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The foregoing sections have provided a conceptual framework for the inflammatory response, specifically, the hemodynamic alterations, mechanisms of specific leukocyte–endothelial adhesive interactions, chemotaxis, leukocyte activation, phagocytosis, intracellular microbial killing mechanisms, active termination/resolution of the acute inflammatory response, and the contributions of M1 and M2 macrophages to inflammation and tissue repair. The many steps that constitute this paradigm are regulated by soluble mediators produced by endothelial cells and leukocytes at a site of inflammation, by other resident cells (e.g., tissue macrophages, fibroblasts, mast cells) and as byproducts of bloodborne proteins (e.g., complement system, coagulation cascade; Table 19–3). There are many examples of “crosstalk” among regulatory systems (e.g., proteinase-activated receptors), complex regulatory networks (e.g., proinflammatory and antiinflammatory cytokine balance), and pleiotropism exhibited by individual mediators (e.g., TNF-α and IL-1β).
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REACTIVE OXYGEN INTERMEDIATES
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Since the early 1970s it has been recognized that activated phagocytes exhibit a transient but marked increase in oxygen consumption and the mechanistically coupled generation of reduced oxygen metabolites.32 Although small quantities of reactive oxygen intermediates are produced as byproducts of several metabolic pathways, the chief source is the leukocyte cytosol and membrane-associated NADPH oxidase, an enzyme complex that is defective in most patients with chronic granulomatous disease (Chap. 66). Reactive oxygen intermediates include superoxide anion (O2−), hydrogen peroxide (H2O2), hydroxyl radical (HO•), and singlet oxygen (1O2).32 These reduced oxygen products play a major role in intraphagolysosomal killing of microorganisms, and when released extracellularly, are directly or indirectly responsible for a variety of proinflammatory processes, including endothelial cell lysis, extracellular matrix degradation, activation of latent proteolytic enzymes (collagenase, gelatinase), inactivation of antiproteases, interaction with metabolites of l-arginine, and generation of chemotactic factors from arachidonic acid and the complement component, C5.33 In addition to their role in endothelial cytotoxicity, reactive oxygen intermediates are cytotoxic to fibroblasts, erythrocytes, tumor cells and many types of parenchymal cells.33 Implicated biochemical mechanisms include lipid peroxidation, formation of carbonyl moieties and nitrosylation products, intracellular enzyme inactivation, protein oxidation, and oxidant-mediated DNA damage. Reactive oxygen intermediates (e.g., O2−) can also undergo reactions with reactive nitrogen intermediates (e.g., NO; see “Reactive Nitrogen Intermediates” below) to generate toxic NO derivatives.33 Within limits, host cells are protected by antioxidant defense systems (e.g., superoxide dismutase, catalase, reduced glutathione).33
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REACTIVE NITROGEN INTERMEDIATES
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Described in 1980 as “endothelium-derived relaxing factor,” NO is the soluble, gaseous, short-acting biosynthetic product of l-arginine, O2, NADPH, and NO synthase (NOS).34 As suggested by its original name, NO mediates vascular smooth muscle relaxation. NO binds to the heme moiety of guanylyl cyclase to trigger the generation of intracytoplasmic cyclic guanosine monophosphate (cGMP) and, through the activation of a series of kinases, induces smooth-muscle relaxation and vasodilation.35 Three different forms of NOS have been characterized: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS).35 Nitric oxide can be produced either constitutively (eNOS, nNOS) or induced (iNOS) in a wide variety of cell types (e.g., endothelial cells, neurons, macrophages, respectively). Nitric oxide produced by eNOS plays a particularly important role in the localized regulation of vascular tone, whereas NO derived from nNOS is important in neuronal signal transduction. NO also plays important roles in the inhibition of smooth-muscle proliferation and in inflammation. The roles of NO in inflammation include inhibition of cell-mediated inflammation, reduction in platelet aggregation and adhesion, and as a regulator of leukocyte recruitment. Specifically, NO produced by cytokine-iNOS reduces leukocyte recruitment into sites of inflammation. NO can react with reactive oxygen intermediates to form both reactive oxygen and nitrogen species (e.g., NO + O2− → NO2− + HO•); it can inhibit DNA synthesis; it can directly kill microbes and tumor cells; and it can inactivate cytosolic glutathione and other sulfhydryl enzymes. NO and its generating enzymes, eNOS, nNOS, and iNOS, represent a regulatory system that has varied effects on the inflammatory response depending upon location and setting.
