Neutrophil Extracellular Traps, Platelets and Endothelial Cells Cooperatively Contribute to Hypercoagulability in Non-Small Cell Lung Cancer

Abstract

Background

Thromboembolism is the second leading cause of death among patients with non-small cell lung cancer (NSCLC), but the precise mechanisms of thrombogenesis in NSCLC remain largely unknown. Our objectives were to evaluate the definitive role of neutrophil extracellular traps (NETs) in the hypercoagulability in NSCLC and to explore its interactions with platelets and endothelial cells (ECs).

Methods

The levels of NET markers in samples from 100 NSCLC patients and 30 healthy controls were measured by ELISA. NET formation was detected using immunofluorescence. Procoagulant activity was assessed based on purified coagulation complex, thrombin, clotting time, and fibrin formation assays.

Results

The plasma levels of NETs were increased in a stage-dependent manner in NSCLC patients and were markedly higher than those in controls. Neutrophils from NSCLC patients were more prone to form NETs, resulting in shortened coagulation time, significantly increased thrombin–antithrombin complexes and fibrin compared to controls. Moreover, NETs generation was mediated by High Mobility Group Box 1 from activated platelets in NSCLC patients. Conversely, NETs from NSCLC patients also induce phosphatidylserine exposure on platelets, leading to markedly enhanced procoagulant activity (PCA). Furthermore, NETs can damage endothelial cells and convert them to a procoagulant phenotype. The administration of NETs inhibitors (DNase I/activated protein C) could markedly diminish the PCA of NETs, activated platelets, and ECs.

Conclusion

Our results suggest that NETs contribute to hypercoagulability and may represent a potential therapeutic target to prevent cancer-associated thrombosis in NSCLC patients.

Keywords

neutrophil extracellular traps - non-small cell lung cancer - platelet - endothelial cells - procoagulant activity

Ethical Approval Statement

This study was approved by the Ethics Committee of Qingdao Municipal Hospital according to the Helsinki Declaration and written informed consent was obtained from all participants during enrollment.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author, upon reasonable request.

Authors' Contribution

J.H.L. and Y.Z. are responsible for the study design and experiment adjustment. D.X.T. and W.H.S. performed the experiments involved, drafted the manuscript, and conducted statistical analysis. Y.G., J.Y., and Y.L. collected the blood sample and clinical information of patients. All the authors read and approved the final manuscript.

