Interactions between the innate immune and blood coagulation systems
Authors: Charles T. Esmon
Journal: Trends in Immunology (2004)

Blood coagulation and inflammation are universal responses to infection and there is crosstalk between inflammation and coagulation that can either amplify or dampen the responses. Loss of appropriate interactions between these systems probably contributes to morbidity and mortality in infectious diseases. For instance, inflammatory cytokines and leukocyte elastase can downregulate natural anticoagulant proteins that help to maintain endothelial-cell integrity, control clotting, inhibit vasoactive peptides and dampen leukocyte infiltration into the vessel wall. This Review will summarize our current understanding of the mechanisms involved in the crosstalk between these two important systems. Blood coagulation and inflammation are universal responses to infection and there is crosstalk between inflammation and coagulation that can either amplify or dampen the responses. Loss of appropriate interactions between these systems probably contributes to morbidity and mortality in infectious diseases. For instance, inflammatory cytokines and leukocyte elastase can downregulate natural anticoagulant proteins that help to maintain endothelial-cell integrity, control clotting, inhibit vasoactive peptides and dampen leukocyte infiltration into the vessel wall. This Review will summarize our current understanding of the mechanisms involved in the crosstalk between these two important systems. When infectious agents, regardless of their nature, initiate the innate immune response, it in turn triggers blood coagulation [1Opal S.M. et al.Systemic host responses in severe sepsis analyzed by causative microorganism and treatment effects of drotrecogin alfa (activated).Clin. Infect. Dis. 2003; 37: 50-58Crossref PubMed Scopus (142) Google Scholar]. For instance, as viral infections, such as severe acute respiratory syndrome (SARS), progress into severe sepsis, markers of blood coagulation are increased markedly [2Lee N. et al.A major outbreak of severe acute respiratory syndrome in Hong Kong.N. Engl. J. Med. 2003; 348: 1986-1994Crossref PubMed Scopus (1986) Google Scholar]. Blood coagulation components are not simply bystanders but can either amplify or inhibit the inflammatory response. Blood clotting can be initiated when inflammatory cytokines and endotoxin induce the de novo synthesis of tissue factor on leukocytes [3Rauch U. et al.Transfer of tissue factor from leukocytes to platelets is mediated by CD15 and tissue factor.Blood. 2000; 96: 170-175Crossref PubMed Google Scholar]. Exposure of tissue factor to the blood then triggers the coagulation cascade. Complement activation can lead to the formation of plasma membrane surfaces enriched in negatively charged phospholipids that amplify the coagulation reactions [4Bevers E.M. et al.Transmembrane phospholipid distribution in blood cells: control mechanisms and pathophysiological significance.Biol. Chem. 1998; 379: 973-986PubMed Google Scholar, 5Sims P.J. et al.Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane. Studies in Scott Syndrome: An isolated defect in platelet procoagulant activity.J. Biol. Chem. 1989; 264: 17049-17057Abstract Full Text PDF PubMed Google Scholar]. Opposing this coagulation cascade are the natural anticoagulant pathways. These pathways not only limit the coagulation response but also dampen the inflammatory response by minimizing leukocyte chemotaxis [6Sturn D.H. et al.Expression and function of the endothelial protein C receptor in human neutrophils.Blood. 2003; 102: 1499-1505Crossref PubMed Scopus (170) Google Scholar] and endothelial-cell interactions [7Isobe H. et al.Activated protein C prevents endotoxin-induced hypotension in rats by inhibiting excessive production of nitric oxide.Circulation. 2001; 104: 1171-1175Crossref PubMed Scopus (107) Google Scholar], and by suppressing apoptosis [8Cheng T. et al.Activated protein C blocks p53-mediated apoptosis in ischemic human brain endothelium and is neuroprotective.Nat. Med. 2003; 9: 338-342Crossref PubMed Scopus (543) Google Scholar, 9Joyce D.E. et al.Gene expression profile of antithrombotic protein C defines new mechanisms modulating inflammation and apoptosis.J. Biol. Chem. 2001; 276: 11199-11203Crossref PubMed Scopus (599) Google Scholar] and reducing cytokine expression [10Oelschläger C. et al.Antithrombin III inhibits nuclear factor κB activation in human monocytes and vascular endothelial cells.Blood. 