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From neutrophil extracellular traps release to thrombosis: an overshooting host‐defense mechanism?

2011; Elsevier BV; Volume: 9; Issue: 9 Linguagem: Inglês

10.1111/j.1538-7836.2011.04425.x

1538-7933

Julian Ilcheff Borissoff, Hugo Ten Cate,

Blood disorders and treatments

Innate immunity and blood coagulation are evolutionary entangled in an intricate network of molecular and cellular interactions, thus forming an integral part of the host‐defense system [1Delvaeye M. Conway E.M. Coagulation and innate immune responses: can we view them separately?.Blood. 2009; 114: 2367-74Crossref PubMed Scopus (210) Google Scholar]. Polymorphonuclear cells, in particular neutrophils, are essential for the primary innate immune response against local and systemic infections or tissue injury [2Nathan C. Neutrophils and immunity: challenges and opportunities.Nat Rev Immunol. 2006; 6: 173-82Crossref PubMed Scopus (2072) Google Scholar], but are also major cellular mediators supporting inflammation–coagulation interactions [3Ruf W. Ruggeri Z.M. Neutrophils release brakes of coagulation.Nat Med. 2010; 16: 851-2Crossref PubMed Scopus (37) Google Scholar]. Upon inflammation, multiple chemotactic stimuli (cytokines, chemokines, etc.) are released to promote neutrophil activation, extravazation and migration towards the infectious foci. Neutrophils exert their bactericidal capacity by phagocytizing the disseminating pathogens, releasing numerous cytotoxic mediators and promoting cell death. Scientific evidence suggests that activation of blood coagulation, leading to subsequent fibrin deposition at the sites of inflammation, is an additional protective mechanism serving against microbial dissemination [4Degen J.L. Bugge T.H. Goguen J.D. Fibrin and fibrinolysis in infection and host defense.J Thromb Haemost. 2007; 5: 24-31Crossref PubMed Scopus (150) Google Scholar, 5Massberg S. Grahl L. von Bruehl M.L. Manukyan D. Pfeiler S. Goosmann C. Brinkmann V. Lorenz M. Bidzhekov K. Khandagale A.B. Konrad I. Kennerknecht E. Reges K. Holdenrieder S. Braun S. Reinhardt C. Spannagl M. Preissner K.T. Engelmann B. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases.Nat Med. 2010; 16: 887-96Crossref PubMed Scopus (797) Google Scholar]. However, a persisting neutrophil hyper‐responsiveness may trigger a pronounced oxidative stress and proteolysis through an enhanced synthesis of enzymatic proteins such as myeloperoxidase (MPO), neutrophil elastase and cathepsin G. These molecular mechanisms can result in the inactivation and degradation of important anti‐coagulant proteins such as antithrombin, thrombomodulin (TM), protein C and tissue factor pathway inhibitor (TFPI), thus inducing a strong inflammation‐driven local or systemic pro‐coagulant response [6Levi M. van der Poll T. Buller H.R. Bidirectional relation between inflammation and coagulation.Circulation. 2004; 109: 2698-704Crossref PubMed Scopus (691) Google Scholar]. Persisting inflammation may trigger an over‐reactive host defense response over time, thus disrupting the immune balance, contributing to tissue injury and thrombosis [5Massberg S. Grahl L. von Bruehl M.L. Manukyan D. Pfeiler S. Goosmann C. Brinkmann V. Lorenz M. Bidzhekov K. Khandagale A.B. Konrad I. Kennerknecht E. Reges K. Holdenrieder S. Braun S. Reinhardt C. Spannagl M. Preissner K.T. Engelmann B. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases.Nat Med. 2010; 16: 887-96Crossref PubMed Scopus (797) Google Scholar, 7Borissoff J.I. Spronk H.M. ten Cate H. The hemostatic system as a modulator of atherosclerosis.N Engl J Med. 2011; 364: 1746-60Crossref PubMed Scopus (420) Google Scholar]. In fact, neutrophils have been indicated to play a role in the pathophysiology of several pathologic conditions including venous thrombosis, acute coronary syndromes and stroke [8Segel G.B. Halterman M.W. Lichtman M.A. The paradox of the neutrophil’s role in tissue injury.J Leukoc Biol. 2011; 89: 359-72Crossref PubMed Scopus (219) Google Scholar, 9Baetta R. Corsini A. Role of polymorphonuclear neutrophils in atherosclerosis: current state and future perspectives.Atherosclerosis. 