**Immunoregulatory Properties of Acute Phase Proteins — Specific Focus on α1-Antitrypsin**

S. Janciauskiene, S. Wrenger and T. Welte

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56393

## **1. Introduction**

Activation of innate immune cells in response to various insults is a part of the host defence. However, if uncontrolled, this inflammatory response induces persistent hyper-expression of pro-inflammatory mediators and tissue damage. Tight control of pro-inflammatory pathways is therefore critical for immune homeostasis and host survival.

A complex network of activating and regulatory pathways controls innate immune responses; thehepaticacute-phaseresponseisoneofthecrucialcontributorstothisregulation.Forexample, in response to infection or tissue injury within few hours the pattern of protein synthesis by the liver is drastically altered, i.e. increased expression of the so called positive acute phase pro‐ teins (APPs) like C-reactive protein (CRP), alpha1-antitrypsin (AAT) or alpha1-acid glycopro‐ tein (AGP) and decreased expression of transthyretin, retinol binding protein, cortisol binding globulin, transferrin and albumin, which represent the group of negative APPs. This produc‐ tion of APPs in hepatocytes is controlled by a variety of cytokines released during inflamma‐ tionwhereas leadingregulators are IL-1- andIL-6-type cytokineshavingadditive,inhibitory,or synergistic effects. For instance, IL-1β is shown to almost completely abrogate IL-6-induced production of α2-macroglobulin and α1-antichymotrypsin but, in contrast, to enhance produc‐ tion of CRP and serum amyloid A. No doubt, this specific regulation of AAPs expression plays a critical role in the regulation of the host innate immune responses.

## **2. Alpha1-antitrypsin and the acute phase response**

AAT, also referred to as alpha1-proteinase inhibitor or SERPINA1, is the most abundant serine protease inhibitor in human blood. AAT consists of a single polypeptide chain of 394 amino

© 2013 Janciauskiene et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

acid residues containing one free cysteine residue and three asparagines-linked carbohydrate side-chains. AAT is mainly produced by liver cells but can also be synthesized by blood monocytes, macrophages, pulmonary alveolar cells, and by intestinal and corneal epithelium (Geboes et al., 1982; Perlmutter et al., 1985; Ray et al., 1982). In terms of tissue expression AAT has been demonstrated in the kidney, stomach, small intestine, pancreas, spleen, thymus, adrenal glands, ovaries and testes. De novo synthesis of AAT has also been demonstrated in human cancer cell lines. These observations indicate that AAT transcription is relatively widespread. In fact, tissue-specific promoter activity for AAT has been reported in the liver, the major source of AAT, and alternative promotors for other tissues that express the protein (Kalsheker et al., 2002; Tuder et al., 2010). Interestingly, AAT expression also shows some degree of substrate and/or auto-regulation: upon exposure to neutrophil and pancreatic elastases, either alone or as a complex of AAT, enhanced synthesis of AAT was observed (Perlmutter et al., 1988).

induced nasal inflammation, AAT might be a byproduct of the activated inflammatory cells,

Immunoregulatory Properties of Acute Phase Proteins — Specific Focus on α1-Antitrypsin

http://dx.doi.org/10.5772/56393

3

According to recent studies, activated neutrophils and eosinophils can store and secrete AAT, which plays a role in protection of tissues at local inflammation sites (Johansson et al., 2001; Paakko et al., 1996). Furthermore, Clemmensen and coworkers found that the mRNA for AAT increases during maturity of the myeloid cell precursors and is even higher in blood neutro‐ phils. This in itself is quite remarkable as blood neutrophils are generally considered tran‐ scriptionally inactive, but it is even more striking that the transcriptional activity of the AAT gene increases further when neutrophils migrate into tissues (Clemmensen et al., 2011). Moreover, circulating AAT produced by liver cells can enter granulocytes and is stored in the

**3. Protective anti-inflammatory, immunomodulatory and antimicrobial**

Findings from different experimental models provide clear evidence that AAT expresses broad anti-inflammatory and immunoregulatory activities (Figure 1). AAT has been reported to inhibit neutrophil superoxide production, adhesion, and chemotaxis, to enhance insulininduced mitogenesis in cell lines and to induce IL-1 receptor antagonist, a negative regulator to IL-1 signalling, in blood monocytes and neutrophils (Tuder et al., 2010). Findings that AAT enhances the synthesis of transferrin receptor and ferritin revealed a role of AAT in iron metabolism (Graziadei et al., 1997). In murine models, exogenous human AAT protects islet cell allografts from rejection and increase survival in an allogeneic marrow transplantation models. In other models AAT therapy protects against TNF-α / endotoxin induced lethality, cigarette smoke induced emphysema and inflammation and even suppressed bacterial proliferation during infections ((Lewis, 2012), review). Furthermore, human AAT given to mice during renal ischemia–reperfusion (I/R) injury lessens tissue injury and attenuated organ

These beneficial impacts of AAT are incompletely understood, although exscinding knowl‐ edge suggests that AAT promotes a switch from pro-inflammatory to anti-inflammatory

AAT has long been thought of as a main inhibitor of neutrophil elastase, proteinase 3, and other serine proteases released from activated human neutrophils during an inflammatory response. In fact, the rate of formation of the AAT/neutrophil elastase inhibitory complex is one of the fastest known for serpins (6.5x 107 M-1 s-1) (Gettins, 2002). The structure of AAT consists of three β-sheets (A, B, C) and 9 α-helices (A-I). The inhibitory active conformation of AAT like for other serine protease inhibitors represents a metastable state, characterized by an exposed reactive center loop that acts as bait for the target enzyme (Stocks et al., 2012). Cleavage of the scissile bond in the loop results in a large conformational change in which the reactive site loop migrates and is inserted into the pre-existing β-sheet A forming a very stable complex

and is thus implicated in the allergic immune response (Shin et al., 2011).

secretory vesicles (Borregaard et al., 1992).