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LYSOSOMAL GRANULE CONSTITUENTS
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The activation of neutrophils, monocytes and macrophages results in the release, either through exocytosis or as the result of cell death, of a wide variety of proinflammatory mediators that have important roles in the inflammatory response. Neutrophils contain three major types of granules and also secretory vesicles (Chap. 60). Large, primary (azurophilic) granules contain myeloperoxidase, lysozyme, a variety of cationic proteins, defensins, phospholipase, acid hydrolases and neutral proteases (e.g., proteinase 3, collagenases, elastase). Smaller, secondary (specific) granules contain lactoferrin, lysozyme, type IV collagenase, subunits of NADPH oxidase and the β2-integrin, CD11b/CD18. Tertiary granules contain gelatinase, subunits of NADPH oxidase and CD11b/CD18. Acid proteases function most efficiently within phagolysosomes where the pH is low, whereas neutral proteases can function efficiently within extracellular inflammatory exudates. Lysosomal granule constituents contribute to the inflammatory response and tissue injury through a wide array of mechanisms (e.g., degradation of extracellular matrix, proteolytic generation of chemotactic peptides and catalysis of reactive oxygen metabolite generation).
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CYTOKINES AND CHEMOKINES
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Cytokines are proteins that exhibit a variety of proinflammatory and antiinflammatory effects. They are produced by many cell types and modulate the function of other cell types. Individual cells may produce many different cytokines, and an individual cytokine may exert a wide variety of effects; they are pleiotropic.2,3,6 In addition to their important roles in regulating various aspects of the immune response (e.g., lymphocyte activation, proliferation, and differentiation), many cytokines participate in innate immunity (e.g., TNF-α, IL-1β, IL-6, type I interferons), mediate the acute-phase response (TNF-α, IL1β, IL-6), activate inflammatory cells (e.g., IFN-γ) and participate in hematopoiesis (e.g., IL-3, granulocyte-monocyte colony-stimulating factor, granulocyte colony-stimulating factor, macrophage colony-stimulating factor).2,3 Among the most thoroughly characterized cytokines are TNF-α and IL-1β, which are structurally dissimilar but share many biologic activities and can function as autocrine, paracrine and endocrine mediators of inflammation (Table 19–4).2,3 TNF-α and IL-1β are produced by various cell types and are pleiotropic. Particularly important functions in inflammation include endothelial, leukocyte and fibroblast activation.
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Elevated local (and sometimes systemic) concentrations of TNF-α, IL-1β, and IL-6 are consistently observed during the development of an inflammatory response. Based on their roles in the systemic acute-phase response and in the orchestration of important localized mechanistic steps in inflammation (e.g., induction of endothelial leukocyte adhesion molecules, phagocyte activation, procoagulant mediator induction), these mediators are prototypic “proinflammatory” cytokines. Their expression is regulated by nuclear factor κB (NFκB). NFκB is a transcription factor that exists as a heterodimer complexed with IκB (inhibitor κB) in the cytosol of many different cells types.3,6 When cells are activated by various microbial products, viruses, reactive oxygen intermediates, cytokines, and chemotherapeutic agents, IκB is phosphorylated before it dissociates from NFκB heterodimers. Unbound NFκB translocates into the cell nucleus where it participates in the upregulation of as many as 200 different genes, including TNF-α, IL-1β and IL-6.