2010; 210: 1-13Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 10Horne B.D. Anderson J.L. John J.M. Weaver A. Bair T.L. Jensen K.R. Renlund D.G. Muhlestein J.B. Which white blood cell subtypes predict increased cardiovascular risk?.J Am Coll Cardiol. 2005; 45: 1638-43Crossref PubMed Scopus (716) Google Scholar, 11Tzoulaki I. Murray G.D. Lee A.J. Rumley A. Lowe G.D. Fowkes F.G. Relative value of inflammatory, hemostatic, and rheological factors for incident myocardial infarction and stroke: the Edinburgh Artery Study.Circulation. 2007; 115: 2119-27Crossref PubMed Scopus (193) Google Scholar, 12Brown K.A. Brain S.D. Pearson J.D. Edgeworth J.D. Lewis S.M. Treacher D.F. Neutrophils in development of multiple organ failure in sepsis.Lancet. 2006; 368: 157-69Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 13Falanga A. Marchetti M. Barbui T. Smith C.W. Pathogenesis of thrombosis in essential thrombocythemia and polycythemia vera: the role of neutrophils.Semin Hematol. 2005; 42: 239-47Crossref PubMed Scopus (74) Google Scholar]. Besides the abundant amount of reactive oxygen species (ROS) that neutrophils can generate [14Geering B. Simon H.U. Peculiarities of cell death mechanisms in neutrophils.Cell Death Differ. 2011; 10.1038/cdd.2011.75Crossref Scopus (202) Google Scholar], substantial evidence has emerged over the years showing the potential of neutrophils to kill pathogens also via the release of so‐called neutrophil extracellular traps (NETs) [15Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-5Crossref PubMed Scopus (6095) Google Scholar, 16Yousefi S. Mihalache C. Kozlowski E. Schmid I. Simon H.U. Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps.Cell Death Differ. 2009; 16: 1438-44Crossref PubMed Scopus (607) Google Scholar]. The latter represent extracellular chromatin threads with potent cytotoxic effects, comprised of both histones and granular proteins. Recent studies have shown that NETs formation is a well‐regulated process [17Papayannopoulos V. Metzler K.D. Hakkim A. Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.J Cell Biol. 2010; 191: 677-91Crossref PubMed Scopus (1216) Google Scholar], and not only part of a cell‐death program [18Fuchs T.A. Abed U. Goosmann C. Hurwitz R. Schulze I. Wahn V. Weinrauch Y. Brinkmann V. Zychlinsky A. Novel cell death program leads to neutrophil extracellular traps.J Cell Biol. 2007; 176: 231-41Crossref PubMed Scopus (2162) Google Scholar]. During activation, neutrophil elastase and MPO are released from the azurophilic granules and translocate to the nucleus, where they act in synergy to promote chromatin decondensation and histone degradation [17Papayannopoulos V. Metzler K.D. Hakkim A. Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.J Cell Biol. 2010; 191: 677-91Crossref PubMed Scopus (1216) Google Scholar]. Furthermore, NETs establish a new interface between inflammation and blood coagulation (Figure 1). NETs are able to entrap and kill bacteria, but also to induce numerous pro‐thrombotic effects such as: Both platelets and neutrophils have the potential to activate each other through different molecular mechanisms. Once activated, platelets and neutrophils interact mainly via P‐selectin, β2‐ and β3‐integrin receptors. Besides the classic pathways of activation involving cytokines [e.g. interleukin (IL)‐1β and tumor necrosis factor‐alpha (TNF‐α)], growth factors (e.g. G‐CSF), the complement system (e.g. C5a), etc., neutrophils can be activated by platelets via binding to the TREM‐1 receptor located on the neutrophil surface. The latter can evoke a massive oxidative burst and IL‐8 release [19Haselmayer P. Grosse‐Hovest L. von Landenberg P. Schild H. Radsak M.P. TREM‐1 ligand expression on platelets enhances neutrophil activation.Blood. 2007; 110: 1029-35Crossref PubMed Scopus (164) Google Scholar], thus contributing to the recruitment of even more neutrophils to the site of tissue injury/inflammation. The secretion of neutrophil‐derived proteases (neutrophil elastase and matrix metalloproteinases) can also degrade the proteoglycans; hence, von Willebrand factor (VWF) gets exposed to promote platelet adhesion [20Wohner N. Keresztes Z. Sotonyi P. Szabo L. Komorowicz E. Machovich R. Kolev K. Neutrophil granulocyte‐dependent proteolysis enhances platelet adhesion to the arterial wall under high‐shear flow.J Thromb Haemost. 