**effects of alpha1-antitrypsin**

dysfunction (Daemen et al., 2000).

pathways necessary for the resolution of inflammation.

The normal daily rate of synthesis of AAT is approximately 34 mg/kg body weight and the protein is cleared with a half-life of 3 to 5 days. This results in high plasma concentrations ranging from 0.9 to 2 mg/ml when measured by nephelometry. In addition to high circulating levels in blood, AAT is also present in saliva, tears, milk, semen, urine and bile (Berman et al., 1973; Chowanadisai & Lonnerdal, 2002; Huang, 2004; Janciauskiene et al., 1996; Poortmans & Jeanloz, 1968). The distribution of the protein in the tissues is not uniform. For example, in the epithelial lining fluid of the lower respiratory tract its concentration is approximately 10% of plasma levels (Janciauskiene, 2001).

As an acute-phase reactant, circulating AAT levels increase rapidly (3 to 4 fold) in response to inflammation or infection. The concentration of AAT in plasma also increases during oral contraceptive therapy and pregnancy. During an inflammatory response, tissue concentra‐ tions of AAT may increase as much as 11-fold as a result of local synthesis by resident or invading inflammatory cells (Boskovic & Twining, 1997). Blood monocytes and alveolar macrophages can contribute to tissue AAT levels in response to inflammatory cytokines (IL-6, IL-1 and TNFα) and endotoxins (Knoell et al., 1998; Perlmutter & Punsal, 1988). Recent data demonstrate that AAT expression by alpha and delta cells of human islets (Bosco et al., 2005) and intestinal epithelial cells (Faust et al., 2001) is also enhanced by pro-inflammatory cytokines. AAT synthesis by corneal epithelium, on the other hand, appears to be under the influence of retinol, IL-2, fibroblast growth factor-2, and insulin-like growth factor-I (Boskovic & Twining, 1997; Boskovic & Twining, 1998). Oncostatin M, a member of the IL-6 family was shown to induce AAT production by human bronchial epithelial cells. This effect of oncostatin M was in turn modulated by TGF-β and IFN-γ at both the protein and mRNA level. IFN-γ decreased oncostatin M-induced AAT production whilst TGF-β induced a significant and synergistic up-regulation of AAT that was not observed in a hepatocyte cell line (Boutten et al., 1998). Study by Shin and coworkers (Shin et al., 2011) have demonstrated that nasal lavage fluids from the patients with allergic rhinitis contains AAT and that the levels of nasal AAT markedly increase in response to allergenic stimulation. This response seems to be closely associated with the activation of eosinophils induced by allergen-specific IgA. In allergeninduced nasal inflammation, AAT might be a byproduct of the activated inflammatory cells, and is thus implicated in the allergic immune response (Shin et al., 2011).

acid residues containing one free cysteine residue and three asparagines-linked carbohydrate side-chains. AAT is mainly produced by liver cells but can also be synthesized by blood monocytes, macrophages, pulmonary alveolar cells, and by intestinal and corneal epithelium (Geboes et al., 1982; Perlmutter et al., 1985; Ray et al., 1982). In terms of tissue expression AAT has been demonstrated in the kidney, stomach, small intestine, pancreas, spleen, thymus, adrenal glands, ovaries and testes. De novo synthesis of AAT has also been demonstrated in human cancer cell lines. These observations indicate that AAT transcription is relatively widespread. In fact, tissue-specific promoter activity for AAT has been reported in the liver, the major source of AAT, and alternative promotors for other tissues that express the protein (Kalsheker et al., 2002; Tuder et al., 2010). Interestingly, AAT expression also shows some degree of substrate and/or auto-regulation: upon exposure to neutrophil and pancreatic elastases, either alone or as a complex of AAT, enhanced synthesis of AAT was observed

The normal daily rate of synthesis of AAT is approximately 34 mg/kg body weight and the protein is cleared with a half-life of 3 to 5 days. This results in high plasma concentrations ranging from 0.9 to 2 mg/ml when measured by nephelometry. In addition to high circulating levels in blood, AAT is also present in saliva, tears, milk, semen, urine and bile (Berman et al., 1973; Chowanadisai & Lonnerdal, 2002; Huang, 2004; Janciauskiene et al., 1996; Poortmans & Jeanloz, 1968). The distribution of the protein in the tissues is not uniform. For example, in the epithelial lining fluid of the lower respiratory tract its concentration is approximately 10% of