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The proinflammatory cytokines and their activities are counterbalanced by a wide variety of “antiinflammatory” cytokines, including IL-4, IL-10, IL-11, IL-13, TGFβ, IL-1ra, and several soluble cytokine receptors.2,3,7 IL-4, a 20-kDa peptide produced by CD4 Th2 cells, inhibits IL-1β synthesis and induces IL-1ra (IL-1 receptor antagonist). Soluble IL-1ra binds IL-1β (and IL-1α), preventing their binding to IL-1 receptors. IL-10 is also secreted by CD4 Th2 cells (and regulatory T cells, monocytes, and macrophages). Acting through its cognate receptor, IL-10 suppresses the expression of proinflammatory cytokines, adhesion molecules, chemokines, and cell-surface activation molecules of neutrophils, monocytes, macrophages, and T lymphocytes.7 IL-10 also induces the shedding of TNF-α receptors, which then function as soluble TNF-α antagonists. IL-11, IL-13, and TGFβ also each exert a set of activities that counter the proinflammatory actions of TNF-α, IL-1β, and IL-6. Recognition of the many counterbalancing actions between proinflammatory and antiinflammatory cytokines has led to the concept of “proinflammatory–antiinflammatory cytokine balance.” This concept is the basis for rational therapeutic strategies to manipulate or “reset” this balance.
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TNF-α, IL-1β, and IL-6 are key proximate mediators of the “acute-phase response.” Stimuli such as bacterial endotoxin (lipopolysaccharide), exotoxins, immune complexes and physical stimuli (e.g., heat or trauma) can induce macrophages (and other cell types) to secrete TNF-α, IL-1β, and IL-6.2,3,7 In turn, TNF-α, IL-1β, and IL-6 mediate fever, somnolence, increased production of proteins such as α1-antitrypsin (α1-antiprotease) and α2-macroglobulin, and decreased production of proteins such as albumin and transferrin. As noted, the acute-phase response is a stereotyped host metabolic response to a wide variety of insults. In addition to the systemic acute-phase response, TNF-α and IL-1β induce endothelial activation marked by increases in leukocyte adherence and a procoagulant state, leukocyte activation marked by cytokine secretion, and fibroblast activation marked by proliferation, collagen synthesis, and collagenase production.2,3,7 These actions are critical components of inflammation and wound healing; they exemplify the linkage between the inflammatory response and the coagulation system.
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TNF-α, originally identified as “cachexin or cachectin” because of its role in the systemic wasting that accompanies some chronic infections and cancer, can induce cytokine production in a variety of cells.2,3 TNF-α can induce neutrophil activation and the expression of adhesion molecules on endothelial cells. In contrast to IL-1β, TNF-α also possesses potent cytotoxic activities for some types of cells. Both IL-1β and TNF-α are produced in response to endotoxemia and both can mediate a systemic shock-like response.
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IL-1β, which exhibits a wide variety of biologic activities, was initially termed endogenous pyrogen because of its ability to induce temperature elevation and the acute-phase response.2,3,36 IL-1β is relevant to acute inflammation because of its ability to induce cytokine production in monocytes, macrophages, fibroblasts and endothelial cells. IL-1β can also induce NOS.36 As noted previously, IL-1β can activate endothelial cells, resulting in the expression of adhesion molecules and a procoagulant phenotype.2,3,36
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IL-6 participates in the acute-phase response through the induction of proinflammatory mediators production by hepatocytes, via the differentiation of CD4 T lymphocytes that produce IL-17 and through the induction of marrow neutrophil production.2,3 IL-6 is produced by a variety of cell types following activation by TNF-α, IL-1β, and pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharides (endotoxin), mannans, flagellin, and microbial nucleic acids.3,6
