AbstractPancreatic cancer is associated with a high incidence of venous thromboembolism. Neutrophils have been shown to contribute to thrombosis in part by releasing neutrophil extracellular traps (NET). A recent study showed that increased plasma levels of the NET biomarker, citrullinated histone H3 (H3Cit), are associated with venous thromboembolism in patients with pancreatic and lung cancer but not in those with other types of cancer, including breast cancer. In this study, we examined the contribution of neutrophils and NET to venous thrombosis in nude mice bearing human pancreatic tumors. We found that tumor-bearing mice had increased circulating neutrophil counts and levels of granulocyte-colony stimulating factor, neutrophil elastase, H3Cit and cell-free DNA compared with controls. In addition, thrombi from tumor-bearing mice contained increased levels of the neutrophil marker Ly6G, as well as higher levels of H3Cit and cell-free DNA. Thrombi from tumor-bearing mice also had denser fibrin with thinner fibers consistent with increased thrombin generation. Importantly, either neutrophil depletion or administration of DNase I reduced the thrombus size in tumor-bearing but not in control mice. Our results, together with clinical data, suggest that neutrophils and NET contribute to venous thrombosis in patients with pancreatic cancer.
Cancer patients have a 4- to 7-fold increased risk of venous thromboembolism (VTE) compared with the general population.1 However, the rates of VTE vary in different cancer types. For instance, breast cancer has a low rate whereas pancreatic cancer has a high rate of VTE.2 This variability suggests that there may be cancer type-specific mechanisms of VTE.3 For instance, we found an association between levels of circulating extracellular vesicle tissue factor activity and VTE in pancreatic cancer in two studies and a borderline significance in a third study.64 Circulating tumor-derived, tissue factor-positive extracellular vesicles are also observed in mice bearing human pancreatic tumors.107 Importantly, we have shown that these tumor-derived, human tissue factor-positive extracellular vesicles enhance venous thrombosis in mice.10
Leukocytosis is often observed in cancer patients, particularly patients with lung and colorectal cancer.3 Leukocytosis is also associated with VTE in cancer patients, and is a component of the Khorana Risk Score for predicting chemotherapy-associated thrombosis in ambulatory cancer patients.1311 In addition, some patients have increased circulating levels of hematopoietic cytokines, such as granulocyte-colony stimulating factor (G-CSF).14 The coagulation cascade is activated by pathogens as part of the innate immune system to limit dissemination of infection.15 Recently, the term “immunothrombosis” was introduced to describe the contribution of immune cells to thrombus.16 Activated monocytes can trigger thrombosis by expressing tissue factor.17 Activated neutrophils release proteases, such as neutrophil elastase (NE), which enhance thrombosis by degrading the anticoagulant protein tissue factor pathway inhibitor.18 In addition, neutrophils release neutrophil extracellular traps (NET). NET are composed of extracellular chromatin components and neutrophil granule proteins that enhance thrombosis by capturing platelets and procoagulant extracellular vesicles.2219 NET are present in both arterial and venous thrombi.242319 NET can also obstruct smaller blood vessels in a coagulation-independent manner.25 Interestingly, two studies showed that neutrophils contribute to thrombosis in the mouse inferior vena cava (IVC) stenosis model, although this was not observed in a third study.272620 In contrast, neutrophil depletion did not affect thrombosis in the IVC stasis model.28