As an acute-phase reactant, circulating AAT levels increase rapidly (3 to 4 fold) in response to inflammation or infection. The concentration of AAT in plasma also increases during oral contraceptive therapy and pregnancy. During an inflammatory response, tissue concentra‐ tions of AAT may increase as much as 11-fold as a result of local synthesis by resident or invading inflammatory cells (Boskovic & Twining, 1997). Blood monocytes and alveolar macrophages can contribute to tissue AAT levels in response to inflammatory cytokines (IL-6, IL-1 and TNFα) and endotoxins (Knoell et al., 1998; Perlmutter & Punsal, 1988). Recent data demonstrate that AAT expression by alpha and delta cells of human islets (Bosco et al., 2005) and intestinal epithelial cells (Faust et al., 2001) is also enhanced by pro-inflammatory cytokines. AAT synthesis by corneal epithelium, on the other hand, appears to be under the influence of retinol, IL-2, fibroblast growth factor-2, and insulin-like growth factor-I (Boskovic & Twining, 1997; Boskovic & Twining, 1998). Oncostatin M, a member of the IL-6 family was shown to induce AAT production by human bronchial epithelial cells. This effect of oncostatin M was in turn modulated by TGF-β and IFN-γ at both the protein and mRNA level. IFN-γ decreased oncostatin M-induced AAT production whilst TGF-β induced a significant and synergistic up-regulation of AAT that was not observed in a hepatocyte cell line (Boutten et al., 1998). Study by Shin and coworkers (Shin et al., 2011) have demonstrated that nasal lavage fluids from the patients with allergic rhinitis contains AAT and that the levels of nasal AAT markedly increase in response to allergenic stimulation. This response seems to be closely associated with the activation of eosinophils induced by allergen-specific IgA. In allergen-

(Perlmutter et al., 1988).

2 Acute Phase Proteins

plasma levels (Janciauskiene, 2001).

According to recent studies, activated neutrophils and eosinophils can store and secrete AAT, which plays a role in protection of tissues at local inflammation sites (Johansson et al., 2001; Paakko et al., 1996). Furthermore, Clemmensen and coworkers found that the mRNA for AAT increases during maturity of the myeloid cell precursors and is even higher in blood neutro‐ phils. This in itself is quite remarkable as blood neutrophils are generally considered tran‐ scriptionally inactive, but it is even more striking that the transcriptional activity of the AAT gene increases further when neutrophils migrate into tissues (Clemmensen et al., 2011). Moreover, circulating AAT produced by liver cells can enter granulocytes and is stored in the secretory vesicles (Borregaard et al., 1992).

## **3. Protective anti-inflammatory, immunomodulatory and antimicrobial effects of alpha1-antitrypsin**

Findings from different experimental models provide clear evidence that AAT expresses broad anti-inflammatory and immunoregulatory activities (Figure 1). AAT has been reported to inhibit neutrophil superoxide production, adhesion, and chemotaxis, to enhance insulininduced mitogenesis in cell lines and to induce IL-1 receptor antagonist, a negative regulator to IL-1 signalling, in blood monocytes and neutrophils (Tuder et al., 2010). Findings that AAT enhances the synthesis of transferrin receptor and ferritin revealed a role of AAT in iron metabolism (Graziadei et al., 1997). In murine models, exogenous human AAT protects islet cell allografts from rejection and increase survival in an allogeneic marrow transplantation models. In other models AAT therapy protects against TNF-α / endotoxin induced lethality, cigarette smoke induced emphysema and inflammation and even suppressed bacterial proliferation during infections ((Lewis, 2012), review). Furthermore, human AAT given to mice during renal ischemia–reperfusion (I/R) injury lessens tissue injury and attenuated organ dysfunction (Daemen et al., 2000).

These beneficial impacts of AAT are incompletely understood, although exscinding knowl‐ edge suggests that AAT promotes a switch from pro-inflammatory to anti-inflammatory pathways necessary for the resolution of inflammation.

AAT has long been thought of as a main inhibitor of neutrophil elastase, proteinase 3, and other serine proteases released from activated human neutrophils during an inflammatory response. In fact, the rate of formation of the AAT/neutrophil elastase inhibitory complex is one of the fastest known for serpins (6.5x 107 M-1 s-1) (Gettins, 2002). The structure of AAT consists of three β-sheets (A, B, C) and 9 α-helices (A-I). The inhibitory active conformation of AAT like for other serine protease inhibitors represents a metastable state, characterized by an exposed reactive center loop that acts as bait for the target enzyme (Stocks et al., 2012). Cleavage of the scissile bond in the loop results in a large conformational change in which the reactive site loop migrates and is inserted into the pre-existing β-sheet A forming a very stable complex between the inhibitor and the protease. This reaction results in a rapid and irreversible inactivation of both AAT and its target protease.

reported that oxidized AAT (again without elastase inhibitory activity) reduces endotoxininduced TNF-α, IL-8, MCP-1, and IL-1 release in human monocytes *in vitro* (Janciauskiene et al., 2004). We also found that the pattern of gene expression regulated in human primary lung endothelial cells by native and oxidized AAT was similar with neither inducing pro-inflam‐

Immunoregulatory Properties of Acute Phase Proteins — Specific Focus on α1-Antitrypsin

http://dx.doi.org/10.5772/56393

5

Moreover, a specific short carboxyl terminal peptide of AAT which doesn´t inhibit elastase is a more potent inhibitor of LPS-induced TNF-α and IL-8 production than native AAT