2010; 8: 1624-31Crossref PubMed Scopus (22) Google Scholar]. During severe inflammatory conditions such as sepsis, lipopolysaccharide (LPS) binds to toll‐like receptor 4 (TLR4) on platelets, hence stimulating neutrophil activation and NETs formation [21Clark S.R. Ma A.C. Tavener S.A. McDonald B. Goodarzi Z. Kelly M.M. Patel K.D. Chakrabarti S. McAvoy E. Sinclair G.D. Keys E.M. Allen‐Vercoe E. Devinney R. Doig C.J. Green F.H. Kubes P. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood.Nat Med. 2007; 13: 463-9Crossref PubMed Scopus (1589) Google Scholar]. TLR2 and TLR4 receptors are also involved in the histone‐induced platelet activation and increased platelet pro‐coagulant activity. Histones trigger platelet aggregation, P‐selectin, phosphatidylserine and factor V/Va expression [22Semeraro F. Ammollo C.T. Morrissey J.H. Dale G.L. Friese P. Esmon N.L. Esmon C.T. Extracellular histones promote thrombin generation through platelet‐dependent mechanisms: involvement of platelet TLR2 and 4.Blood. 2011; Crossref Scopus (565) Google Scholar]. Of note, experimental studies have shown that NETs can serve as a surface for red blood cell and platelet adhesion, platelet activation and aggregation, thus resulting in thrombosis [23Fuchs T.A. Brill A. Duerschmied D. Schatzberg D. Monestier M. Myers Jr, D.D. Wrobleski S.K. Wakefield T.W. Hartwig J.H. Wagner D.D. Extracellular DNA traps promote thrombosis.Proc Natl Acad Sci U S A. 2010; 107: 15880-5Crossref PubMed Scopus (1556) Google Scholar]. Neutrophils can actively participate in clotting through expression of tissue factor (TF) [24de Waard V. Hansen H.R. Spronk H.H. Timmerman J.J. Pannekoek H. Florquin S. Reitsma P.H. ten Cate H. Differential expression of tissue factor mRNA and protein expression in murine sepsis. The role of the granulocyte revisited.Thromb Haemost. 2006; 95: 348-53Crossref PubMed Scopus (34) Google Scholar]. Neutrophil‐derived externalized nucleosomes can trigger both TF‐ and contact activation pathways of coagulation [5Massberg S. Grahl L. von Bruehl M.L. Manukyan D. Pfeiler S. Goosmann C. Brinkmann V. Lorenz M. Bidzhekov K. Khandagale A.B. Konrad I. Kennerknecht E. Reges K. Holdenrieder S. Braun S. Reinhardt C. Spannagl M. Preissner K.T. Engelmann B. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases.Nat Med. 2010; 16: 887-96Crossref PubMed Scopus (797) Google Scholar]. These pro‐coagulant actions can be exerted either through the formation of NETs or through direct effects of the neutrophil serine proteases (neutrophil elastase, cathepsin G) on blood coagulation, e.g. proteolytic cleavage of C1 inhibitor, TFPI, etc. [5Massberg S. Grahl L. von Bruehl M.L. Manukyan D. Pfeiler S. Goosmann C. Brinkmann V. Lorenz M. Bidzhekov K. Khandagale A.B. Konrad I. Kennerknecht E. Reges K. Holdenrieder S. Braun S. Reinhardt C. Spannagl M. Preissner K.T. Engelmann B. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases.Nat Med. 2010; 16: 887-96Crossref PubMed Scopus (797) Google Scholar, 25Brower M.S. Harpel P.C. Proteolytic cleavage and inactivation of alpha 2‐plasmin inhibitor and C1 inactivator by human polymorphonuclear leukocyte elastase.J Biol Chem. 1982; 257: 9849-54Abstract Full Text PDF PubMed Google Scholar]. Besides the pro‐coagulant nature of cathepsin G, the latter can also function as a potent platelet activator [26Ferrer‐Lopez P. Renesto P. Schattner M. Bassot S. Laurent P. Chignard M. Activation of human platelets by C5a‐stimulated neutrophils: a role for cathepsin G.Am J Physiol. 