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Chemokines, or “intercrines,” are small proteins, which, in addition to many of the general properties of cytokines, exhibit prominent chemotactic activities.3,6,37 Chemokines are grouped into four classes based on the amino acid sequence positions of conserved cysteine (C) residues in mature peptides.6,37 The four classes include CC, CXC, XC, and CX3C chemokines. There are four families of corresponding chemokine receptors: CCR, CXCR, XCR, and CX3CR, respectively. Nomenclature of chemokines is based on the locations of N-terminal cysteine residues whereby “CC” indicates two adjacent residues, “CXC” indicates two cysteine residues separated by one intervening amino acid, and so on. Individual chemokines contain the letter “L” for ligand, followed by individual numbers (e.g., CCL1, CCL2, CCL3, etc.); more than 40 have been identified. The two most studied subfamilies include the alpha, or “CXC” chemokines, and the beta, or “CC” chemokines. Alpha chemokines, of which IL-8 (CXCL8) is the prototype, consistently exhibit neutrophil chemotactic activity, whereas the beta, or “CC” chemokines, of which MCP-1 (CCL2) is the prototype, exhibit monocyte chemotactic activity (Table 19–5).37,38 Both in vitro and in vivo studies have provided insight into the roles of chemokines in inflammation. For example, MCP-1 knockout mice (MCP-1 −/−) exhibit reductions in monocyte influx into sites of experimentally induced peritonitis and delayed-type hypersensitivity.39 Complementary studies using knockout mice devoid of the MCP-1 receptor CCR2 do not form typical granulomas.39 These types of studies, as well as many that have employed specific chemokine-neutralizing antibodies or soluble chemokine receptor antagonists, have provided valuable insight into the pathophysiology of inflammation. Seemingly contradictory experimental results suggest that leukocyte recruitment mechanisms are multiple, overlapping or redundant, and not completely understood. Chemokine receptors noted above (CCR, CXCR, etc.) activate leukocytes through membrane receptors (sometimes called “serpentine” receptors) that contain seven transmembrane domains and are linked to cytosolic heterotrimeric G proteins.3,6
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Lipid mediators of inflammation, commonly derived from cell membrane precursor molecules, can act either intracellularly or extracellularly, the latter in a short-lived, localized manner.41 Arachidonic acid, a 20-carbon polyunsaturated fatty acid (5,8,11,14-eicosatetraenoic acid) derived either from dietary sources or by conversion from linoleic acid, is maintained in cell membranes as an esterified phospholipid. Three families of inflammatory mediators derived from arachidonic acid are generated via the cyclooxygenase and lipoxygenase pathways. Arachidonic acid is released from membrane phospholipids via cellular phospholipases such as phospholipase A2. Phospholipase activation is triggered by mechanical/physical or chemical stimuli. Arachidonic acid can be metabolized via the cyclooxygenase pathway to prostaglandins (e.g., PGG2, PGH2, PGD2, PGE2, PGF2), prostacyclin (PGI2) or thromboxane (TXA2).41 Prostacyclin mediates vasodilation and the inhibition of platelet aggregation; thromboxane has the opposite effects; and PGD2, PGE2, and PGF2 mediate vasodilation and edema. Activation of the lipoxygenase pathway results in the synthesis of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which is a potent chemoattractant of neutrophils and can be enzymatically modified to yield a series of other leukotrienes. LTB4 induces neutrophil chemotaxis, aggregation, degranulation, and adherence, while LTC4, LTD4, and LTE4 trigger smooth-muscle constriction, increases in vascular permeability and bronchoconstriction.41 Members of both of these families of lipid-derived mediators and their catabolites have been detected in inflammatory exudates. There are two important branches within the lipoxygenase pathway.41,42 Lipoxins (A4 [LXA4] and B4 [LXB4]) are generated via the 12-lipooxygenase branch of the lipoxygenase pathway in conjunction with a unique transcellular biosynthetic pathway.42 Neutrophils generate LTA4 via the 5-lipoxygenase pathway branch; in turn, lipoxins (LXA4 and LXB4) are generated through the action of platelet 12-lipooxygense on neutrophil LTA4. Prevention of neutrophil-platelet binding interrupts this pathway. Lipoxins inhibit neutrophil chemotaxis and adhesion to endothelium.42 As noted above, resolvins and protectins each encompass several molecular species—all derived from omega-3 polyunsaturated fatty acids.26,27