Recently, we examined the effects of plasma purified AAT in LPS-induced acute lung injury in wild-type (WT) and neutrophil elastase-deficient mice as well as in neutrophils isolated from the bone marrow of WT and elastase-deficient mice. Analyses of lung lavage fluids and tissues revealed that, regardless from the mouse strain, AAT induced a 50% decrease in LPSinduced neutrophil counts as well as a reduction in the lavage fluid levels of IL-8 and TNF-α. Furthermore, AAT inhibited the ability of LPS to increase TNF-α, DNA damage-inducible transcript 3 and X-box binding protein 1 gene expression in the lung parenchyma (Jonigk et

These findings provide clear evidence that inhibition of elastase is not the sole mechanism behind the anti-inflammatory and immunoregulatory activities of AAT. The responsible

**4. Interaction with other macromolecules and cell surface 'receptors' and**

AAT shows the property to interact with other proteins. For example, in sera from patients with myeloma and Bence-Jones proteinemia complexes between AAT and the kappa light chain of immunoglobulins were detected (Laurell & Thulin, 1975). In plasma from diabetic subjects, complexes between AAT and factor Xia, AAT and heat shock protein-70 as well as AAT and glucose were detected (Murakami et al., 1993; Finotti & Pagetta, 2004; Hall et al., 1986). Moreover, complexes between AAT and immunoglobulin A have been detected in the sera and synovial fluid of patients with rheumatoid arthritis, systemic lupus erythematosus and ankylosing spondylitis (Adam & Bieth, 1996). Localization of AAT-low-density-lipopro‐ tein (LDL) complexes in atherosclerotic lesions and enhanced degradation of AAT-LDL by macrophages suggested the involvement of the complex in atherogenesis (Mashiba et al., 2001). Earlier studies have shown that cellular internalization and degradation of AAT-elastase-, or AAT-trypsin-complexes, but not of the native form of AAT, is mediated by serpin-enzyme complex (SEC) receptor (Perlmutter et al., 1990), low-density lipoprotein receptor related protein (Poller et al., 1995) and very-low-density lipoprotein receptor which require intact raft

Recent studies provide new evidence that clathrin-mediated endocytosis (Sohrab et al., 2009) and the caveolar pathway (Aldonyte et al., 2008) and Fc receptor(s) (Bergin et al., 2010) might

matory gene expression (Subramaniyam et al., 2008).

(Amelinckx et al., 2011; Subramaniyam et al., 2006)

molecular mechanisms remain to be elucidated.

lipid environment (Wu & Gonias, 2005; Yoon et al., 2007).

al., PNAS, in press).

**signalling mechanisms**

As an inhibitor, AAT also shows true substrate-like behaviour and cleavage without complex formation. Novel studies show that AAT, without forming complexes, inhibits the activity of gelatinase B (MMP9) and caspases-1 and -3 that play an essential role in cell apoptosis. AAT also inactivates the catalytic domain of matriptase, a cell surface serine protease involved in the activation of epithelial sodium channels. In addition, recent evidence has emerged on the ability of AAT to inhibit the matrix metalloprotease, ADAM-17 (Bergin et al., 2010) and aspartic-cysteine protease, calpain I (Al-Omari et al., 2011). Calpain I activity has been implicated in neutrophil apoptosis (Chen et al., 2006), chemotaxis (Lokuta et al., 2003) and adhesion (Wiemer et al., 2010). In fact, AAT inhibits neutrophil adhesion, chemotaxis (Al-Omari et al., 2011; Bergin et al., 2010) and apoptosis (Zhang et al., 2007). The mechanism behind these latter effects of AAT might be directly linked to its ability to inhibit calpain I activity.

So far, it is assumed that anti-inflammatory and immunomodulatory functions of AAT are dependent on its metastable native conformation (with inhibitory activity); however, this has not been proven. Earlier studies by Churg and collaborators have demonstrated that oxidized AAT (without elastase inhibitory activity) is effective in preventing neutrophil influx and lung tissue damage in a silica-induced inflammation model in mice (Churg et al., 2001). We also

**Figure 1.** Selected anti-inflammatory and immunoregulatory activities of AAT.

reported that oxidized AAT (again without elastase inhibitory activity) reduces endotoxininduced TNF-α, IL-8, MCP-1, and IL-1 release in human monocytes *in vitro* (Janciauskiene et al., 2004). We also found that the pattern of gene expression regulated in human primary lung endothelial cells by native and oxidized AAT was similar with neither inducing pro-inflam‐ matory gene expression (Subramaniyam et al., 2008).

between the inhibitor and the protease. This reaction results in a rapid and irreversible

As an inhibitor, AAT also shows true substrate-like behaviour and cleavage without complex formation. Novel studies show that AAT, without forming complexes, inhibits the activity of gelatinase B (MMP9) and caspases-1 and -3 that play an essential role in cell apoptosis. AAT also inactivates the catalytic domain of matriptase, a cell surface serine protease involved in the activation of epithelial sodium channels. In addition, recent evidence has emerged on the ability of AAT to inhibit the matrix metalloprotease, ADAM-17 (Bergin et al., 2010) and aspartic-cysteine protease, calpain I (Al-Omari et al., 2011). Calpain I activity has been implicated in neutrophil apoptosis (Chen et al., 2006), chemotaxis (Lokuta et al., 2003) and adhesion (Wiemer et al., 2010). In fact, AAT inhibits neutrophil adhesion, chemotaxis (Al-Omari et al., 2011; Bergin et al., 2010) and apoptosis (Zhang et al., 2007). The mechanism behind these latter effects of AAT might be directly linked to its ability to inhibit calpain I activity. So far, it is assumed that anti-inflammatory and immunomodulatory functions of AAT are dependent on its metastable native conformation (with inhibitory activity); however, this has not been proven. Earlier studies by Churg and collaborators have demonstrated that oxidized AAT (without elastase inhibitory activity) is effective in preventing neutrophil influx and lung tissue damage in a silica-induced inflammation model in mice (Churg et al., 2001). We also

inactivation of both AAT and its target protease.

