracellular traps (NETs), enucleated neutrophil cytoplasts were evident in the lungs. Surprisingly, allergen-driven airway neutrophilia was decreased in peptidyl arginine deiminase 4-deficient mice with defective NETosis but not by deoxyribonuclease treatment, implicating the cytoplasts for the non-type 2 immune responses to allergen. Neutrophil cytoplasts were also present in mediastinal lymph nodes, and the cytoplasts activated lung dendritic cells in vitro to trigger antigen-specific interleukin-17 (IL-17) production from naïve CD4 T cells. Bronchoalveolar lavage fluid from patients with severe asthma and high neutrophil counts had detectable NETs and cytoplasts that were positively correlated with IL-17 levels. Together, these translational findings have identified neutrophil cytoplast formation in asthmatic lung inflammation and linked the cytoplasts to T helper 17-mediated neutrophilic inflammation in severe asthma.
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http://dx.doi.org/10.1126/sciimmunol.aao4747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320225PMC
August 2018

Increased neutrophil extracellular trap formation promotes thrombosis in myeloproliferative neoplasms.

Sci Transl Med 2018 04;10(436)

Division of Hematology, Brigham and Women's Hospital, Boston, MA 02115, USA.

Thrombosis is a major cause of morbidity and mortality in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), clonal disorders of hematopoiesis characterized by activated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling. Neutrophil extracellular trap (NET) formation, a component of innate immunity, has been linked to thrombosis. We demonstrate that neutrophils from patients with MPNs are primed for NET formation, an effect blunted by pharmacological inhibition of JAK signaling. Mice with conditional knock-in of , the most common molecular driver of MPN, have an increased propensity for NET formation and thrombosis. Inhibition of JAK-STAT signaling with the clinically available JAK2 inhibitor ruxolitinib abrogated NET formation and reduced thrombosis in a deep vein stenosis murine model. We further show that expression of PAD4, a protein required for NET formation, is increased in -expressing neutrophils and that PAD4 is required for -driven NET formation and thrombosis in vivo. Finally, in a population study of more than 10,000 individuals without a known myeloid disorder, -positive clonal hematopoiesis was associated with an increased incidence of thrombosis. In aggregate, our results link expression to NET formation and thrombosis and suggest that JAK2 inhibition may reduce thrombosis in MPNs through cell-intrinsic effects on neutrophil function.
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http://dx.doi.org/10.1126/scitranslmed.aan8292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442466PMC
April 2018

ADAMTS13 Deficiency Worsens Colitis and Exogenous ADAMTS13 Administration Decreases Colitis Severity in Mice.

TH Open 2017 Jun;1(1):e11-e23

Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, United States.

Background: Inflammatory bowel disease (IBD) affects 1.6 million people in the United States. IBD is associated with an increased risk of thrombosis, which rises with disease activity. The pathogenesis of IBD and its increased thrombotic risk is not completely understood. Ultra large von Willebrand factor (ULVWF) multimers are secreted from activated endothelium, leading to recruitment of platelets and leukocytes. A disintegrin and metalloproteinase with thrombospondin type I repeats motif 13 (ADAMTS13) cleaves highly adhesive ULVWF into smaller, less bioactive, multimers, releasing them into circulation. Mice deficient in ADAMTS13 (ADAMTS13) have heightened inflammatory and thrombotic responses.

Objectives: We hypothesized that upon colitis induction, ADAMTS13 mice would have more severe symptoms compared with wild-type (WT) mice, and rhADAMTS13 administration to mice with colitis would improve their condition.

Results: Dextran sodium sulfate-induced colitis was worse in ADAMTS13 mice than WT. ADAMTS13 showed increased weight loss, worse anemia, and increased clinical and histologic colitis severity, compared with WT mice. ADAMTS13 mice had increased VWF release, with accumulation at inflamed colonic sites. Also, the majority of mice showed one or more submucosal colonic thrombi. ADAMTS13 deficiency worsened colitis and propagated intestinal inflammation, most likely through increased platelet-leukocyte recruitment by VWF. Treatment of WT mice with rhA-DAMTS13 decreased colitis severity without worsening anemia. Additionally, several immune-mediated chronic murine colitis models, and inflamed colon tissue specimens from IBD patients, showed increased VWF release at inflamed sites, suggesting a generalizability of our findings.