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PAF is a potent proinflammatory lipid produced by a variety of cell types, including neutrophils, monocytes, endothelial cells and IgE-sensitized basophils.43 Derived from the cell membrane constituent, choline phosphoglyceride, PAF is an acetyl glycerol ether phosphocholine that is synthesized following the activation of phospholipase A2. PAF triggers platelet aggregation and degranulation, increases vascular permeability, and promotes leukocyte accumulation and activation. In vivo studies using specific PAF antagonists have suggested a role for PAF in a variety of acute inflammatory lesions.43
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The kinin system is activated by contact activation of clotting factor XII (Hageman factor) (Chaps. 113 and 114).44 Activation of the kinin system results in the generation of bradykinin, a nine-amino-acid vasoactive peptide. Bradykinin possesses several activities, including the capacity to increase vascular permeability, induce smooth-muscle contraction, trigger vasodilation, and cause pain.44 Activated Hageman factor (factor XIIa), also known as the prekallikrein activator, converts plasma prekallikrein to kallikrein. In turn, kallikrein cleaves high-molecular-weight kininogen to produce bradykinin. Models of septic shock reveal decreases in plasma kininogen that parallel decreases in peripheral arterial resistance.44
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Histamine and serotonin (5-hydroxytryptamine) are low-molecular-weight vasoactive amines. Histamine is contained in mast cell and basophil granules, whereas platelets are a chief source of serotonin.45 Localized release of histamine results in wheal formation as a consequence of increases in vascular permeability. Histamine induces the formation of reversible openings in endothelial tight junctions, triggers the formation of prostacyclin by endothelial cells and induces NO release from the endothelium. In addition, histamine, like thrombin, can induce the rapid upregulation of endothelial P-selectin.45 Serotonin, acting through receptors on vascular smooth-muscle cells, is responsible for vasoconstriction, whereas interaction with endothelial receptors results in vasodilation (via release of NO) and increased permeability.2 Release of histamine and serotonin from mast cells and platelets can be triggered by IgE-mediated type I hypersensitivity reactions, directly by C3a or C5a, and directly by neutrophil granule-derived cationic proteins.
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The complement system, including its soluble and cell membrane-associated regulators, consists of nearly two dozen plasma proteins that give rise to mediators of chemotaxis, increased vascular permeability, opsonic activity, phagocyte activation, and cytolysis.46 In a manner analogous to coagulation, the complement system is activated through a cascade of proteolytic cleavage reactions. There are three convergent pathways (Fig. 19–3). The first of these, the “classical pathway,” is initiated primarily (but not exclusively) by complement-fixing immune complexes (IgG subclasses 1 to 3 and IgM), whereas the second, the “alternative pathway,” is triggered by a variety of substances that include IgA aggregates, endotoxin, cobra venom factor, and polysaccharide moieties found on some bacterial and fungal cell walls. The third pathway, the “mannan-binding” lectin (MBL) pathway, is activated when MBL binds to a microorganism coated with certain carbohydrate moieties (e.g., mannans). Upon binding, MBL activates MBL-associated serine proteases (e.g., MASP-1, MASP-2) which function in a manner analogous to C1r and C1s of the classical pathway. MBL recognizes carbohydrate moieties infrequently present in mammalian hosts, thus constituting a system for recognizing foreign particulates. As such, the alternative and MBL pathways are considered to be part of the innate system of host defense.6 The classical pathway is initiated by the fixation of C1 (C1qr2s2) by the Fc portion of surface-bound IgG or IgM immunoglobulins. Activated C1 (C1qr2s2) cleaves C2 and C4, which leads to the formation of the “classical pathway” C3 convertase, C4b2a. Activation of the alternative pathway results in the formation of an “alternative pathway” C3 convertase following direct cleavage of C3 and subsequent interactions of C3b with factors B and D in the presence of Mg2+. The resulting complex, C3bBb, is stabilized by properdin, leading to the stable C3 convertase, C3bBbP. C3 convertases generated via any of the three pathways efficiently cleave C3 to form C3a and C3b.