There is a wide range of agonists that can induce NET formation.29 In neutrophils histone citrullination by peptidylarginine deiminases (PAD), including PAD4, is considered a driver of chromatin decondensation and subsequent NET formation.30 PAD4 is also expressed by the human breast cancer cell line MCF7.31 Citrullinated histones, such as citrullinated histone H3 (H3Cit), are therefore widely used as a biomarker of NET formation. In mice, it has been proposed that PAD4 is required for NET formation.32 Indeed, PAD4 mice have smaller thrombi in the IVC stenosis model.33 However, a recent study found that inhibition of PAD did not affect human neutrophil NET formation induced by a variety of pathogens,29 suggesting that certain forms of NET formation can occur without PAD. Interestingly, a recent study found an association between plasma levels of H3Cit and VTE in patients with pancreatic and lung cancer but not in those with other types of cancer, such as breast cancer.34 In another study plasma levels of nucleosomes and cell-free DNA (cfDNA) were higher in cancer patients than in healthy controls, but these are not NET-specific biomarkers.35
Neutrophilia was observed in mice bearing murine breast 4T1 tumors and human pancreatic BxPc-3 tumors.383610 In addition, mice bearing 4T1 breast tumors had increased levels of circulating markers of neutrophil activation and NET, such as H3Cit and myeloperoxidase.3837 Furthermore, tumor-bearing mice had more rapid thrombotic occlusion in a jugular vein Rose Bengal/laser-induced injury model.38 Interestingly, administration of DNase I to degrade cfDNA and NET did not affect thrombotic occlusion in control mice but provided protection from the enhanced venous thrombosis observed in tumor-bearing mice.38 These studies suggest that neutrophils and NET contribute to venous thrombosis in a murine breast cancer model.
In the light of recent clinical data suggesting a role of NET in VTE in patients with pancreatic cancer,34 we investigated the contribution of neutrophils and NET to venous thrombosis in mice bearing human pancreatic BxPc-3 tumors.
Cells and the mouse tumor model
We used a human pancreatic cancer cell line BxPc-3 expressing the firefly luciferase reporter.10 BxPc-3 tumors were grown in the pancreas of Crl:NU-Foxn1nu male mice (nude mice) and monitored by measuring luciferase expression.10 We used mice with tumors weighing from 1.5 to 3.9 grams. All animal studies were approved by the University of North Carolina at Chapel Hill Animal Care and Use Committee, and complied with National Institutes of Health guidelines.
Measurement of blood cells
A Hemavet HV950FS (Drew Scientific, Miami Lakes, FL, USA) was used to count neutrophils.
Preparation of plasma and measurement of plasma biomarkers
Blood was collected39 and plasma was prepared by centrifuging the blood at 4500 × g for 15 min. CfDNA was quantified as described elsewhere.40 Mouse G-CSF and NE were measured using commercially available enzyme-linked immunosorbent assays (R&D systems, Minneapolis, MN, USA). H3Cit was measured using an in-house enzyme-linked immunosorbent assay.41
We used the IVC stasis model of thrombosis.10
Western blot analysis of thrombus samples
Processing of thrombi and detection of primary antibodies is described in the Online Supplementary Methods. Membranes were probed with 2 μg/mL anti-Ly6G (BioXCell, West Lebanon, NH, USA), a 1,000-fold dilution of anti-β-actin (Abgent, San Diego, CA, USA), a 2,000-fold dilution of anti-PAD4 (Abcam), 1 μg/mL anti-H3Cit (Abcam) or 0.5 μg/mL anti-histone H3 (Abcam) primary antibody.
Analysis of thrombi by immunofluorescence is described in the Online Supplementary Methods. Areas of different fluorescent signals were quantified using Image J software.
Scanning electron microscopy
Analysis of thrombi by scanning electron microscopy is described in the Online Supplementary Methods.
Neutrophils were depleted by intravenous administration of 5 mg/kg anti-Ly6G antibody (BioXCell) 24 h and 1 h before IVC stasis. A rat IgG (Sigma-Aldrich) was used as a control.
DNase I treatment
DNase I (50 U/mouse (Genentech, South San Francisco, CA, USA) or phosphate-buffered saline was intravenously administered to mice 1 h before and 24 h after IVC stasis.