Conclusion: Measuring VWF/ADAMTS13 levels could have clinical utility. When applicable, the administration of ADAMTS13, in addition to primary treatment, may improve outcomes for IBD patients.
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http://dx.doi.org/10.1055/s-0037-1603927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5782810PMC
June 2017

Recombinant Human ADAMTS13 Treatment Improves Myocardial Remodeling and Functionality After Pressure Overload Injury in Mice.

J Am Heart Assoc 2018 01 24;7(3). Epub 2018 Jan 24.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA

Background: A disintegrin-like metalloproteinase with thrombospondin motif type 1 member 13 (ADAMTS13), the von Willebrand factor-cleaving enzyme, decreases leukocyte and platelet recruitment and, thus, reduces thrombosis and inflammation. Recombinant human ADAMTS13 (rhADAMTS13) is a novel drug candidate for ischemia/reperfusion injury and has shown short-term benefits in mouse models of myocardial injury, but long-term outcome has not been investigated.

Methods And Results: We evaluated the impact of rhADAMTS13 on cardiac remodeling, scarring, and contractile function, under chronic left ventricular pressure overload. The role of von Willebrand factor and the effect of rhADAMTS13 treatment were studied. This model of heart failure, based on ascending aortic constriction, produces a coronary inflammatory response and microvascular dysfunction, resulting in fibrotic remodeling and cardiac failure. Mice were treated with either rhADAMTS13 or vehicle and assessed for coronary vascular inflammation and ventricular function at several postsurgical time points, as well as for cardiac fibrosis after 4 weeks. Early upon induction of pressure overload under rhADAMTS13 treatment, we detected less endothelial-lumen-associated von Willebrand factor, fewer platelet aggregates, and decreased activated transforming growth factor-β1 levels than in vehicle-treated mice. We observed significant preservation of cardiac function and decrease in fibrotic remodeling as a result of rhADAMTS13 administration.

Conclusions: Herein, we show that rhADAMTS13 decreases coronary vascular dysfunction and improves cardiac remodeling after left ventricular pressure overload in mice. We propose that this effect may, at least in part, be the result of decreased von Willebrand factor-mediated recruitment of platelets, a major source of the activated profibrotic cytokine transforming growth factor-β1. Our study further supports the therapeutic potential of rhADAMTS13 for conditions characterized by inflammatory cardiac damage that results in fibrosis.
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http://dx.doi.org/10.1161/JAHA.117.007004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5850234PMC
January 2018

Sirt3 deficiency does not affect venous thrombosis or NETosis despite mild elevation of intracellular ROS in platelets and neutrophils in mice.

PLoS One 2017 13;12(12):e0188341. Epub 2017 Dec 13.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, United States of America.

Inflammation is a common denominator in chronic diseases of aging. Yet, how inflammation fuels these diseases remains unknown. Neutrophils are the primary leukocytes involved in the early phase of innate immunity and inflammation. As part of their anti-microbial defense, neutrophils form extracellular traps (NETs) by releasing decondensed chromatin lined with cytotoxic proteins. NETs have been shown to induce tissue injury and thrombosis. Here, we demonstrated that Sirt3, a nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase, an enzyme linked to human longevity, was expressed in mouse neutrophils and platelets. Using Sirt3-/- mice as a model of accelerated aging, we investigated the effects of Sirt3 deficiency on NETosis and platelet function, aiming to detect enhancement of thrombosis. More mitochondrial reactive oxygen species (ROS) were generated in neutrophils and platelets of Sirt3-/- mice compared to WT, when stimulated with a low concentration of phorbol 12-myristate 13-acetate (PMA) and a high concentration of thrombin, respectively. There were no differences in in vitro NETosis, with or without stimulation. Platelet aggregation was mildly augmented in Sirt3-/- mice compared to WT mice, when stimulated with a low concentration of collagen. The effect of Sirt3 deficiency on platelet and neutrophil activation in vivo was examined by the venous thrombosis model of inferior vena cava stenosis. Elevation of plasma DNA concentration was observed after stenosis in both genotypes, but no difference was shown between the two genotypes. The systemic response to thrombosis was enhanced in Sirt3-/- mice with significantly elevated neutrophil count and reduced platelet count. However, no differences were observed in incidence of thrombus formation, thrombus weight and thrombin-antithrombin complex generation between WT and Sirt3-/- mice. We conclude that Sirt3 does not considerably impact NET formation, platelet function, or venous thrombosis in healthy young mice.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0188341PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728566PMC
January 2018

