Phosphotidylserine exposure and neutrophil extracellular traps enhance procoagulant activity in patients with inflammatory bowel disease

Summary

Inflammatory bowel disease (IBD)-associated thromboembolic event often lacks precise aetiology. The aim of this study was to investigate the contribution of phosphatidylserine (PS) exposure and neutrophil extracellular traps (NETs) towards the hypercoagulable state in IBD. We demonstrated that the levels of PS exposed MPs and the sources of MP-origin, platelets, erythrocytes, leukocytes and cultured endothelial cells (ECs) were higher in IBD groups than in healthy controls using flow cytometry and confocal microscopy. Wright-Giemsa and immunofluorescence staining demonstrated that the elevated NETs were released by activated IBD neutrophils or by control neutrophils treated with IBD sera obtained from patients with the active disease. MPs and MP-origin cells in IBD groups, especially in active stage, markedly shortened coagulation time and had increased levels of fibrin, thrombin and FXa production as assessed by coagulation function assays. Importantly, we found that on stimulated ECs, PS rich membranes provided binding sites for FXa and FVa, promoting fibrin formation while TNF blockage or IgG depletion attenuated this effect. Treatment of control neutrophils with TNF and isolated IgG from PR3-ANCA-positive active IBD patients also resulted in the release of NETs. Blockade of PS with lactadherin prolonged coagulation time, decreased fibrin formation to control levels, and inhibited the procoagulant enzymes production in the MPs and MP-origin cells. NET cleavage by DNase I partly decreased PCA in IBD or stimulated neutrophils. Our study reveals a previously unrecognised link between hypercoagulable state and PS exposure or NETs, and may further explain the epidemiological association of thrombosis within IBD patients.

Keywords

IBD hypercoagulable disorders - phosphatidylserine - Neutrophil Extracellular Traps - procoagulant activity

^* Equal contribution.