Data are shown as mean ± standard error of the means for normally distributed data or median ± interquartile range for non-normally distributed data. The Shapiro-Wilk test was used to determine normality. For the majority of the studies two-group comparisons, the unpaired two-tailed Student t-test or the Mann-Whitney U-test was used depending on the data distribution. For the ultrasound data, two-way analysis of variance with the Sidak multiple comparison test was used. These statistical analyses were performed with GraphPad PRISM version 7.03 (GraphPad Software, La Jolla, CA, USA). For immunofluorescence data and scanning electron microscopy data, bootstrap simulations of the chosen quantiles of the distribution of the measured values were performed42 followed by a Bootstrap Kuiper test for identity of the quantile distributions.43 These statistical analyses are described in detail in the Online Supplementary Material. P values <0.05 were considered statistically significant for all experiments.
Measurement of circulating neutrophil counts and different biomarkers in mice bearing human pancreatic tumors
We measured the numbers of neutrophils in whole blood and various circulating biomarkers in the plasma of mice bearing human BxPc-3 pancreatic tumors (1.5-3.9 grams) and in control mice. We observed significant increases in neutrophil numbers and mouse G-CSF levels in tumor-bearing mice compared with those in controls (Figure 1A, B). We did not observe an increase in human G-CSF (data not shown). We found a significant correlation between circulating neutrophil count and mouse G-CSF level (r = 0.83, P=0.02, Spearman test). In addition, we found significant increases in the neutrophil bio-marker NE and the NET biomarker H3Cit in tumor-bearing mice compared with controls (Figure 1C, D). Finally, the amount of cfDNA was significantly greater in tumor-bearing mice than in controls (Figure 1E).
The human breast cancer cell line MCF7 expresses PAD4.31 We therefore determined whether BxPc-3 cells also express PAD4. Western blotting showed that the BxPc-3 cells did express PAD4 protein (data not shown). Tumor cells may, therefore, also contribute to plasma H3Cit.
Measurement of neutrophils, histone H3 and citrullinated histone H3 in thrombi from mice bearing human pancreatic tumors
We generated thrombi in control and tumor-bearing mice using the IVC stasis model. Using western blotting, we measured levels of Ly6G (a neutrophil marker), β-actin (a housekeeping gene), histone H3 (a marker of cellular content), and H3Cit (a NET biomarker) in thrombi from mice bearing human pancreatic tumors and control mice (Figure 2A). Thrombi from tumor-bearing mice had higher levels of Ly6G, β-actin, histone H3, and H3Cit compared with the levels in thrombi from control mice (Figure 2 B-E). The H3Cit/H3 ratio for thrombi from control mice was ~1 (Figure 2F). Interestingly, there was significant variation in the H3Cit/H3 ratio in thrombi from tumor-bearing mice (Figure 2F).
Next, we measured the cfDNA and H3Cit content in thrombi from mice bearing human pancreatic tumors and in thrombi from controls using immunofluorescence. Consistent with the data from the western blot analysis, thrombi from tumor-bearing mice had increased levels of cfDNA and H3Cit compared with levels of these bio-markers in thrombi from control mice (Figure 3).
Finally, we analyzed the composition of thrombi from control and tumor-bearing mice by scanning electron microscopy. Red blood cells were decreased in thrombi from tumor-bearing mice compared with thrombi from control mice (Figure 4A). In addition, we observed an increase in fibrin density and a decrease in fibrin fiber thickness in thrombi from tumor-bearing mice compared with thrombi from controls (Figure 4B). Previous studies have shown that increased levels of thrombin lead to the generation of denser fibrin with thinner fibers.44
Effect of neutrophil depletion on thrombus size in mice bearing human pancreatic tumors
Thrombi in the group of tumor-bearing mice treated with IgG weighed significantly more than those in controls [controls vs. tumor-bearing mice (median [range]): 16.4 [13.8-22.4] g vs. 31.9 [29.1-36.5] g; P<0.001, n=4-5]. Next, we investigated the role of neutrophils in thrombus formation in tumor-bearing mice and control mice by depleting neutrophils using the anti-Ly6G antibody 1A8. Administration of 1A8 significantly decreased levels of circulating neutrophils in both control mice [IgG vs. 1A8 (median [range]): 2.9 [1.9-4.4] × 103/μL vs. 0.3 [0.2-1.3] × 103/μL; P<0.05, n=4-5] and mice bearing human pancreatic tumors [IgG vs. 1A8 (median [range]): 6.7 [3.8-8.5] × 103/μL vs. 1.1 [1.0-3.7] × 103/μL; P<0.05, n=4-5]. Depletion of neutrophils decreased thrombus weight in tumor-bearing mice but not in control mice (Figure 5).