1990; 258: C1100-7Crossref PubMed Google Scholar, 27LaRosa C.A. Rohrer M.J. Benoit S.E. Rodino L.J. Barnard M.R. Michelson A.D. Human neutrophil cathepsin G is a potent platelet activator.J Vasc Surg. 1994; 19: 306-18Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 28Goel M.S. Diamond S.L. Neutrophil cathepsin G promotes prothrombinase and fibrin formation under flow conditions by activating fibrinogen‐adherent platelets.J Biol Chem. 2003; 278: 9458-63Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar]. Furthermore, externalized nucleosomes have the potential to activate factor (F)XII directly most probably via the negatively charged surfaces of the nucleic acids rather than the histone components [5Massberg S. Grahl L. von Bruehl M.L. Manukyan D. Pfeiler S. Goosmann C. Brinkmann V. Lorenz M. Bidzhekov K. Khandagale A.B. Konrad I. Kennerknecht E. Reges K. Holdenrieder S. Braun S. Reinhardt C. Spannagl M. Preissner K.T. Engelmann B. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases.Nat Med. 2010; 16: 887-96Crossref PubMed Scopus (797) Google Scholar, 29Kannemeier C. Shibamiya A. Nakazawa F. Trusheim H. Ruppert C. Markart P. Song Y. Tzima E. Kennerknecht E. Niepmann M. von Bruehl M.L. Sedding D. Massberg S. Gunther A. Engelmann B. Preissner K.T. Extracellular RNA constitutes a natural procoagulant cofactor in blood coagulation.Proc Natl Acad Sci USA. 2007; 104: 6388-93Crossref PubMed Scopus (410) Google Scholar]. Histones accelerate thrombin formation in platelet‐rich plasma possibly via platelet‐derived polyphosphates [22Semeraro F. Ammollo C.T. Morrissey J.H. Dale G.L. Friese P. Esmon N.L. Esmon C.T. Extracellular histones promote thrombin generation through platelet‐dependent mechanisms: involvement of platelet TLR2 and 4.Blood. 2011; Crossref Scopus (565) Google Scholar], but this process seems independent of FXII as it has been demonstrated previously [30Muller F. Mutch N.J. Schenk W.A. Smith S.A. Esterl L. Spronk H.M. Schmidbauer S. Gahl W.A. Morrissey J.H. Renne T. Platelet polyphosphates are proinflammatory and procoagulant mediators in vivo.Cell. 2009; 139: 1143-56Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar]. In vitro evidence suggests that neutrophil elastase can inactivate both antithrombin and TFPI [31Higuchi D.A. Wun T.C. Likert K.M. Broze Jr, G.J. The effect of leukocyte elastase on tissue factor pathway inhibitor.Blood. 1992; 79: 1712-9Crossref PubMed Google Scholar, 32Jordan R.E. Kilpatrick J. Nelson R.M. Heparin promotes the inactivation of antithrombin by neutrophil elastase.Science. 1987; 237: 777-9Crossref PubMed Scopus (88) Google Scholar]. Neutrophil‐derived externalized nucleosomes promote recruitment and proteolytic deactivation of TFPI in vivo, mediated by both the action of the extracellular DNA fragments but also the serine proteases [5Massberg S. Grahl L. von Bruehl M.L. Manukyan D. Pfeiler S. Goosmann C. Brinkmann V. Lorenz M. Bidzhekov K. Khandagale A.B. Konrad I. Kennerknecht E. Reges K. Holdenrieder S. Braun S. Reinhardt C. Spannagl M. Preissner K.T. Engelmann B. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases.Nat Med. 2010; 16: 887-96Crossref PubMed Scopus (797) Google Scholar]. TM is a cellular transmembrane protein, predominantly expressed on vascular endothelium, but also found in other compartments of the vasculature [33Borissoff J.I. Heeneman S. Kilinc E. Kassak P. van Oerle R. Winckers K. Govers‐Riemslag J.W. Hamulyak K. Hackeng T.M. Daemen M.J. ten Cate H. Spronk H.M. Early atherosclerosis exhibits an enhanced procoagulant state.Circulation. 2010; 122: 821-30Crossref PubMed Scopus (159) Google Scholar]. TM protects against neutrophil‐induced tissue damage by attenuating several molecular pathways, which mediate pro‐inflammatory effects [34Conway E.M. van de Wouwer M. Pollefeyt S. Jurk K. van Aken H. De Vriese A. Weitz J.I. Weiler H. Hellings P.W. Schaeffer P. Herbert J.M. Collen D. Theilmeier G. The lectin‐like domain of thrombomodulin confers protection from neutrophil‐mediated tissue damage by suppressing adhesion molecule expression via nuclear factor kappaB and mitogen‐activated protein kinase pathways.J Exp Med. 2002; 196: 565-77Crossref PubMed Scopus (304) Google Scholar], whereas activated protein C (APC) can cleave histones reducing histone‐induced cytotoxicity [35Xu J. Zhang X. Pelayo R. Monestier M. Ammollo C.T. Semeraro F. Taylor F.B. Esmon N.L. Lupu F. Esmon C.T. Extracellular histones are major mediators of death in sepsis.Nat Med. 2009; 15: 1318-21Crossref PubMed Scopus (1045) Google Scholar]. In this issue of the Journal of Thrombosis and Hemostasis, Ammollo et al. [36Ammollo C.T. Semeraro F. Xu J. Esmon N.L. Esmon C.T. Extracellular histones increase plasma thrombin generation by impairing thrombomodulin‐dependent protein C activation.J Thromb Haemost. 