4 Acute Phase Proteins

**Figure 1.** Selected anti-inflammatory and immunoregulatory activities of AAT.

Moreover, a specific short carboxyl terminal peptide of AAT which doesn´t inhibit elastase is a more potent inhibitor of LPS-induced TNF-α and IL-8 production than native AAT (Amelinckx et al., 2011; Subramaniyam et al., 2006)

Recently, we examined the effects of plasma purified AAT in LPS-induced acute lung injury in wild-type (WT) and neutrophil elastase-deficient mice as well as in neutrophils isolated from the bone marrow of WT and elastase-deficient mice. Analyses of lung lavage fluids and tissues revealed that, regardless from the mouse strain, AAT induced a 50% decrease in LPSinduced neutrophil counts as well as a reduction in the lavage fluid levels of IL-8 and TNF-α. Furthermore, AAT inhibited the ability of LPS to increase TNF-α, DNA damage-inducible transcript 3 and X-box binding protein 1 gene expression in the lung parenchyma (Jonigk et al., PNAS, in press).

These findings provide clear evidence that inhibition of elastase is not the sole mechanism behind the anti-inflammatory and immunoregulatory activities of AAT. The responsible molecular mechanisms remain to be elucidated.

## **4. Interaction with other macromolecules and cell surface 'receptors' and signalling mechanisms**

AAT shows the property to interact with other proteins. For example, in sera from patients with myeloma and Bence-Jones proteinemia complexes between AAT and the kappa light chain of immunoglobulins were detected (Laurell & Thulin, 1975). In plasma from diabetic subjects, complexes between AAT and factor Xia, AAT and heat shock protein-70 as well as AAT and glucose were detected (Murakami et al., 1993; Finotti & Pagetta, 2004; Hall et al., 1986). Moreover, complexes between AAT and immunoglobulin A have been detected in the sera and synovial fluid of patients with rheumatoid arthritis, systemic lupus erythematosus and ankylosing spondylitis (Adam & Bieth, 1996). Localization of AAT-low-density-lipopro‐ tein (LDL) complexes in atherosclerotic lesions and enhanced degradation of AAT-LDL by macrophages suggested the involvement of the complex in atherogenesis (Mashiba et al., 2001).

Earlier studies have shown that cellular internalization and degradation of AAT-elastase-, or AAT-trypsin-complexes, but not of the native form of AAT, is mediated by serpin-enzyme complex (SEC) receptor (Perlmutter et al., 1990), low-density lipoprotein receptor related protein (Poller et al., 1995) and very-low-density lipoprotein receptor which require intact raft lipid environment (Wu & Gonias, 2005; Yoon et al., 2007).

Recent studies provide new evidence that clathrin-mediated endocytosis (Sohrab et al., 2009) and the caveolar pathway (Aldonyte et al., 2008) and Fc receptor(s) (Bergin et al., 2010) might be responsible for the interaction and entry of native AAT into the cell. Experimental studies have shown that various APPs, like C-reactive protein (CRP), interact with lipid rafts (Ji et al., 2009) and therefore, gave support for the hypothesis that APPs-lipid raft interaction may be a putative mechanism responsible for the diverse activities of APPs during inflammation.

unknown. As a matter of fact, AAT is shown to interact with the transferrin receptor (Graziadei et al., 1994) which is constantly internalized via endocytic vesicles that fuse with early endosomes and returns to the plasma membrane through recycling endosomes (Harding et al., 1983). Thus it cannot be excluded that excess of internalized AAT trafficking occurs via

Immunoregulatory Properties of Acute Phase Proteins — Specific Focus on α1-Antitrypsin

Nevertheless, incorporation of AAT into membranes and transient depletion of cholesterol can affect recruitment of TLRs into lipid rafts subsequently desensitizing signalling by bacterial endotoxins and resulting in consequent reduction of the pro-inflammatory response. In support, human innate immune cells stimulated with bacterial lipopolysaccharide (LPS) in the presence of AAT show suppressed TNFα, IL-8, IL-12 and IL-1β, but enhanced IL-10 production

> **ILͲ1, ILͲ6, ILͲ8, ILͲ12, TNFͲɲ ILͲ10, ILͲ1Ra**

**AAT TLR**

MyD88

**IRAK1 TRAF6 ERK1/2 Akt IKK NFͲʃB**

lipid droplets **LPS** LBP

CD14

http://dx.doi.org/10.5772/56393

7

transferrin receptor pathway.

**LPS** LBP

CD14

LPS signalling and cell activation.

(Figure 2) (Janciauskiene et al., 2004; Nita et al., 2005).