• References

• 1 Grainge MJ, West J, Card TR. Venous thromboembolism during active disease and remission in inflammatory bowel disease: a cohort study. Lancet 2010; 375: 657-663.
• 2 Tan VP, Chung A, Yan BP. et al. Venous and arterial disease in inflammatory bowel disease. J Gastroenterol Hepatol 2013; 28: 1095-1113.
• 3 Twig G, Zandman-Goddard G, Szyper-Kravitz M. et al. Systemic thromboembolism in inflammatory bowel disease: mechanisms and clinical applications. Ann NY Acad Sci 2005; 1051: 166-173.
• 4 Deutschmann A, Schlagenhauf A, Leschnik B. et al. Increased procoagulant function of microparticles in pediatric inflammatory bowel disease: role in increased thrombin generation. J Pediatr Gastroenterol Nutr 2013; 56: 401-407.
• 5 Scaldaferri F, Sans M, Vetrano S. et al. Crucial role of the protein C pathway in governing microvascular inflammation in inflammatory bowel disease. J Clin Invest 2007; 117: 1951-1960.
• 6 Undas A, Owczarek D, Gissel M. et al. Activated factor XI and tissue factor in inflammatory bowel disease. Inflamm Bowel Dis 2010; 16: 1447-1478.
• 7 Chamouard P, Grunebaum L, Wiesel ML. et al. Prothrombin fragment 1 + 2 and thrombin-antithrombin III complex as markers of activation of blood coagulation in inflammatory bowel diseases. Eur J Gastroenterol Hepatol 1995; 07: 1183-1188.
• 8 Smith CJ, Haire WD, Kaufman SS. et al. Determination of prothrombin activation fragments in young patients with inflammatory bowel disease. Am J Gastroenterol 1996; 91: 1221-1225.
• 9 Owczarek D, Cibor D, Salapa K. et al. Reduced plasma fibrin clot permeability and susceptibility to lysis in patients with inflammatory bowel disease: a novel prothrombotic mechanism. Inflamm Bowel Dis 2013; 19: 2616-2624.
• 10 Gao C, Xie R, Yu C. et al. Procoagulant activity of erythrocytes and platelets through phosphatidylserine exposure and microparticles release in patients with nephrotic syndrome. Thromb Haemost 2012; 107: 681-689.
• 11 Tan X, Shi J, Fu Y. et al. Role of erythrocytes and platelets in the hypercoagulable status in polycythemia vera through phosphatidylserine exposure and micro-particle generation. Thromb Haemost 2013; 109: 1025-1032.
• 12 Trappenburg MC, van Schilfgaarde M, Marchetti M. et al. Elevated procoagulant microparticles expressing endothelial and platelet markers in essential thrombocythemia. Haematologica 2009; 94: 911-918.
• 13 Morel O, Jesel L, Freyssinet JM. et al. Cellular mechanisms underlying the formation of circulating microparticles. Arterioscler Thromb Vasc Biol 2011; 31: 15-26.
• 14 Zwaal RF, Schroit AJ. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 1997; 89: 1121-1132.
• 15 Morel O, Toti F, Hugel B. et al. Procoagulant microparticles: disrupting the vascular homeostasis equation?. Arterioscler Thromb Vasc Biol 2006; 26: 2594-2604.
• 16 Yan SL, Russell J, Granger DN. Platelet activation and platelet-leukocyte aggregation elicited in experimental colitis are mediated by interleukin-6. Inflamm Bowel Dis 2014; 20: 353-362.
• 17 Yoshida H, Granger DN. Inflammatory bowel disease: a paradigm for the link between coagulation and inflammation. Inflamm Bowel Dis 2009; 15: 1245-1255.
• 18 Brinkmann V, Reichard U, Goosmann C. et al. Neutrophil extracellular traps kill bacteria. Science 2004; 303: 1532-1535.
• 19 Martinod K, Wagner DD. Thrombosis: tangled up in NETs. Blood 2014; 123: 2768-2776.
• 20 Clark SR, Ma AC, Tavener SA. et al. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med 2007; 13: 463-469.
• 21 Bennike TB, Carlsen TG, Ellingsen T. et al. Neutrophil extracellular traps in ul-cerative Colitis: A proteome analysis of intestinal biopsies. Inflamm Bowel Dis 2015; 21: 2052-2067.
• 22 Yan SL, Russell J, Harris NR. et al. Platelet abnormalities during colonic inflammation. Inflamm Bowel Dis 2013; 19: 1245-1253.
• 23 Danese S, Papa A, Saibeni S. et al. Inflammation and coagulation in inflammatory bowel disease: The clot thickens. Am J Gastroenterol 2007; 102: 174-186.
• 24 Campbell RA, Overmyer KA, Selzman CH. et al. Contributions of extravascular and intravascular cells to fibrin network formation, structure, and stability. Blood 2009; 114: 4886-4896.
• 25 Esmon CT. Targeting factor Xa and thrombin: impact on coagulation and beyond. Thromb Haemost 2014; 111: 625-633.
• 26 Murdoch T, O’Donnell S, Silverberg MS. et al. Biomarkers as potential treatment targets in inflammatory bowel disease: A systematic review. Can J Gastroenterol Hepatol 2015; 29: 203-208.
• 27 Slebioda TJ, Kmiec Z. Tumour necrosis factor superfamily members in the pathogenesis of inflammatory bowel disease. Mediators Inflamm 2014; 2014: 325129.