Effect of DNase I administration on thrombus size in mice bearing human pancreatic tumors
Thrombus weight was significantly increased in the vehicle treatment group of tumor-bearing mice compared with that of control mice [controls vs. tumor-bearing mice (median [range]): 15.7 [12.1-25.2] g vs. 27.25 [22.8-47.8] g; P<0.05, n=5-6]. We determined the effect of DNase I administration on the size of thrombi in mice bearing human pancreatic tumors. DNase I degrades both cfDNA and NET. We found that administration of DNase I significantly reduced thrombus weight in tumor-bearing mice but did not affect thrombus weight in control mice (Figure 6).
We observed a striking increase in neutrophils in the circulation of nude mice bearing human pancreatic tumors compared with controls. In addition, we found increased levels of plasma biomarkers that either increase neutrophil counts, such as G-CSF, or are biomarkers of activated neutrophils and NET, such as NE and H3Cit, in tumor-bearing mice compared with controls. We also observed increased levels of the neutrophil biomarker Ly6G and the NET biomarker H3Cit in thrombi from tumor-bearing mice. Finally, depletion of neutrophils reduced thrombus size in tumor-bearing mice but not in control mice. The lack of effect on thrombus size in neutrophil-depleted control mice is consistent with a previous study showing that neutrophil depletion did not reduce thrombus size in mice in an IVC stasis model.28 Taken together, these data suggest that neutrophils contribute to increased venous thrombosis observed in tumor-bearing mice.
We found that tumor-bearing mice have increased levels of mouse G-CSF and there was a significant positive correlation between levels of mouse G-CSF and neutrophil numbers. This suggests that increased levels of G-CSF drive the increase in neutrophil numbers. At present, we do not know the cellular source of G-CSF in tumor-bearing mice but this cytokine is normally expressed by endothelial cells, macrophages and immune cells.45 In support of this, mice with murine breast 4T1 tumors exhibit increased plasma levels of G-CSF and administration of an anti-G-CSF antibody reduces neutrophil counts.37 G-CSF level and neutrophil count may, therefore, represent novel biomarkers of VTE risk in cancer patients.
It has been hypothesized that G-CSF primes neutrophils to undergo NET formation in tumor-bearing mice.4637 In one study it was found that the percentage of H3Cit-positive neutrophils was increased in tumor-bearing mice compared with controls.37 Furthermore, neutrophils isolated from mice bearing murine 4T1 tumors treated with an anti-G-CSF antibody had significantly less NET formation compared with those from tumor-bearing mice treated with an isotype control.37 In addition, administration of recombinant G-CSF to mice bearing murine melanoma B16 tumors, which do not produce G-CSF, increased H3Cit in tumors in a PAD4-dependent manner.4746 We observed a large variation in the H3Cit/H3 ratio in thrombi from tumor-bearing mice which appears to be due to different levels of H3Cit in the thrombi. At present, we do not know the reason for this range of H3Cit in thrombi from tumor-bearing mice but it may reflect differences in G-CSF levels and the degree of neutrophil priming in the different tumor-bearing mice.