A key role for Rac and Pak signaling in neutrophil extracellular traps (NETs) formation defines a new potential therapeutic target.

Am J Hematol 2018 02 6;93(2):269-276. Epub 2017 Dec 6.

Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA; Department of Pediatrics, Harvard Medical School, Boston, Massachusetts.

NET formation in mice (NETosis) is supported by reactive oxygen species (ROS) production by NADPH oxidase and histone hypercitrullination by peptidylarginine deiminase 4 (PAD4). Rac1 and Rac2, expressed in polymorphonuclear neutrophils (PMNs), regulate the cytoskeleton, cell shape, adhesion, and migration and are also essential components of the NADPH oxidase complex. We aimed to explore the role of the Rac signaling pathway including the upstream guanosine exchange factor (GEF) activator, Vav, and a downstream effector, the p21-activated kinase, Pak, on NETosis in PMNs using a previously described flow-cytometry-based assay. Rac2 PMNs showed reduced levels of citrullinated histone H3 (H3Cit)-positive cells and defective NETosis. Rac1 ; Rac2 PMNs demonstrated a further reduction in PMA-induced H3Cit levels and a more profound impairment of NETosis than deletion of Rac2 alone, suggesting an overlapping role of these two highly related proteins. Genetic knockouts of Vav1, or Vav2, did not impair H3Cit response to phorbol myristate ester (PMA) or NETosis. Combined, Vav1 and Vav3 deletions decreased H3Cit response and caused a modest but significant impairment of NETosis. Pharmacologic inhibition of Pak by two inhibitors with distinct mechanisms of action, led to reduced H3Cit levels after PMA stimulation, as well as significant inhibition of NETosis. We validated the importance of Pak using Pak2 PMNs, which demonstrated significantly impaired histone H3 citrullination and NETosis. These data confirm and more comprehensively define the key role of the Rac signaling pathway in PMN NETosis. The Rac signaling cascade may represent a valuable target for inhibition of NETosis and related pathological processes.
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http://dx.doi.org/10.1002/ajh.24970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760446PMC
February 2018

Peptidylarginine deiminase 4 promotes age-related organ fibrosis.

J Exp Med 2017 02 28;214(2):439-458. Epub 2016 Dec 28.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115

Aging promotes inflammation, a process contributing to fibrosis and decline in organ function. The release of neutrophil extracellular traps (NETs [NETosis]), orchestrated by peptidylarginine deiminase 4 (PAD4), damages organs in acute inflammatory models. We determined that NETosis is more prevalent in aged mice and investigated the role of PAD4/NETs in age-related organ fibrosis. Reduction in fibrosis was seen in the hearts and lungs of aged PAD4 mice compared with wild-type (WT) mice. An increase in left ventricular interstitial collagen deposition and a decline in systolic and diastolic function were present only in WT mice, and not in PAD4 mice. In an experimental model of cardiac fibrosis, cardiac pressure overload induced NETosis and significant platelet recruitment in WT but not PAD4 myocardium. DNase 1 was given to assess the effects of extracellular chromatin. PAD4 deficiency or DNase 1 similarly protected hearts from fibrosis. We propose a role for NETs in cardiac fibrosis and conclude that PAD4 regulates age-related organ fibrosis and dysfunction.
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http://dx.doi.org/10.1084/jem.20160530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5294849PMC
February 2017

Priming of neutrophils toward NETosis promotes tumor growth.