2002; 99: 4015-4020Crossref PubMed Scopus (151) Google Scholar, 11Yasui H. et al.Intratracheal administration of activated protein C inhibits bleomycin-induced lung fibrosis in the mouse.Am. J. Respir. Crit. Care Med. 2001; 163: 1660-1668Crossref PubMed Scopus (132) Google Scholar, 12Okajima K. Regulation of inflammatory responses by natural anticoagulants.Immunol. Rev. 2001; 184: 258-274Crossref PubMed Scopus (129) Google Scholar]. However, the acute inflammatory response can lead to suppression of the natural anticoagulant pathways by consumption, proteolytic inactivation and downregulation of protein expression [13Opal S.M. Esmon C.T. Bench-to-bedside review: Functional relationships between coagulation and the innate immune response and their respective roles in the pathogenesis of sepsis.Crit. Care. 2003; 7: 23-38Crossref PubMed Scopus (261) Google Scholar]. Loss of the coordinate control of the coagulation and inflammation pathways due to impaired function of the natural anticoagulant systems probably contributes to organ failure in severe sepsis. The blood clotting process is extremely complex (the more biochemical details are reviewed in Refs [14Furie B. Furie B.C. The molecular basis of blood coagulation.Cell. 1988; 53: 505-518Abstract Full Text PDF PubMed Scopus (1108) Google Scholar, 15Esmon C.T. Blood coagulation.in: Nathan D.G. Nathan and Oski's Hematology of Infancy and Childhood. W.B. Saunders Company, 2003: 1475-1496Google Scholar]). Briefly, blood coagulation is initiated when tissue factor comes into contact with blood (Figure 1). Under normal circumstances, there is little tissue factor in the intravascular compartment. Most of the tissue factor is found constitutively expressed on extravascular cells, where it surrounds the vessels providing a mechanism to rapid seal breaches in vessel integrity [16Drake T.A. et al.Selective cellular expression of tissue factor in human tissues: Implications for disorders of hemostasis and thrombosis.Am. J. Pathol. 1989; 134: 1087-1097PubMed Google Scholar]. Recent studies have indicated that there is a circulating form of tissue factor, probably localized on leukocyte-derived microparticles [17Celi A. et al.Thrombus formation: direct real-time observation and digital analysis of thrombus assembly in a living mouse by confocal and widefield intravital microscopy.J. Thromb. Haemost. 2003; 1: 60-68Crossref PubMed Scopus (107) Google Scholar, 18Giesen P.L.A. et al.Blood-borne tissue factor: another view of thrombosis.Proc. Natl. Acad. Sci. U. S. 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Chem. 1989; 264: 17049-17057Abstract Full Text PDF PubMed Google Scholar], thus promoting both the tissue factor–factor VIIa-mediated factor X and IX activation, and enabling the propagation of coagulation by the factor Xa–factor Va activation of prothrombin and the factor IXa–factor VIIIa activation of factor X. Potent platelet agonists, such as thrombin in combination with collagen or complement C5b-9, can further augment the availability of negatively charged phospholipid membrane surfaces on cells, particularly platelets, thus enabling further amplification of the coagulation system [5Sims P.J. et al.Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane. Studies in Scott Syndrome: An isolated defect in platelet procoagulant activity.J. Biol. Chem. 1989; 264: 17049-17057Abstract Full Text PDF PubMed Google Scholar, 20Zwaal R.F.A. Schroit A.J. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells.Blood. 1997; 89: 1121-1132Crossref PubMed Google Scholar]. Once initiated, the coagulation pathway has the potential to generate ∼100 times more thrombin than is required to form a rapid, firm clot. Under most conditions, this is prevented by the natural anticoagulant mechanism. The major inhibitory mechanisms involve the protein C anticoagulant pathway, antithrombin–heparin, and the tissue factor pathway inhibitor (TFPI). The protein C pathway is triggered when thrombin binds to thrombomodulin on the endothelial-cell surface. This complex, in concert with protein C bound to the endothelial-cell protein C receptor (EPCR), generates activated protein C. Once activated, protein C dissociates from the EPCR and can bind to protein S on membrane surfaces. The activated protein C–protein S complex then inactivates factors Va and VIIIa. Without these cofactors, factor Xa and factor IXa have much less than 1% of the capacity to activate the downstream zymogen. Practically, this means that inactivation of the cofactors shuts off the coagulation system completely. In addition, relative to thrombin in solution, thrombin bound to thrombomodulin is inhibited much more rapidly by antithrombin and protein C inhibitor [21Rezaie A.R. et al.Protein C inhibitor is a potent inhibitor of the thrombin-thrombomodulin complex.J. Biol. Chem. 1995; 270: 25336-25339Crossref PubMed Scopus (177) Google Scholar]. The antithrombin–heparin mechanism neutralizes factor Xa, thrombin and factor IXa. It also inactivates factor VIIa but only when the factor VIIa is bound to tissue factor [22Lawson J.H. et al.Complex-dependent inhibition of factor VIIa by antithrombin III and heparin.J. Biol. Chem. 1993; 268: 767-770Abstract Full Text PDF PubMed Google Scholar, 23Rosenberg R.D. Regulation of the hemostatic mechanism.in: Stamatoyannopoulos G. The Molecular Basis of Blood Diseases. W.B. Saunders Company, 1987: 534-574Google Scholar]. Antithrombin inhibition of the factor VIIa–tissue factor complex, factor IXa, factor Xa and thrombin are all thought to be accelerated by vascular heparin-like proteoglycans [24Marcum J.A. Rosenberg R.D. Anticoagulantly active heparin-like molecules from the vascular tissue.Biochemistry. 