Plasma cfDNA and the NET biomarker, H3Cit, were increased in tumor-bearing mice compared with controls. Tumors are known to release cfDNA into the blood.48 In addition, we observed that BxPc-3 cells express PAD4 and therefore tumors may also contribute to the plasma levels of H3Cit. Thrombi from tumor-bearing mice also had increased levels cfDNA and H3Cit compared with thrombi from controls. Importantly, administration of DNase I reduced thrombus size in tumor-bearing mice but not in control mice. Similarly, a previous study showed that DNase I did not affect jugular vein occlusion times in control mice but prolonged the time to occlusion in 4T1 tumor-bearing mice.38 We observed that DNase I reduced thrombus size more effectively than depletion of neutrophils. It is possible that DNase I is more effective than neutrophil depletion because it digests not only NET but also cfDNA which may also be released by cancer cells and may enhance thrombosis by activating factor XII.4940
In the present study, we observed decreased numbers of red blood cells in thrombi from tumor-bearing mice compared with controls. This is consistent with our recent study that showed a decrease in red blood cell-rich areas and an increase in inflammatory cell-rich areas in thrombi from tumor-bearing mice compared with controls.10 In the current study, we did not observe an increase in neutrophils in thrombi of tumor-bearing mice because these cells were not preserved during the preparation of the samples for scanning electron microscopy. In addition, thrombi from tumor-bearing mice had a denser fibrin network with thinner fibrin fibers compared with thrombi from control mice. In vitro experiments showed that higher thrombin produces thrombi with a denser fibrin network with thinner fibrin fibers.44 In our model of cancer-associated thrombosis, it is likely that tumor-derived, tissue factor-positive extracellular vesicles increase the thrombin concentration in thrombi and this results in the formation of a denser fibrin network with thinner fibers. Indeed, in our previous study,10 we observed that thrombi from BxPc-3 tumor-bearing mice had increased levels of human tissue factor activity derived from extracellular vesicles released from BxPc-3 tumors. In addition, a previous study showed that extracellular vesicles bind to NET via a phosphatidylserine-histone interaction.50
In summary, we have demonstrated a contribution of neutrophils to venous thrombosis in a mouse model of pancreatic cancer-associated venous thrombosis. Our data, taken together with a clinical study showing an association between circulating H3Cit and VTE in pancreatic cancer, support the notion that neutrophils and NET formation enhance venous thrombosis in pancreatic cancer.
The Animal Surgery Core Laboratory of the McAllister Heart Institute at UNC performed the thrombosis experiments. This work was supported by grants from the National Institutes of Health (to YH T32 HL007149), the John C. Parker Professorship (to NM), the Jochnick Foundation (to CT and HW), the Hungarian National Research, Development and Innovation Office (NKFIH) (129528, to KK) and the Higher Education Institutional Excellence Programme of the Ministry of Human Capacities in Hungary for the Molecular Biology thematic programme of Semmelweis University (to KK).
- Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/105/1/218
- Received January 18, 2019.
- Accepted April 24, 2019.