Oncoimmunology 2016 May 18;5(5):e1134073. Epub 2016 Feb 18.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA; Department of Pediatrics, Harvard Medical School, Boston, MA, USA; Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA, USA.

Neutrophils play a major role in cancer biology and both pro- and antitumoral functions of tumor-infiltrating neutrophils have been described. We have shown that tumors, by releasing G-CSF into the bloodstream, prime circulating neutrophils to form neutrophil extracellular traps (NETs) and we have detected the presence of NETs within the tumor microenvironment. Here, we report, using PAD4-deficient mice with a defect in neutrophil chromatin decondensation and NET formation, that the priming of neutrophils toward NETosis favors tumor growth. Interestingly, in a tumor model that does not release G-CSF and in which neutrophils are not primed for NETosis, PAD4-deficiency did not reduce tumor growth. However, supplying exogenous G-CSF to the wild-type (WT) host promoted intratumoral NETosis and tumor growth. Taken together, our results suggest that the priming of neutrophils for NETosis by the tumor or its environment leads to the accumulation of intratumoral NETs and a growth advantage to the tumor. Our work unveiled a pro-tumoral role for NETs which strengthens their potential as a new target in the fight against cancer.
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http://dx.doi.org/10.1080/2162402X.2015.1134073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4910712PMC
May 2016

Mx1 reveals innate pathways to antiviral resistance and lethal influenza disease.

Science 2016 Apr;352(6284):463-6

Department of Immunobiology, Yale School of Medicine, New Haven, CT 06520, USA. Howard Hughes Medical Institute, Yale School of Medicine, New Haven, CT 06520, USA.

Influenza A virus (IAV) causes up to half a million deaths worldwide annually, 90% of which occur in older adults. We show that IAV-infected monocytes from older humans have impaired antiviral interferon production but retain intact inflammasome responses. To understand the in vivo consequence, we used mice expressing a functional Mx gene encoding a major interferon-induced effector against IAV in humans. In Mx1-intact mice with weakened resistance due to deficiencies in Mavs and Tlr7, we found an elevated respiratory bacterial burden. Notably, mortality in the absence of Mavs and Tlr7 was independent of viral load or MyD88-dependent signaling but dependent on bacterial burden, caspase-1/11, and neutrophil-dependent tissue damage. Therefore, in the context of weakened antiviral resistance, vulnerability to IAV disease is a function of caspase-dependent pathology.
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http://dx.doi.org/10.1126/science.aaf3926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465864PMC
April 2016

Flow cytometric assay for direct quantification of neutrophil extracellular traps in blood samples.

Am J Hematol 2015 Dec 6;90(12):1155-8. Epub 2015 Oct 6.

Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.

Neutrophil extracellular traps (NETs) contribute to innate immunity as well as numerous diseases processes such as deep vein thrombosis, myocardial ischemia, and autoimmune disease. To date, most knowledge on NETs formation has been gathered via the qualitative microscopic examination of individual neutrophils in vitro, or aggregate structures in vivo. Here we describe a novel flow cytometry (FLOW)-based assay to identify and quantify NETs using antibodies against key NETs constituents, specifically DNA, modified histones, and granular enzymes. This method is applicable to both murine and human samples for the assessment of induced NETs in vitro, or detection of NETosis in vivo in blood samples. This FLOW-based method was validated by comparison with the well-established microscopy assay using two genetic mouse models previously demonstrated to show defective NETosis. It was then used on healthy human neutrophils for detection of ex vivo induced NETs and on blood samples from patients with sepsis for direct assessment of in vivo NET-forming neutrophils. This new methodology allows rapid and robust assessment of several thousand cells per sample and is independent of potential observer-bias, the two main limitations of the microscopic quantification. Using this new technology facilitates the direct detection of in vivo circulating NETs in blood samples and purification of NETting neutrophils by fluorescence-activated cell sorting (FACS) for further analysis.
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http://dx.doi.org/10.1002/ajh.24185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4715743PMC
December 2015

Diabetes primes neutrophils to undergo NETosis, which impairs wound healing.