Publication History

Received: 06 February 2024

Accepted: 28 November 2024

Accepted Manuscript online:
29 November 2024

Article published online:
27 December 2024

• References

• 1 Ay C, Pabinger I, Cohen AT. Cancer-associated venous thromboembolism: Burden, mechanisms, and management. Thromb Haemost 2017; 117 (02) 219-230
• 2 Farge D, Bounameaux H, Brenner B. et al. International clinical practice guidelines including guidance for direct oral anticoagulants in the treatment and prophylaxis of venous thromboembolism in patients with cancer. Lancet Oncol 2016; 17 (10) e452-e466
• 3 Siegel R, Ward E, Brawley O, Jemal A. Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA Cancer J Clin 2011; 61 (04) 212-236
• 4 Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019; 69 (01) 7-34
• 5 Connolly GC, Menapace L, Safadjou S, Francis CW, Khorana AA. Prevalence and clinical significance of incidental and clinically suspected venous thromboembolism in lung cancer patients. Clin Lung Cancer 2013; 14 (06) 713-718
• 6 Tagalakis V, Levi D, Agulnik JS, Cohen V, Kasymjanova G, Small D. High risk of deep vein thrombosis in patients with non-small cell lung cancer: a cohort study of 493 patients. J Thorac Oncol 2007; 2 (08) 729-734
• 7 Kadlec B, Skrickova J, Merta Z, Dusek L, Jarkovsky J. The incidence and predictors of thromboembolic events in patients with lung cancer. ScientificWorldJournal 2014; 2014: 125706
• 8 Elyamany G, Alzahrani AM, Bukhary E. Cancer-associated thrombosis: an overview. Clin Med Insights Oncol 2014; 8: 129-137
• 9 Li R, Hermann G, Baldini E. et al. Advanced nodal stage predicts venous thromboembolism in patients with locally advanced non-small cell lung cancer. Lung Cancer 2016; 96: 41-47
• 10 Shen Q, Dong X, Tang X, Zhou J. Risk factors and prognosis value of venous thromboembolism in patients with advanced non-small cell lung cancer: a case-control study. J Thorac Dis 2017; 9 (12) 5068-5074
• 11 Hisada Y, Mackman N. Cancer-associated pathways and biomarkers of venous thrombosis. Blood 2017; 130 (13) 1499-1506
• 12 Döring Y, Soehnlein O, Weber C. Neutrophil extracellular traps in atherosclerosis and atherothrombosis. Circ Res 2017; 120 (04) 736-743
• 13 von Brühl ML, Stark K, Steinhart A. et al. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. J Exp Med 2012; 209 (04) 819-835
• 14 Brinkmann V, Reichard U, Goosmann C. et al. Neutrophil extracellular traps kill bacteria. Science 2004; 303 (5663) 1532-1535
• 15 Demers M, Wagner DD. NETosis: a new factor in tumor progression and cancer-associated thrombosis. Semin Thromb Hemost 2014; 40 (03) 277-283
• 16 Cedervall J, Zhang Y, Huang H. et al. Neutrophil extracellular traps accumulate in peripheral blood vessels and compromise organ function in tumor bearing animals. Cancer Res 2015; 75 (13) 2653-2662
• 17 Wang Y, Liu F, Chen L. et al. Neutrophil extracellular traps (NETs) promote non-small cell lung cancer metastasis by suppressing lncRNA MIR503HG to activate the NF-κB/NLRP3 inflammasome pathway. Front Immunol 2022; 13: 867516
• 18 Lee J, Lee D, Lawler S, Kim Y. Role of neutrophil extracellular traps in regulation of lung cancer invasion and metastasis: structural insights from a computational model. PLOS Comput Biol 2021; 17 (02) e1008257
• 19 Rayes RF, Mouhanna JG, Nicolau I. et al. Primary tumors induce neutrophil extracellular traps with targetable metastasis promoting effects. JCI Insight 2019; 5 (16) e128008
• 20 Semeraro F, Ammollo CT, Morrissey JH. et al. Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4. Blood 2011; 118 (07) 1952-1961
• 21 Abdol Razak N, Elaskalani O, Metharom P. Pancreatic cancer-induced neutrophil extracellular traps: a potential contributor to cancer-associated thrombosis. Int J Mol Sci 2017; 18 (03) 487
• 22 Ma R, Bi Y, Kou J, Zhou J, Shi J. Enhanced procoagulant activity of platelets after chemotherapy in non-small cell lung cancer. Cancer Biol Ther 2017; 18 (08) 627-634
• 23 Saffarzadeh M, Juenemann C, Queisser MA. et al. Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones. PLoS One 2012; 7 (02) e32366
