Neutrophils undergo a cell death pathway that results in release of chromatin coated with antimicrobial proteins forming a web called neutrophil extracellular traps (NETs). NETs trap and kill microbes. Our preliminary results show that NETs recruit and activate platelets. We have evidence of extracellular DNA/chromatin presence in pathological specimens from deep vein thrombosis and in plasma of patients with thrombotic disease. The central hypothesis of this application is that NETs provide a scaffold that, together with fibrin, increases thrombus stability and promotes thrombus growth and microparticle recruitment. We propose that the formation of NETs in pathological circumstances could initiate thrombotic events and contribute to conditions such as sepsis, thrombotic microangiopathies and deep vein thrombosis. In addition, we propose that DNase1, which digests NETs, may modulate thrombosis. We will analyze NETs in the context of in vitro and in vivo models of thrombosis. We will employ a flow chamber with video capability using cells from genetically engineered mice. We propose to study several mouse models of thrombosis, all of which are well established in our laboratory, to visualize NETs formation in vivo with sophisticated imaging technology and study the relevance of NETs to thrombus growth and stability. We are particularly keen to investigate the role of NETs in a new inferior vena cava stenosis model of deep vein thrombosis (DVT) and in a mouse model of sepsis. The proposal has 2 specific aims: 1. To study the molecular mechanisms mediating platelet and microparticle interactions with NETs. To evaluate whether NETs promote fibrin generation. In this Aim, we propose to investigate in vitro the molecular mechanisms involved in platelet adhesion to NETs and address whether NETs promote recruitment of pro-coagulant microparticles (MPs) and fibrin deposition. 2. To study NETs formation in vivo and examine the biological relevance of NETs to sepsis-induced thrombocytopenia and to deep vein thrombosis. To measure NETs biomarkers in samples from patients with thrombosis. In this aim, we propose to visualize NETs by intravital microscopy and by histology. We propose to modulate NETs production and circulating DNA levels and see the effects on murine models of sepsis and DVT. We will study NETs as biomarkers in thrombosis. Thrombosis, including deep vein thrombosis/pulmonary embolism is now the biggest killer in the United States. We hope that our studies will broaden understanding of the thrombotic process in general and that we may uncover new molecular targets to treat pathological thrombosis.

Public Health Relevance

Thrombosis is now the biggest killer in the United States. Thrombi are formed by platelets sticking together. White blood cells influence thrombi formation and their stability. Dying white cells actively release DNA producing large spider web-like structures that trap bacteria. We found that platelets bind to these webs and that such webs are present in thrombi. We propose to investigate the role of this DNA scaffold in thrombosis and the possibility of using enzymes to digest the DNA as a new anti- thrombotic approach.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL102101-04
Application #
8494074
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Link, Rebecca P
Project Start
2011-06-15
Project End
2015-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
4
Fiscal Year
2013
Total Cost
$469,753
Indirect Cost
$199,780
Name
Children's Hospital Boston
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115
Gavillet, Mathilde; Martinod, Kimberly; Renella, Raffaele et al. (2018) A key role for Rac and Pak signaling in neutrophil extracellular traps (NETs) formation defines a new potential therapeutic target. Am J Hematol 93:269-276
Zitomersky, Naamah L; Demers, Melanie; Martinod, Kimberly et al. (2017) ADAMTS13 Deficiency Worsens Colitis and Exogenous ADAMTS13 Administration Decreases Colitis Severity in Mice. TH Open 1:e11-e23
Hayashi, Hideki; Cherpokova, Deya; Martinod, Kimberly et al. (2017) Sirt3 deficiency does not affect venous thrombosis or NETosis despite mild elevation of intracellular ROS in platelets and neutrophils in mice. PLoS One 12:e0188341
Martinod, Kimberly; Witsch, Thilo; Erpenbeck, Luise et al. (2017) Peptidylarginine deiminase 4 promotes age-related organ fibrosis. J Exp Med 214:439-458
Shaw, Maureen A; Kombrinck, Keith W; McElhinney, Kathryn E et al. (2016) Limiting prothrombin activation to meizothrombin is compatible with survival but significantly alters hemostasis in mice. Blood 128:721-31
Demers, Mélanie; Wong, Siu Ling; Martinod, Kimberly et al. (2016) Priming of neutrophils toward NETosis promotes tumor growth. Oncoimmunology 5:e1134073
Pillai, Padmini S; Molony, Ryan D; Martinod, Kimberly et al. (2016) Mx1 reveals innate pathways to antiviral resistance and lethal influenza disease. Science 352:463-6
Martinod, K; Witsch, T; Farley, K et al. (2016) Neutrophil elastase-deficient mice form neutrophil extracellular traps in an experimental model of deep vein thrombosis. J Thromb Haemost 14:551-8
Erpenbeck, Luise; Demers, Melanie; Zsengellér, Zsuzsanna K et al. (2016) ADAMTS13 Endopeptidase Protects against Vascular Endothelial Growth Factor Inhibitor-Induced Thrombotic Microangiopathy. J Am Soc Nephrol 27:120-31
Thålin, Charlotte; Demers, Melanie; Blomgren, Bo et al. (2016) NETosis promotes cancer-associated arterial microthrombosis presenting as ischemic stroke with troponin elevation. Thromb Res 139:56-64

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