Nat Med 2015 Jul 15;21(7):815-9. Epub 2015 Jun 15.

1] Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA. [2] Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA. [3] Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA.

Wound healing is impaired in diabetes, resulting in significant morbidity and mortality. Neutrophils are the main leukocytes involved in the early phase of healing. As part of their anti-microbial defense, neutrophils form extracellular traps (NETs) by releasing decondensed chromatin lined with cytotoxic proteins. NETs, however, can also induce tissue damage. Here we show that neutrophils isolated from type 1 and type 2 diabetic humans and mice were primed to produce NETs (a process termed NETosis). Expression of peptidylarginine deiminase 4 (PAD4, encoded by Padi4 in mice), an enzyme important in chromatin decondensation, was elevated in neutrophils from individuals with diabetes. When subjected to excisional skin wounds, wild-type (WT) mice produced large quantities of NETs in wounds, but this was not observed in Padi4(-/-) mice. In diabetic mice, higher levels of citrullinated histone H3 (H3Cit, a NET marker) were found in their wounds than in normoglycemic mice and healing was delayed. Wound healing was accelerated in Padi4(-/-) mice as compared to WT mice, and it was not compromised by diabetes. DNase 1, which disrupts NETs, accelerated wound healing in diabetic and normoglycemic WT mice. Thus, NETs impair wound healing, particularly in diabetes, in which neutrophils are more susceptible to NETosis. Inhibiting NETosis or cleaving NETs may improve wound healing and reduce NET-driven chronic inflammation in diabetes.
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http://dx.doi.org/10.1038/nm.3887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631120PMC
July 2015

P-selectin promotes neutrophil extracellular trap formation in mice.

Blood 2015 Jul 15;126(2):242-6. Epub 2015 May 15.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA; Department of Pediatrics, and Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA.

Neutrophil extracellular traps (NETs) can be released in the vasculature. In addition to trapping microbes, they promote inflammatory and thrombotic diseases. Considering that P-selectin induces prothrombotic and proinflammatory signaling, we studied the role of this selectin in NET formation. NET formation (NETosis) was induced by thrombin-activated platelets rosetting with neutrophils and was inhibited by anti-P-selectin aptamer or anti-P-selectin glycoprotein ligand-1 (PSGL-1) inhibitory antibody but was not induced by platelets from P-selectin(-/-) mice. Moreover, NETosis was also promoted by P-selectin-immunoglobulin fusion protein but not by control immunoglobulin. We isolated neutrophils from mice engineered to overproduce soluble P-selectin (P-selectin(ΔCT/ΔCT) mice). Although the levels of circulating DNA and nucleosomes (indicative of spontaneous NETosis) were normal in these mice, basal neutrophil histone citrullination and presence of P-selectin on circulating neutrophils were elevated. NET formation after stimulation with platelet activating factor, ionomycin, or phorbol 12-myristate 13-acetate was significantly enhanced, indicating that the P-selectin(ΔCT/ΔCT) neutrophils were primed for NETosis. In summary, P-selectin, cellular or soluble, through binding to PSGL-1, promotes NETosis, suggesting that this pathway is a potential therapeutic target for NET-related diseases.
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http://dx.doi.org/10.1182/blood-2015-01-624023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4497964PMC
July 2015

PAD4-deficiency does not affect bacteremia in polymicrobial sepsis and ameliorates endotoxemic shock.

Blood 2015 Mar 26;125(12):1948-56. Epub 2015 Jan 26.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA; Department of Pediatrics, Harvard Medical School, Boston, MA; Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA.