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These enzymatic reactions exhibit high activity levels, thus serving to dramatically amplify the cascade. C3b can bind to either the classical or alternative pathway C3 convertase to form a C5 convertase, which cleaves C5 into C5a and C5b. C5a is released into the fluid phase, like C3a, whereas C5b combines first with C6 and then C7 to form C5b-7, which, in turn, binds with C8 and multiple C9 molecules to form C5b-9, the membrane attack complex. In addition to the cell-activating and cytolytic activities of C5b-9, individual complement cleavage products and complexes mediate a variety of specific and potent proinflammatory activities.46 These functions, combined with the rapid amplification in numbers of complement-derived mediators, emphasize the vital role of complement in acute inflammation. The most important activation products of complement appear to be C5a, a major chemotactic factor, and the anaphylatoxins (C3a, C4a, C5a), of which C3a is the most abundant. C5b-9 is a major cytotoxic product, provided that this complex is assembled on the surface of a susceptible cell (e.g., bacterium).
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A series of soluble and cell membrane-associated complement proteins play important roles in the regulation of the complement cascade.46 The pivotal regulator of the proximal arm of classical pathway is C1 esterase inhibitor (C1E-INH), a serine protease inhibitor that covalently bonds to the activated esterase subunits of the C1qrs complex thus preventing activation of the downstream zymogen cascade.46 Defects in C1E-INH, from genetic defects or those acquired (e.g., neutralizing antibodies against C1E-INH), can result in angioedema. Angioedema can manifest in a variety of ways including as life-threatening laryngeal soft tissue swelling.
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The coagulation system is reviewed in detail in Chaps. 113, 114, and 116. The interrelationships among the coagulation system and several inflammatory mediator systems are important in the context of host defense and the pathophysiology of septic shock.4 Activation of the clotting cascade results in the generation of fibrinopeptides which increase vascular permeability and are chemotactic for leukocytes. Thrombin and tissue factor induce endothelial expression of P-selectin, resulting in increased neutrophil adhesion.12 In addition, plasmin is responsible for the activation of Hageman factor, which then can activate the kinin system and can cleave C3 into its active components.44 It can also generate “fibrin-split” or “fibrin-degradation” products. The induction of tissue factor in endothelial cells exposed to TNF-α and IL-1β further links the coagulation system to the inflammatory response.
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PROTEINASE-ACTIVATED RECEPTORS
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Proteinase-activated receptors (PARs) define an important general mechanism that links several seemingly disparate regulatory systems involved in inflammation.47 PARs subsume a G-protein–coupled receptor subfamily defined by a common activation mechanism.47 Individual PARs include an N-terminal extracellular domain, seven transmembrane helices connected by three intracellular and three extracellular loops, and linkage to cytosolic G-protein–mediated signal transduction pathways.47 PARs are activated when extracellular proteinases cleave the N-terminal extracellular domain at a specific site which results in the creation of a “tethered ligand.” The tethered ligand is the residual, now unmasked N-terminal portion of the PAR; it interacts with the nearby nontruncated extracellular PAR domain and activates the receptor. The PAR family possesses of four members: PAR1, PAR2, PAR3, and PAR4. The extracellular domain of each PAR possesses several potential cleavage sites. For example, the canonical PAR1 tethered ligand sequence created after cleavage by thrombin is the amino acid sequence, SFLLRN.47 A wide variety of proteinases that are pivotal in inflammation, thrombosis, hemostasis, and wound healing (as well as in development and cancer progression) can activate PARs. PAR1, PAR3, and PAR4 are susceptible to cleavage by thrombin. Other coagulation system-related proteinases, such as factor Xa, activated protein C, plasmin, and kallikreins can also activate PAR1. Likewise, PAR1 can also be activated by matrix metalloproteinase-1, neutrophil elastase, and neutrophil proteinase-3. Various proteinases cleave the N-terminal extracellular domain of PARs at different, yet specific, sites. Examples of PAR activation relevant to inflammation include thrombin-induced CCL2 expression in osteoblasts and PAR2 and PAR4 activation in animal models of arthritis. A goal of rational therapeutic design is to target crosstalk interactions using paired drugs or bifunctional agents. Although no PAR-targeting compounds have yet come into clinical use, this is a promising area.47