**TLR**

A B

Lipid raft

Cholesterol

**Figure 2.** Hypothesis of the immune modulator effect of AAT. A: LPS signalling and cell activation; B: effects of AAT on

In general, this hypothesis provides the basis for future studies linking bioactivities of acute phase proteins to signalling pathways associated with lipid rafts. Lipid rafts are therapeutic targets for various diseases and studies on physiological significance of interaction between

The clinical relevance of AAT is highlighted in individuals with inherited deficiency in circulating AAT who have an increased susceptibility to early onset pulmonary emphysema, and liver as well as pancreatic diseases. The most interesting AAT variants associated with deficiency are the S and Z genes commonly found in Europeans. Both S and Z AAT result from

**IRAK1 TRAF6 ERK1/2 Akt IKK NFͲʃB**

**ILͲ1, ILͲ6, ILͲ8, ILͲ12, TNFͲɲ ILͲ10, ILͲ1Ra**

acute phase proteins and lipid rafts of great importance.

**5. Diseases associated with AAT deficiency**

MyD88

Lipid rafts are dynamic assemblies of proteins and lipids that play a central role in various cellular processes, including membrane sorting and trafficking, cell polarization, and signal transduction (Baird et al., 1999; Janes et al., 2000; Zhu et al., 2006). Biochemical and cellbiological studies have identified cholesterol as a key factor determining raft and related structure (e.g., caveolae) stability and organization in mammalian cell membranes, and have shown that the equilibrium between free and raft cholesterol plays a critical role in lipid raft function and cell signalling (Golub et al., 2004; Gombos et al., 2006). Many proteins involved in signal transduction, such as Src family kinases, G proteins, growth factor receptors, mitogenactivated protein kinase and protein kinase C are predominantly found in lipid rafts, which act as signaling platforms by bringing together (i.e., colocalizing) various signaling compo‐ nents (Simons & Toomre, 2000).

Our studies on the putative role of lipid rafts and lipid raft cholesterol for AAT entry into monocytes revealed that exogenously added AAT becomes translocated into lipid rafts in the same fraction as the lipid raft marker flotillin (Slaughter et al., 2003; Subramaniyam et al., 2010). It is well documented that plasma membranes of mammalian cells contain a 30–50% molar fraction of cholesterol (Warnock et al., 1993), which is the dynamic glue for the lipid raft assembly (Simons & Toomre, 2000). Taken with the finding that exogenous AAT localizes in the lipid raft prompted us to examine whether altering the integrity of the lipid raft cholesterol would affect AAT-monocyte association. In fact, AAT association with monocytes was remarkably inhibited by various cholesterol depleting/efflux-stimulating agents such as nystatin, filipin, methyl-betacyclodextrin, oxidized low-density lipoprotein and high density lipoproteins, and conversely, enhanced by free cholesterol. We had previously identified that AAT can directly interact with free cholesterol in vitro (Janciauskiene & Eriksson, 1993). In support, we confirmed that AAT /monocyte association *per se* depletes lipid raft cholesterol as characterized by the activation of extracellular signal-regulated kinase 2, increased HMG-CoA reductase expression, formation of cytosolic lipid droplets, and a complete inhibition of oxidized low-density lipoprotein and oxidized phopspholipid uptake by monocytes (Subra‐ maniyam et al., 2010).

Lipid rafts act as platforms, bringing together molecules essential for the activation of immune cells, but also separating such molecules when the conditions for activation are not appropriate (Ehrenstein et al., 2005). We hypothesize that AAT/ lipid raft interaction and cholesterol depletion contributes to re-organize membrane domains and facilitate the formation of compartment-specific signalling platforms. As a consequence, several events can occur intracellularly like transient release of calcium and Na+ /K+ -ATPase-EGFR-Src-caveolin-1 complex formation leading to an increased tyrosine phosphorylation of caveolin-1 and the activation of the Rac1-Cdc42-ERK cascade, and transient activation of hemoxygenase-1. It has been previously reported that exogenous AAT is rapidly internalized into the cells and is localized in the plasma membranes and in the cytoplasm (Sohrab et al., 2009). Whether internalized AAT is further trafficking to the interstitium or remains within the cells is unknown. As a matter of fact, AAT is shown to interact with the transferrin receptor (Graziadei et al., 1994) which is constantly internalized via endocytic vesicles that fuse with early endosomes and returns to the plasma membrane through recycling endosomes (Harding et al., 1983). Thus it cannot be excluded that excess of internalized AAT trafficking occurs via transferrin receptor pathway.

be responsible for the interaction and entry of native AAT into the cell. Experimental studies have shown that various APPs, like C-reactive protein (CRP), interact with lipid rafts (Ji et al., 2009) and therefore, gave support for the hypothesis that APPs-lipid raft interaction may be a putative mechanism responsible for the diverse activities of APPs during inflammation.