• 28 Arias-Loste MT, Bonilla G, Moraleja I. et al. Presence of anti-proteinase 3 anti-neutrophil Cytoplasmic antibodies (anti-PR3 ANCA) as serologic markers in inflammatory bowel disease. Clin Rev Allergy Immunol 2013; 45: 109-116.
• 29 Kessenbrock K, Krumbholz M, Schonermarck U. et al. Netting neutrophils in autoimmune small-vessel vasculitis. Nat Med 2009; 15: 623-625.
• 30 Neuman MG. Immune dysfunction in inflammatory bowel disease. Transl Pes 2007; 149173-149186.
• 31 Allocca M, Fiorino G, Vermeire S. et al. Blockade of lymphocyte trafficking in inflammatory bowel diseases therapy: importance of specificity of endothelial target. Expert Rev Clin Immunol 2014; 10: 885-895.
• 32 Collins CE, Cahill MR, Newland AC. et al. Platelets circulate in an activated state in inflammatory bowel disease. Gastroenterology 1994; 106: 840-845.
• 33 Zilberman L, Rogowski O, Rozenblat M. et al. Inflammation-related erythrocyte aggregation in patients with inflammatory bowel disease. Dig Dis Sci 2005; 50: 677-683.
• 34 Vowinkel T, Wood KC, Stokes KY. et al. Mechanisms of platelet and leukocyte recruitment in experimental colitis. Am J Physiol Gastrointest Liver Physiol 2007; 293: G1054-G1060.
• 35 Leonetti D, Reimund JM, Tesse A. et al. Circulating microparticles from Crohn’s disease patients cause endothelial and vascular dysfunctions. PLoS One 2013; 08: e73088.
• 36 Macedo-Ribeiro S, Bode W, Huber R. et al. Crystal structures of the membrane-binding C2 domain of human coagulation factor V. Nature 1999; 402: 434-439.
• 37 Shi J, Gilbert GE. Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid-binding sites. Blood 2003; 101: 2628-2636.
• 38 Massberg S, Grahl L, von Bruehl ML. et al. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases. Nat Med 2010; 16: 887-896.
• 39 Fuchs TA, Brill A, Duerschmied D. et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci USA 2010; 107: 15880-15885.
• 40 Malickova K, Duricova D, Bortlik M. et al. Impaired deoxyribonuclease I activity in patients with inflammatory bowel diseases. Autoimmune Dis 2011; 2011: 945861-945865.
• 41 Jimenez-Alcazar M, Napirei M, Panda R. et al. Impaired DNase1-mediated degradation of neutrophil extracellular traps is associated with acute thrombotic microangiopathies. J Thromb Haemost 2015; 13: 732-742.
• 42 Brill A, Fuchs TA, Savchenko AS. et al. Neutrophil extracellular traps promote deep vein thrombosis in mice. J Thromb Haemost 2012; 10: 136-144.
• 43 Cooper PR, Palmer LJ, Chapple IL. Neutrophil extracellular traps as a new paradigm in innate immunity: friend or foe?. Periodontology 2000 2013; 63: 165-197.
• 44 Abraham C, Medzhitov R. Interactions between the host innate immune system and microbes in inflammatory bowel disease. Gastroenterology 2011; 140: 1729-1737.
• 45 Komatsu M, Kobayashi D, Saito K. et al. Tumor necrosis factor-alpha in serum of patients with inflammatory bowel disease as measured by a highly sensitive immuno-PCR. Clin Chem 2001; 47: 1297-1301.
• 46 Hong Y, Eleftheriou D, Hussain AA. et al. Anti-neutrophil cytoplasmic antibodies stimulate release of neutrophil microparticles. J Am Soc Nephrol 2012; 23: 49-62.
• 47 Borensztajn K, Peppelenbosch MP, Spek CA. Coagulation factor Xa signaling: the link between coagulation and inflammatory bowel disease?. Trends Pharmacol Sci 2009; 30: 8-16.
• 48 Cenac N, Coelho AM, Nguyen C. et al. Induction of intestinal inflammation in mouse by activation of proteinase-activated receptor-2. Am J Pathol 2002; 161: 1903-1915.
• 49 Vergnolle N, Cellars L, Mencarelli A. et al. A role for proteinase-activated recep-tor-1 in inflammatory bowel diseases. J Clin Invest 2004; 114: 1444-1456.
• 50 Semeraro F, Ammollo CT, Esmon NL. et al. Histones induce phosphatidylserine exposure and a procoagulant phenotype in human red blood cells. J Thromb Haemost 2014; 12: 1697-1702.
• 51 Carty E, Rampton DS, Schneider H. et al. Lack of efficacy of ridogrel, a thromb-oxane synthase inhibitor, in a placebo-controlled, double-blind, multi-centre clinical trial in active Crohn’s disease. Aliment Pharmacol Ther 2001; 15: 1323-1329.
• 52 Shak S, Capon DJ, Hellmiss R. et al. Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum. Proc Natl Acad Sci USA 1990; 87: 9188-9192.
• 53 Davis Jr. JC, Manzi S. et al. Recombinant human Dnase I (rhDNase) in patients with lupus nephritis. Lupus 1999; 08: 68-76.
• 54 Papayannopoulos V, Staab D, Zychlinsky A. Neutrophil elastase enhances sputum solubilisation in cystic fibrosis patients receiving DNase therapy. PLoS One 2011; 06: e28526.