- Timp JF, Braekkan SK, Versteeg HH, Cannegieter SC. Epidemiology of cancer-associated venous thrombosis. Blood. 2013; 122(10):1712-1723. PubMedhttps://doi.org/10.1182/blood-2013-04-460121Google Scholar
- Horsted F, West J, Grainge MJ. Risk of venous thromboembolism in patients with cancer: a systematic review and meta-analysis. PLoS Med. 2012; 9(7):e1001275. PubMedhttps://doi.org/10.1371/journal.pmed.1001275Google Scholar
- Hisada Y, Mackman N. Cancer-associated pathways and biomarkers of venous thrombosis. Blood. 2017; 130(13):1499-1506. PubMedhttps://doi.org/10.1182/blood-2017-03-743211Google Scholar
- Khorana AA, Francis CW, Menzies KE. Plasma tissue factor may be predictive of venous thromboembolism in pancreatic cancer. J Thromb Haemost. 2008; 6(11):1983-1985. PubMedhttps://doi.org/10.1111/j.1538-7836.2008.03156.xGoogle Scholar
- Thaler J, Ay C, Mackman N. Microparticle-associated tissue factor activity, venous thromboembolism and mortality in pancreatic, gastric, colorectal and brain cancer patients. J Thromb Haemost. 2012; 10(7):1363-1370. PubMedhttps://doi.org/10.1111/j.1538-7836.2012.04754.xGoogle Scholar
- Bharthuar A, Khorana AA, Hutson A. Circulating microparticle tissue factor, thromboembolism and survival in pancreaticobiliary cancers. Thromb Res. 2013; 132(2):180-184. Google Scholar
- Davila M, Amirkhosravi A, Coll E. Tissue factor-bearing microparticles derived from tumor cells: impact on coagulation activation. J Thromb Haemost. 2008; 6(9):1517-1524. PubMedhttps://doi.org/10.1111/j.1538-7836.2008.02987.xGoogle Scholar
- Wang JG, Geddings JE, Aleman MM. Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer. Blood. 2012; 119(23):5543-5552. PubMedhttps://doi.org/10.1182/blood-2012-01-402156Google Scholar
- Geddings JE, Hisada Y, Boulaftali Y. Tissue factor-positive tumor microvesicles activate platelets and enhance thrombosis in mice. J Thromb Haemost. 2016; 14(1):153-166. Google Scholar
- Hisada Y, Ay C, Auriemma AC, Cooley BC, Mackman N. Human pancreatic tumors grown in mice release tissue factor-positive microvesicles that increase venous clot size. J Thromb Haemost. 2017; 15(11):2208-2217. Google Scholar
- Khorana AA, Kuderer NM, Culakova E, Lyman GH, Francis CW. Development and validation of a predictive model for chemotherapy-associated thrombosis. Blood. 2008; 111(10):4902-4907. PubMedhttps://doi.org/10.1182/blood-2007-10-116327Google Scholar
- Pabinger I, Posch F. Flamethrowers: blood cells and cancer thrombosis risk. Hematology. 2014; 2014(1):410-417. PubMedhttps://doi.org/10.1182/asheducation-2014.1.410Google Scholar
- Blix K, Jensvoll H, Braekkan SK, Hansen JB. White blood cell count measured prior to cancer development is associated with future risk of venous thromboembolism--the Tromso study. PLoS One. 2013; 8(9):e73447. PubMedhttps://doi.org/10.1371/journal.pone.0073447Google Scholar
- Kasuga I, Makino S, Kiyokawa H, Katoh H, Ebihara Y, Ohyashiki K. Tumor-related leukocytosis is linked with poor prognosis in patients with lung carcinoma. Cancer. 2001; 92(9):2399-2405. PubMedhttps://doi.org/10.1002/1097-0142(20011101)92:9<2399::AID-CNCR1588>3.0.CO;2-WGoogle Scholar
- Esmon CT. Interactions between the innate immune and blood coagulation systems. Trends Immunol. 2004; 25(10):536-542. PubMedhttps://doi.org/10.1016/j.it.2004.08.003Google Scholar
- Engelmann B, Massberg S. Thrombosis as an intravascular effector of innate immunity. Nat Rev Immunol. 2013; 13(1):34-45. PubMedhttps://doi.org/10.1038/nri3345Google Scholar