Neutrophil extracellular traps (NETs), consisting of nuclear DNA with histones and microbicidal proteins, are expelled from activated neutrophils during sepsis. NETs were shown to trap microbes, but they also fuel cardiovascular, thrombotic, and autoimmune disease. The role of NETs in sepsis, particularly the balance between their antimicrobial and cytotoxic actions, remains unclear. Neutrophils from peptidylarginine deiminase 4-(PAD4(-/-)) deficient mice, which lack the enzyme allowing for chromatin decondensation and NET formation, were evaluated. We found that neutrophil functions involved in bacterial killing, other than NETosis, remained intact. Therefore, we hypothesized that prevention of NET formation might not have devastating consequences in sepsis. To test this, we subjected the PAD4(-/-) mice to mild and severe polymicrobial sepsis produced by cecal ligation and puncture. Surprisingly, under septic conditions, PAD4(-/-) mice did not fare worse than wild-type mice and had comparable survival. In the presence of antibiotics, PAD4-deficiency resulted in slightly accelerated mortality but bacteremia was unaffected. PAD4(-/-) mice were partially protected from lipopolysaccharide-induced shock, suggesting that PAD4/NETs may contribute to the toxic inflammatory and procoagulant host response to endotoxin. We propose that preventing NET formation by PAD4 inhibition in inflammatory or thrombotic diseases is not likely to increase host vulnerability to bacterial infections.
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http://dx.doi.org/10.1182/blood-2014-07-587709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4366625PMC
March 2015

Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation.

Nat Chem Biol 2015 Mar 26;11(3):189-91. Epub 2015 Jan 26.

1] EpiNova DPU, Immuno-Inflammation Therapy Area, GlaxoSmithKline,Medicines Research Centre, Stevenage, Hertfordshire, UK. [2] AstraZeneca, Oncology iMed, Cambridge Science Park, Cambridge, UK (R.J.S. and D.M.W.); Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA (D.J.S.); University of Massachussetts Medical School, Worcester, Massachusetts, USA (P.R.T.).

PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases through clinical genetics and gene disruption in mice. New selective PAD4 inhibitors binding a calcium-deficient form of the PAD4 enzyme have validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation for, to our knowledge, the first time. The therapeutic potential of PAD4 inhibitors can now be explored.
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http://dx.doi.org/10.1038/nchembio.1735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4397581PMC
March 2015

Dietary omega-3 alpha-linolenic acid does not prevent venous thrombosis in mice.

Thromb Haemost 2015 Jan 18;113(1):177-84. Epub 2014 Sep 18.

Prof. Dr. med. Juerg Hans Beer, M D, FACP, Head of the Department of Medicine, Cantonal Hospital Baden, Im Ergel 1, CH-5404 Baden, Switzerland and Laboratory for Platelet Research, University of Zurich, Switzerland, Tel.: +41 56 486 25 02, Fax: +41 56 486 25 09, E-mail:

Venous thromboembolism (VTE) is a leading cause of cardiovascular death. Omega-3 fatty acids (n-3 FA) exhibit protective effects against cardiovascular disease. Others and our group have reported that the plant-derived n-3 FA alpha-linolenic acid (ALA) displays antiinflammatory, anticoagulant and antiplatelet effects, thereby reducing atherosclerosis and arterial thrombosis in mice fed a high ALA diet. Since procoagulant factors such as tissue factor and fibrin as well as platelets and leukocytes are crucially involved in the development of VTE, we investigated possible protective effects of dietary ALA on venous thrombus formation in a mouse model of stenosis- and furthermore, in a mouse model of endothelial injury-induced venous thrombosis. Four week old C57BL/6 mice underwent four weeks of high (7.3g%) or low ALA (0.03g%) treatment before being exposed to inferior vena cava (IVC) stenosis for 48 hours or laser injury of the endothelium of the internal jugular vein (IJV). Thrombus generation frequency, thrombus size and composition (IVC stenosis group) and time to thrombus formation (endothelial injury group) were assessed. In addition, plasma glycocalicin, a marker of platelet activation, platelet P-selectin and activated integrin expression as well as plasma thrombin generation was determined, but did not reveal any significant differences between the groups. Despite its protective properties against arterial thrombus formation, dietary ALA did not protect against venous thrombosis neither in the IVC stenosis nor the endothelial injury model, further indicating that the biological processes involved in arterial and venous thrombosis are different.
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http://dx.doi.org/10.1160/TH14-03-0200DOI Listing
January 2015

Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.