1984; 23: 1730-1737Crossref PubMed Scopus (182) Google Scholar]. TFPI inactivates factor VIIa bound to tissue factor using a unique mechanism. The inhibitor has two functional inhibitory Kunitz domains. The second domain binds to and inhibits factor Xa, which, as a result of its ability to bind negatively charged membranes, concentrates the TFPI–factor Xa complex on the membrane surface, at which time the first Kunitz domain of the inhibitor reversibly neutralizes factor VIIa. The physiological significance of these pathways is demonstrated by gene experiments in mice, which result in embryonic or neonatal lethality when any single pathway is disrupted [25Ishiguro K. et al.Complete antithrombin deficiency in mice results in embryonic lethality.J. Clin. Invest. 2000; 106: 873-878Crossref PubMed Scopus (180) Google Scholar, 26Gu J-M. et al.Disruption of the endothelial cell protein C receptor gene in mice causes placental thrombosis and early embryonic lethality.J. Biol. Chem. 2002; 277: 43335-43343Crossref PubMed Scopus (124) Google Scholar, 27Huang Z-F. et al.Tissue factor pathway inhibitor gene disruption produces intrauterine lethality in mice.Blood. 1997; 90: 944-951Crossref PubMed Google Scholar, 28Weiler-Guettler H. et al.A targeted point mutation in thrombomodulin generates viable mice with a prethrombotic state.J. Clin. 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Antithrombin inhibitory activity decreases markedly during severe sepsis, often to <50% of normal levels [31Levi M. ten Cate H. Disseminated intravascular coagulation.N. Engl. J. Med. 1999; 341: 586-592Crossref PubMed Scopus (965) Google Scholar]. Because the rates of inhibition of the target proteases are strongly dependent on the antithrombin concentration, this decrease in inhibitor concentration would contribute to increased stability of the coagulation enzymes and hence favor intravascular coagulation. There is evidence that the vascular heparin-like molecules are reduced by inflammatory cytokines and neutrophil activation products [32Klein N.J. et al.Alteration in glycosaminoglycan metabolism and surface charge on human umbilical vein endothelial cells induced by cytokines, endotoxin and neutrophils.J. Cell Sci. 1992; 102: 821-832PubMed Google Scholar]. 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Once tight control of thrombin and other coagulation enzymes is lost, they can participate in promoting the inflammatory response (Figure 3). The enhanced inflammatory response enhances the cell-associated coagulation activities as described earlier. Therefore, anticoagulants that dampen the cellular inflammatory response also dampen the coagulant response [40Souter P.J. et al.Antithrombin inhibits lipopolysaccharide-induced tissue factor and interleukin-6 production by mononuclear cells, human umbilical vein endothelial cells, and whole blood.Crit. Care Med. 2001; 29: 134-139Crossref PubMed Scopus (108) Google Scholar]. The antithrombin–heparin pathway can modulate inflammatory responses not only by inhibiting coagulant enzyme-mediated cell signaling through the protease activated receptors but also by modulating cellular responses [12Okajima K. Regulation of inflammatory responses by natural anticoagulants.Immunol. Rev. 2001; 184: 258-274Crossref PubMed Scopus (129) Google Scholar]. 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Infusion of the isolated lectin domain can reverse this response [52Conway E.M. et al.The lectin-like domain of thrombomodulin confers protection from neutrophil-mediated tissue damage by suppressing adhesion molecule expression via nuclear factor κB and mitogen-activated protein kinase pathways.J. Exp. Med. 2002; 196: 565-577Crossref PubMed Scopus (315) Google Scholar]. Cell culture studies demonstrated that this domain dampened the mitogen activated kinase and NF-κB responses in endothelium [51Esmon C.T. The roles of protein C and thrombomodulin in the regulation of blood coagulation.J. Biol. Chem. 1989; 264: 4743-4746Abstract Full Text PDF PubMed Google Scholar]. 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Activated protein C can also decrease endothelial-cell apoptosis [9Joyce D.E. et al.Gene expression profile of antithrombotic protein C defines new mechanisms modulating inflammation and apoptosis.J. Biol. Chem. 2001; 276: 11199-11203Crossref PubMed Scopus (599) Google Scholar]. This function is dependent on the EPCR and apparently involves activation of PAR1 [58Riewald M. et al.Activation of endothelial cell protease activated receptor 1 by the protein C pathway.Science. 