- Osterud B. Tissue factor expression by monocytes: regulation and pathophysiological roles. Blood Coagul Fibrinolysis. 1998; 9(Suppl 1):S9-14. PubMedGoogle Scholar
- Massberg S, Grahl L, von Bruehl ML. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases. Nat Med. 2010; 16(8):887-896. PubMedhttps://doi.org/10.1038/nm.2184Google Scholar
- Fuchs TA, Brill A, Duerschmied D. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A. 2010; 107(36):15880-15885. PubMedhttps://doi.org/10.1073/pnas.1005743107Google Scholar
- Brill A, Fuchs TA, Savchenko AS. Neutrophil extracellular traps promote deep vein thrombosis in mice. J Thromb Haemost. 2012; 10(1):136-144. PubMedhttps://doi.org/10.1111/j.1538-7836.2011.04544.xGoogle Scholar
- Fuchs TA, Brill A, Wagner DD. Neutrophil extracellular trap (NET) impact on deep vein thrombosis. Arterioscler Thromb Vasc Biol. 2012; 32(8):1777-1783. PubMedhttps://doi.org/10.1161/ATVBAHA.111.242859Google Scholar
- Noubouossie DF, Reeves BN, Strahl BD, Key NS. Neutrophils: back in the thrombosis spotlight. Blood. 2019; 133(23):2529-2541. PubMedhttps://doi.org/10.1182/blood.2019000424Google Scholar
- Longstaff C, Varju I, Sotonyi P. Mechanical stability and fibrinolytic resistance of clots containing fibrin, DNA, and histones. J Biol Chem. 2013; 288(10):6946-6956. PubMedhttps://doi.org/10.1074/jbc.M112.404301Google Scholar
- Mangold A, Alias S, Scherz T. Coronary neutrophil extracellular trap burden and deoxyribonuclease activity in ST-elevation acute coronary syndrome are predictors of ST-segment resolution and infarct size. Circ Res. 2015; 116(7):1182-1192. PubMedhttps://doi.org/10.1161/CIRCRESAHA.116.304944Google Scholar
- Jimenez-Alcazar M, Rangaswamy C, Panda R. Host DNases prevent vascular occlusion by neutrophil extracellular traps. Science. 2017; 358(6367):1202-1206. PubMedhttps://doi.org/10.1126/science.aam8897Google Scholar
- von Bruhl ML, Stark K, Steinhart A. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. J Exp Med. 2012; 209(4):819-835. PubMedhttps://doi.org/10.1084/jem.20112322Google Scholar
- Meng H, Yalavarthi S, Kanthi Y. In vivo tole of neutrophil extracellular traps in antiphospholipid antibody-mediated venous thrombosis. Arthritis Rheumatol. 2017; 69(3):655-667. Google Scholar
- El-Sayed OM, Dewyer NA, Luke CE. Intact Toll-like receptor 9 signaling in neu-trophils modulates normal thrombogenesis in mice. J Vasc Surg. 2016; 64(5):1450-1458.e1. Google Scholar
- Kenny EF, Herzig A, Kruger R. Diverse stimuli engage different neutrophil extracellular trap pathways. Elife. 2017; 6Google Scholar
- Li P, Li M, Lindberg MR, Kennett MJ, Xiong N, Wang Y. PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps. J Exp Med. 2010; 207(9):1853-1862. PubMedhttps://doi.org/10.1084/jem.20100239Google Scholar
- Stadler SC, Vincent CT, Fedorov VD. Dysregulation of PAD4-mediated citrullination of nuclear GSK3beta activates TGF-beta signaling and induces epithelial-to-mesenchymal transition in breast cancer cells. Proc Natl Acad Sci U S A. 2013; 110(29):11851-11856. PubMedhttps://doi.org/10.1073/pnas.1308362110Google Scholar
- Wang Y, Li M, Stadler S. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol. 2009; 184(2):205-213. PubMedhttps://doi.org/10.1083/jcb.200806072Google Scholar
- Martinod K, Demers M, Fuchs TA. Neutrophil histone modification by peptidylarginine deiminase 4 is critical for deep vein thrombosis in mice. Proc Natl Acad Sci U S A. 2013; 110(21):8674-8679. PubMedhttps://doi.org/10.1073/pnas.1301059110Google Scholar