• 24 Detterbeck FC, Boffa DJ, Kim AW, Tanoue LT. The eighth edition lung cancer stage classification. Chest 2017; 151 (01) 193-203
• 25 Zhou P, Sun Q, Song G. et al. Radiomics features from perihematomal edema for prediction of prognosis in the patients with basal ganglia hemorrhage. Front Neurol 2022; 13: 982928
• 26 Zhao L, Bi Y, Kou J, Shi J, Piao D. Phosphatidylserine exposing-platelets and microparticles promote procoagulant activity in colon cancer patients. J Exp Clin Cancer Res 2016; 35: 54
• 27 Yao Z, Wang L, Wu X. et al. Enhanced procoagulant activity on blood cells after acute ischemic stroke. Transl Stroke Res 2017; 8 (01) 83-91
• 28 Stein PD, Beemath A, Meyers FA, Skaf E, Sanchez J, Olson RE. Incidence of venous thromboembolism in patients hospitalized with cancer. Am J Med 2006; 119 (01) 60-68
• 29 Blom JW, Doggen CJ, Osanto S, Rosendaal FR. Malignancies, prothrombotic mutations, and the risk of venous thrombosis. JAMA 2005; 293 (06) 715-722
• 30 Noubouossie DF, Whelihan MF, Yu YB. et al. In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps. Blood 2017; 129 (08) 1021-1029
• 31 Brill A, Fuchs TA, Savchenko AS. et al. Neutrophil extracellular traps promote deep vein thrombosis in mice. J Thromb Haemost 2012; 10 (01) 136-144
• 32 Martinod K, Wagner DD. Thrombosis: tangled up in NETs. Blood 2014; 123 (18) 2768-2776
• 33 Demers M, Wong SL, Martinod K. et al. Priming of neutrophils toward NETosis promotes tumor growth. OncoImmunology 2016; 5 (05) e1134073
• 34 Fuchs TA, Brill A, Duerschmied D. et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A 2010; 107 (36) 15880-15885
• 35 Clark SR, Ma AC, Tavener SA. et al. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med 2007; 13 (04) 463-469
• 36 Ma Y, Feng Q, Han B, Yu R, Jin Z. Elevated HMGB1 promotes the malignant progression and contributes to cisplatin resistance of non-small cell lung cancer. Hereditas 2023; 160 (01) 33
• 37 Wu XJ, Chen YY, Guo WW. et al. HMGB1 regulates SNAI1 during NSCLC metastasis, both directly, through transcriptional activation, and indirectly, in a RSF1-IT2-dependent manner. Mol Oncol 2020; 14 (06) 1348-1364
• 38 Li J, Tong D, Song B. et al. Inflammatory cytokines induce neutrophil extracellular traps interaction with activated platelets and endothelial cells exacerbate coagulation in moderate and severe essential hypertension. J Hypertens 2022; 40 (11) 2219-2229
• 39 Hudock KM, Collins MS, Imbrogno M. et al. Neutrophil extracellular traps activate IL-8 and IL-1 expression in human bronchial epithelia. Am J Physiol Lung Cell Mol Physiol 2020; 319 (01) L137-L147
• 40 Melero I, Villalba-Esparza M, Recalde-Zamacona B. et al. Neutrophil extracellular traps, local IL-8 expression, and cytotoxic T-lymphocyte response in the lungs of patients with fatal COVID-19. Chest 2022; 162 (05) 1006-1016
• 41 Guney N, Soydinc HO, Derin D. et al. Serum levels of intercellular adhesion molecule ICAM-1 and E-selectin in advanced stage non-small cell lung cancer. Med Oncol 2008; 25 (02) 194-200
• 42 Bombeli T, Karsan A, Tait JF, Harlan JM. Apoptotic vascular endothelial cells become procoagulant. Blood 1997; 89 (07) 2429-2442
• 43 Hisada Y, Grover SP, Maqsood A. et al. Neutrophils and neutrophil extracellular traps enhance venous thrombosis in mice bearing human pancreatic tumors. Haematologica 2020; 105 (01) 218-225
• 44 Dyer MR, Chen Q, Haldeman S. et al. Deep vein thrombosis in mice is regulated by platelet HMGB1 through release of neutrophil-extracellular traps and DNA. Sci Rep 2018; 8 (01) 2068
• 45 Thomson AH. Human recombinant DNase in cystic fibrosis. J R Soc Med 1995; 88 (Suppl. 25) 24-29
• 46 Várady CBS, Oliveira AC, Monteiro RQ, Gomes T. Recombinant human DNase I for the treatment of cancer-associated thrombosis: a pre-clinical study. Thromb Res 2021; 203: 131-137
• 47 Colón DF, Wanderley CW, Franchin M. et al. Neutrophil extracellular traps (NETs) exacerbate severity of infant sepsis. Crit Care 2019; 23 (01) 113