2002; 296: 1880-1882Crossref PubMed Scopus (767) Google Scholar]. In mouse models of stroke, activated protein C minimizes damage at least in part by inhibiting apoptosis through downregulation of P53 [59Shibata M. et al.Anti-inflammatory, antithrombotic, and neuroprotective effects of activated protein C in a murine model of focal ischemic stroke.Circulation. 2001; 103: 1799-1805Crossref PubMed Scopus (184) Google Scholar]. This inhibition of P53 expression was also dependent on EPCR and PAR1. How activating PAR1 generates anti-inflammatory activities remains an area of active investigation. The recent determination of the EPCR structure suggests additional potential roles of the protein C pathway in regulating the immune response. EPCR shares considerable sequence identity to the MHC I–CD1 family of molecules [35Fukudome K. Esmon C.T. Identification, cloning and regulation of a novel endothelial cell protein C/activated protein C receptor.J. Biol. Chem. 1994; 269: 26486-26491Abstract Full Text PDF PubMed Google Scholar]. The crystal structure of EPCR reveals that, like the CD1 family, EPCR has a tightly bound lipid, in this case phospholipid, located in a region virtually identical to the antigen-presenting groove in the CD1 family [60Oganesyan V. et al.The crystal structure of the endothelial protein C receptor and a bound phospholipid.J. Biol. Chem. 2002; 277: 24851-24854Crossref PubMed Scopus (175) Google Scholar] (Figure 6). 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Blood (in press)Google Scholar], consistent with its involvement in immune regulation. Platelets are also involved in the linking of inflammation and coagulation. Inflammatory mediators, such as IL-6, not only increase platelet production, but the platelets that are generated are more thrombogenic, demonstrating an increased sensitivity to platelet agonists, such as thrombin [64Burstein S.A. Cytokines, platelet production and hemostasis.Platelets. 1997; 8: 93-104PubMed Google Scholar]. Platelets contain high concentrations of the proinflammatory mediator CD40 ligand. On platelet activation, CD40 ligand is released. This protein then induces tissue factor synthesis [65Pendurthi U.R. et al.Binding of factor VIIa to tissue factor induces alterations in gene expression in human fibroblast cells: up-regulation of poly(A) polymerase.Proc. Natl. Acad. Sci. U. S. 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Severe acute inflammatory challenges can downregulate the natural anticoagulants, thereby diminishing their ability to dampen the coagulant and inflammatory responses. When this happens, a progressive cycle can evolve in which the coagulation increases the inflammatory response, thereby increasing the coagulant response until severe vascular and organ injury occurs. Modulation of this complex series of interactive events promises to offer an attractive method for intervening clinically in diseases involving acute inflammatory challenges, such as severe sepsis. The recent successful demonstration that recombinant activated protein C can reduce the 28-day all cause mortality in patients with severe sepsis [69Bernard G.R. et al.Efficacy and safety of recombinant human activated protein C for severe sepsis.N. Engl. J. Med. 2001; 344: 699-709Crossref PubMed Scopus (5184) Google Scholar] provides support for this proposal.Questions for future researchThe exact signaling mechanisms responsible for the protective effects of natural anticoagulants need to be characterized more fully. At the preclinical level, differences in the protective effects of natural anticoagulants need to be explored more fully, particularly in situations where disease has already progressed, as would almost always be the case in patients. Future research will also need to address the impact of common disease processes, such as diabetes, on both the innate immune response and the ability of natural anticoagulant pathways to modulate the innate immune response. Combinations of microarray and proteomics approaches might aid in these studies.In addition, although our understanding of the regulation of blood coagulation in systems with artificial membrane surfaces is relatively sophisticated, cellular control of coagulation is still an emerging area. For instance, activated protein C is a potent anticoagulant involving liposomes. By contrast, it is a relatively poor anticoagulant on platelet surfaces. In model systems of blood coagulation, in which platelets are present over endothelium, it appears that activated protein C anticoagulant functions occur on the endothelial cell rather than the platelet surface [76Oliver J.A. et al.Activated protein C cleaves factor Va more efficiently on endothelium than on platelet surfaces.Blood. 2002; 100: 539-546Crossref PubMed Scopus (48) Google Scholar]. The nature of the membrane surface responsible for these differences remains to be elucidated.