Lipid rafts are dynamic assemblies of proteins and lipids that play a central role in various cellular processes, including membrane sorting and trafficking, cell polarization, and signal transduction (Baird et al., 1999; Janes et al., 2000; Zhu et al., 2006). Biochemical and cellbiological studies have identified cholesterol as a key factor determining raft and related structure (e.g., caveolae) stability and organization in mammalian cell membranes, and have shown that the equilibrium between free and raft cholesterol plays a critical role in lipid raft function and cell signalling (Golub et al., 2004; Gombos et al., 2006). Many proteins involved in signal transduction, such as Src family kinases, G proteins, growth factor receptors, mitogenactivated protein kinase and protein kinase C are predominantly found in lipid rafts, which act as signaling platforms by bringing together (i.e., colocalizing) various signaling compo‐

Our studies on the putative role of lipid rafts and lipid raft cholesterol for AAT entry into monocytes revealed that exogenously added AAT becomes translocated into lipid rafts in the same fraction as the lipid raft marker flotillin (Slaughter et al., 2003; Subramaniyam et al., 2010). It is well documented that plasma membranes of mammalian cells contain a 30–50% molar fraction of cholesterol (Warnock et al., 1993), which is the dynamic glue for the lipid raft assembly (Simons & Toomre, 2000). Taken with the finding that exogenous AAT localizes in the lipid raft prompted us to examine whether altering the integrity of the lipid raft cholesterol would affect AAT-monocyte association. In fact, AAT association with monocytes was remarkably inhibited by various cholesterol depleting/efflux-stimulating agents such as nystatin, filipin, methyl-betacyclodextrin, oxidized low-density lipoprotein and high density lipoproteins, and conversely, enhanced by free cholesterol. We had previously identified that AAT can directly interact with free cholesterol in vitro (Janciauskiene & Eriksson, 1993). In support, we confirmed that AAT /monocyte association *per se* depletes lipid raft cholesterol as characterized by the activation of extracellular signal-regulated kinase 2, increased HMG-CoA reductase expression, formation of cytosolic lipid droplets, and a complete inhibition of oxidized low-density lipoprotein and oxidized phopspholipid uptake by monocytes (Subra‐

Lipid rafts act as platforms, bringing together molecules essential for the activation of immune cells, but also separating such molecules when the conditions for activation are not appropriate (Ehrenstein et al., 2005). We hypothesize that AAT/ lipid raft interaction and cholesterol depletion contributes to re-organize membrane domains and facilitate the formation of compartment-specific signalling platforms. As a consequence, several events can occur

complex formation leading to an increased tyrosine phosphorylation of caveolin-1 and the activation of the Rac1-Cdc42-ERK cascade, and transient activation of hemoxygenase-1. It has been previously reported that exogenous AAT is rapidly internalized into the cells and is localized in the plasma membranes and in the cytoplasm (Sohrab et al., 2009). Whether internalized AAT is further trafficking to the interstitium or remains within the cells is

/K+


intracellularly like transient release of calcium and Na+

nents (Simons & Toomre, 2000).

6 Acute Phase Proteins

maniyam et al., 2010).

Nevertheless, incorporation of AAT into membranes and transient depletion of cholesterol can affect recruitment of TLRs into lipid rafts subsequently desensitizing signalling by bacterial endotoxins and resulting in consequent reduction of the pro-inflammatory response. In support, human innate immune cells stimulated with bacterial lipopolysaccharide (LPS) in the presence of AAT show suppressed TNFα, IL-8, IL-12 and IL-1β, but enhanced IL-10 production (Figure 2) (Janciauskiene et al., 2004; Nita et al., 2005).

**Figure 2.** Hypothesis of the immune modulator effect of AAT. A: LPS signalling and cell activation; B: effects of AAT on LPS signalling and cell activation.

In general, this hypothesis provides the basis for future studies linking bioactivities of acute phase proteins to signalling pathways associated with lipid rafts. Lipid rafts are therapeutic targets for various diseases and studies on physiological significance of interaction between acute phase proteins and lipid rafts of great importance.

## **5. Diseases associated with AAT deficiency**

The clinical relevance of AAT is highlighted in individuals with inherited deficiency in circulating AAT who have an increased susceptibility to early onset pulmonary emphysema, and liver as well as pancreatic diseases. The most interesting AAT variants associated with deficiency are the S and Z genes commonly found in Europeans. Both S and Z AAT result from single amino acid substitutions. In the S variant there is a substitution of a valine residue for glutamate at position 264 (Val264Glu) (Curiel et al., 1989). The Z mutation (Glu342Lys) results from the substitution of a positively charged lysine for a negatively charged glutamine at the base of the reactive centre. Severe ZZ deficiency of AAT is characterized by a decrease in serum AAT levels below a protective threshold of 11 mmol/L (Fregonese et al., 2008; Hubbard & Crystal, 1988) and is associated with increased but variable risk for the development of lung emphysema (Janciauskiene et al., 2010).

Inherited AAT deficiency is occasionally associated with antiproteinase-3-associated vasculitis (Wegener's granulomatosis), necrotizing panniculitis and aneurysms of the abdominal aorta and brain arteries. AATD has also been associated with a number of other inflammatory diseases, although the association is only moderate or weak. These include bronchial asthma, bronchiectasis, rheumatoid arthritis, psoriasis, chronic urticaria, glomerulonephritis, pancrea‐ titis and pancreatic tumors, multiple sclerosis, fibromyalgia and other conditions reported

Immunoregulatory Properties of Acute Phase Proteins — Specific Focus on α1-Antitrypsin

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9

Remarkably, associations have been found between reduced plasma AAT levels and HIV-1 infection, hepatitis, diabetes mellitus, systemic vasculitis and necrotizing panniculitis (Lewis,