2011; 9: 1795-803Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar] present novel mechanistic data on the role of histones in modulating the anti‐coagulant protein C pathway. Using the calibrated automated thrombography (CAT) method, the authors show that histones can induce a concentration‐dependent increase in thrombin generation, which appears to be a result of a direct interaction with TM and/or the Gla‐domain of protein C, thus resulting in an impairment of the TM‐dependent protein C activation. This interaction may have profound consequences in conditions such as sepsis [37Zeerleder S. Zwart B. Wuillemin W.A. Aarden L.A. Groeneveld A.B. Caliezi C. van Nieuwenhuijze A.E. van Mierlo G.J. Eerenberg A.J. Lammle B. Hack C.E. Elevated nucleosome levels in systemic inflammation and sepsis.Crit Care Med. 2003; 31: 1947-51Crossref PubMed Scopus (199) Google Scholar], where a deficit of protein C, hence the APC generating potential, occurs in the course of disseminated intravascular coagulation. Experimental studies have clearly shown that diminished PC increases mortality in primates, whereas the administration of (A) PC could prevent mortality from sepsis in the same model [38Taylor F.B. Kinasewitz G. Activated protein C in sepsis.J Thromb Haemost. 2004; 2: 708-17Crossref PubMed Scopus (22) Google Scholar]. While the protective effects of recombinant APC in humans with severe sepsis are still equivocal [39Marti‐Carvajal A.J. Sola I. Lathyris D. Cardona A.F. Human recombinant activated protein C for severe sepsis.Cochrane Database Syst Rev. 2011; 4: CD004388PubMed Google Scholar], the experimental data are quite convincing in establishing a strong link between APC generation and tissue damage in systemic inflammatory conditions such as sepsis. Besides systemic inflammation, histones also appear to be involved in localized tissue damage such as related to ischemia–reperfusion (I/R) injury [40Loubele S.T. Spek C.A. Leenders P. van Oerle R. Aberson H.L. Hamulyak K. Ferrell G. Esmon C.T. Spronk H.M. ten Cate H. Activated protein C protects against myocardial ischemia/reperfusion injury via inhibition of apoptosis and inflammation.Arterioscler Thromb Vasc Biol. 2009; 29: 1087-92Crossref PubMed Scopus (64) Google Scholar, 41Huang H. Evankovich J. Yan W. Nace G. Zhang L. Ross M. Liao X. Billiar T. Xu J. Esmon C.T. Tsung A. Endogenous histones function as alarmins in sterile inflammatory liver injury through toll‐like receptor 9.Hepatology. 2011; Crossref Scopus (271) Google Scholar]. In fact, one may speculate that some of the described protective effects of APC in I/R injury of the brain and the heart may in part be related to compensatory mechanisms for histone‐mediated tissue damage. It raises the question whether antagonizing the effects of histones could be beneficial in limiting organ damage under inflammatory conditions? Several serum proteins and glycosaminoglycans may be able to neutralize histone activity [42Pemberton A.D. Brown J.K. Inglis N.F. Proteomic identification of interactions between histones and plasma proteins: implications for cytoprotection.Proteomics. 2010; 10: 1484-93Crossref PubMed Scopus (47) Google Scholar]. Whether any of these effects can be exploited to improve the management of patients with sepsis or localized organ damage such as stroke remains to be investigated. This will certainly require additional insight into the molecular mechanisms and clinical relevance of NETs formation. These data implicate an important role for NETs in the bidirectional inflammation‐coagulation interplay. Overall, the evidence suggests that NETs may represent a promising therapeutic target against inflammation, tissue injury, multi‐organ failure and thrombosis. The authors state that they have no conflict of interest. J.I. Borissoff is supported by a Marie Curie fellowship (MEST‐CT‐2005‐020706), granted by the European Commission and is the recipient of a Kootstra Talent Fellowship (2011) from Maastricht University.