- Mauracher LM, Posch F, Martinod K. Citrullinated histone H3, a biomarker of neutrophil extracellular trap formation, predicts the risk of venous thromboembolism in cancer patients. J Thromb Haemost. 2018; 16(3):508-518. https://doi.org/10.1111/jth.13951Google Scholar
- Oklu R, Sheth RA, Wong KHK, Jahromi AH, Albadawi H. Neutrophil extracellular traps are increased in cancer patients but does not associate with venous thrombosis. Cardiovasc Diagn Ther. 2017; 7(Suppl 3):S140-S149. Google Scholar
- DuPre SA, Redelman D, Hunter KW. The mouse mammary carcinoma 4T1: characterization of the cellular landscape of primary tumours and metastatic tumour foci. Int J Exp Pathol. 2007; 88(5):351-360. PubMedhttps://doi.org/10.1111/j.1365-2613.2007.00539.xGoogle Scholar
- Demers M, Krause DS, Schatzberg D. Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis. Proc Natl Acad Sci U S A. 2012; 109(32):13076-13081. PubMedhttps://doi.org/10.1073/pnas.1200419109Google Scholar
- Leal AC, Mizurini DM, Gomes T. Tumor-derived exosomes induce the formation of neutrophil extracellular traps: implications for the establishment of cancer-associated thrombosis. Sci Rep. 2017; 7(1):6438. Google Scholar
- Sommeijer DW, van Oerle R, Reitsma PH. Analysis of blood coagulation in mice: pre-analytical conditions and evaluation of a home-made assay for thrombin-antithrombin complexes. Thromb J. 2005; 3:12. PubMedhttps://doi.org/10.1186/1477-9560-3-12Google Scholar
- Noubouossie DF, Whelihan MF, Yu YB. In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps. Blood. 2017; 129(8):1021-1029. PubMedhttps://doi.org/10.1182/blood-2016-06-722298Google Scholar
- Thalin C, Daleskog M, Goransson SP. Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma. Immunol Res. 2017; 65(3):706-712. Google Scholar
- Efron B, Tibshirani RJ. An Introduction to the Bootstrap. Taylor & Francis: New York, NY, USA; 1993. Google Scholar
- Nikolova N, Chai SH, Ivanova SD, Kolev K, Tenekedjiev K. Bootstrap Kuiper testing of the identity of 1D continuous distributions using fuzzy samples. International Journal of Computational Intelligence Systems. 2015; 8:63-75. Google Scholar
- Wolberg AS. Thrombin generation and fibrin clot structure. Blood Rev. 2007; 21(3):131-142. PubMedhttps://doi.org/10.1016/j.blre.2006.11.001Google Scholar
- von Vietinghoff S, Ley K. Homeostatic regulation of blood neutrophil counts. J Immunol. 2008; 181(8):5183-5188. PubMedhttps://doi.org/10.4049/jimmunol.181.8.5183Google Scholar
- Demers M, Wong SL, Martinod K. Priming of neutrophils toward NETosis promotes tumor growth. Oncoimmunology. 2016; 5(5):e1134073. Google Scholar
- Cedervall J, Zhang Y, Huang H. Neutrophil extracellular traps accumulate in peripheral blood vessels and compromise organ function in tumor-bearing animals. Cancer Res. 2015; 75(13):2653-2662. PubMedhttps://doi.org/10.1158/0008-5472.CAN-14-3299Google Scholar
- Schwarzenbach H, Hoon DS, Pantel K. Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer. 2011; 11(6):426-437. PubMedhttps://doi.org/10.1038/nrc3066Google Scholar
- Swystun LL, Mukherjee S, Liaw PC. Breast cancer chemotherapy induces the release of cell-free DNA, a novel procoagulant stimulus. J Thromb Haemost. 2011; 9(11):2313-2321. PubMedhttps://doi.org/10.1111/j.1538-7836.2011.04465.xGoogle Scholar
- Wang Y, Luo L, Braun OO. Neutrophil extracellular trap-microparticle complexes enhance thrombin generation via the intrinsic pathway of coagulation in mice. Sci Rep. 2018; 8(1):4020. Google Scholar