J Immunol 2014 Jun 25;192(11):5390-7. Epub 2014 Apr 25.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115;Division of Hematology-Oncology, Boston Children's Hospital, Boston, MA 02215; andDepartment of Pediatrics, Harvard Medical School, Boston, MA 02115

Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. In this study, we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps in a DNA-dependent manner. Thus, high-affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation.
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http://dx.doi.org/10.4049/jimmunol.1303296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041364PMC
June 2014

Thrombosis: tangled up in NETs.

Blood 2014 May 23;123(18):2768-76. Epub 2013 Dec 23.

Immunology Graduate Program, Division of Medical Sciences, Harvard Medical School, Boston, MA;

The contributions by blood cells to pathological venous thrombosis were only recently appreciated. Both platelets and neutrophils are now recognized as crucial for thrombus initiation and progression. Here we review the most recent findings regarding the role of neutrophil extracellular traps (NETs) in thrombosis. We describe the biological process of NET formation (NETosis) and how the extracellular release of DNA and protein components of NETs, such as histones and serine proteases, contributes to coagulation and platelet aggregation. Animal models have unveiled conditions in which NETs form and their relation to thrombogenesis. Genetically engineered mice enable further elucidation of the pathways contributing to NETosis at the molecular level. Peptidylarginine deiminase 4, an enzyme that mediates chromatin decondensation, was identified to regulate both NETosis and pathological thrombosis. A growing body of evidence reveals that NETs also form in human thrombosis and that NET biomarkers in plasma reflect disease activity. The cell biology of NETosis is still being actively characterized and may provide novel insights for the design of specific inhibitory therapeutics. After a review of the relevant literature, we propose new ways to approach thrombolysis and suggest potential prophylactic and therapeutic agents for thrombosis.
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http://dx.doi.org/10.1182/blood-2013-10-463646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4007606PMC
May 2014

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

Blood 2014 Jan 7;123(1):141-8. Epub 2013 Nov 7.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA;

Innate immune cells play a major role in the early response to myocardial ischemia/reperfusion (MI/R) injury. Recombinant human ADAMTS13 (rhADAMTS13), cleaving von Willebrand factor (VWF), reduces leukocyte recruitment in mice. Death of cardiomyocytes and the possible formation of neutrophil extracellular traps (NETs) may result in chromatin release that is prothrombotic and cytotoxic. We investigated the pathophysiological role of extracellular chromatin during MI/R to evaluate the therapeutic potential of targeting extracellular DNA and VWF by using DNase I with/without rhADAMTS13. Finally, we examined the impact of histone citrullination and NETosis by peptidylarginine deiminase 4 (PAD4) on MI/R. We used a 24-hour MI/R mouse surgical model. MI/R injury caused an increase in plasma nucleosomes, abundant neutrophil infiltration, and the presence of citrullinated histone H3 at the site of injury. Both monotherapies and coadministration of DNase I and rhADAMTS13 revealed a cardioprotective effect, resulting in subsequent improvement of cardiac contractile function. PAD4(-/-) mice, which do not produce NETs, were also significantly protected from MI/R and DNase I treatment had no further beneficial effect. We demonstrate that extracellular chromatin released through NETosis exacerbates MI/R injury. Targeting both VWF-mediated leukocyte recruitment and chromatin removal may be a new therapeutic strategy to reduce ischemia-related cardiac damage.
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http://dx.doi.org/10.1182/blood-2013-07-514992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3879903PMC
January 2014