Given the concept of the protease/antiprotease imbalance role in causing emphysema, augmentation of circulating AAT was introduced 30 years ago to treat emphysema patients with severe ZZ deficiency of AAT. Augmentation therapy with human AAT has been performed for over a decade in the United States and in a number of European countries. Worldwide, more than 4,000 patients are currently receiving regular AAT substitution. Most patients receive weekly intravenous application of 3–5 g AAT (60 mg/kg body weight), which is derived from pooled human plasma. Patients with emphysema may be considered for augmentation if their serum concentration is below 0.8 g/L (≤ 11 µmol/L), if their postbronchodilator FEV1 is between 35% and 60% of predicted, or if their annual decline of FEV1

Substitution therapy has been studied in a few clinical trials, and the evidence for its efficacy is limited. A last double-blind, placebo-controlled trial EXACTLE (the EXAcerbations and computed tomography (CT) scan as Lung Endpoints), was designed to explore the use of CT densitometry as an outcome measure for the assessment of the effect of AAT augmentation therapy on the progression of emphysema in individuals with inherited AAT deficiency (Stockley et al., 2010). This study showed that the rate of lung density decline was reduced by the intravenous augmentation therapy, and although the exacerbation frequency was unal‐

To date fewer than 50 cases of panniculitis associated with various phenotypes of AAT deficiency have been reported (Piliang & Stoller, 2008). The clinical features that distinguish the AAT deficiency-associated panniculitis include higher frequency of ulceration, a vigorous neutrophilic response and histological evidence of both necrosis and elastin breakdown (Smith et al., 1989). In support of AAT deficiency as a contributor to the inflammatory pathogenesis of panniculitis, a few case reports provide evidence that the infusion of purified pooled human AAT induces a rapid clinical resolution of panniculitis (Gross et al., 2009; O'Riordan et al.,

The putative association between AAT deficiency and vasculitis (Wegener's granulomatosis) is based on the fact that AAT deficiency variants occur more frequently among individuals with multisystem vascultitis (anti-neutrophilic cytoplasmic antibodies (C-ANCA) or anti-

tered by this treatment, a reduction in severity of exacerbations was observed.

occasionally (Janciauskiene et al., 2011)

2012; Tuder et al., 2010).

1997; Furey et al., 1996).

**5.1. AAT augmentation therapy**

is more than 100 mL (for review see (Mohanka et al., 2012))

From retrospective studies we also know that up to 25% of those with severe AAT deficiency will suffer from liver cirrhosis and for liver cancer in late adulthood (Propst et al., 1994). Also heterozygous AAT deficiency is a cofactor in the development of chronic liver diseases (Kok et al., 2007). Adults with AAT deficiency–associated genotypes develop liver disease less frequently than pulmonary manifestations. However, AAT is a relevant cause for liver cirrhosis, after viral hepatitis, alcohol abuse, and chronic cholangitis. Other factors that can also predispose AAT-deficient individuals to liver disease are male sex and obesity (Bowlus et al., 2005).The underlying cause may be the intrahepatic accumulation of polymerized AAT molecules. The polymers of Z-mutant AAT can be identified in endoplasmic reticulum by the electron microscopy as diastase resistant inclusion bodies reacting positively with PASstaining (Periodic acid-Schiff). Intracellular inclusion bodies in the liver have also been observed with other polymer-forming phenotypes of AAT deficiency (Mmalton (52Phe del), and Siiyama (Ser53Phe) (Tuder et al., 2010).

**Figure 3.** Schematic diagram depicting the role of polymers of α1-Antitrypsin (AAT) in the development of liver and lung diseases. A) AAT polymers stained with rabbit polyclonal antibody against human AAT, B) AAT polymers stained with mouse monoclonal ATZ11 antibody against human Z polymers.

Inherited AAT deficiency is occasionally associated with antiproteinase-3-associated vasculitis (Wegener's granulomatosis), necrotizing panniculitis and aneurysms of the abdominal aorta and brain arteries. AATD has also been associated with a number of other inflammatory diseases, although the association is only moderate or weak. These include bronchial asthma, bronchiectasis, rheumatoid arthritis, psoriasis, chronic urticaria, glomerulonephritis, pancrea‐ titis and pancreatic tumors, multiple sclerosis, fibromyalgia and other conditions reported occasionally (Janciauskiene et al., 2011)

Remarkably, associations have been found between reduced plasma AAT levels and HIV-1 infection, hepatitis, diabetes mellitus, systemic vasculitis and necrotizing panniculitis (Lewis, 2012; Tuder et al., 2010).

#### **5.1. AAT augmentation therapy**

single amino acid substitutions. In the S variant there is a substitution of a valine residue for glutamate at position 264 (Val264Glu) (Curiel et al., 1989). The Z mutation (Glu342Lys) results from the substitution of a positively charged lysine for a negatively charged glutamine at the base of the reactive centre. Severe ZZ deficiency of AAT is characterized by a decrease in serum AAT levels below a protective threshold of 11 mmol/L (Fregonese et al., 2008; Hubbard & Crystal, 1988) and is associated with increased but variable risk for the development of lung

From retrospective studies we also know that up to 25% of those with severe AAT deficiency will suffer from liver cirrhosis and for liver cancer in late adulthood (Propst et al., 1994). Also heterozygous AAT deficiency is a cofactor in the development of chronic liver diseases (Kok et al., 2007). Adults with AAT deficiency–associated genotypes develop liver disease less frequently than pulmonary manifestations. However, AAT is a relevant cause for liver cirrhosis, after viral hepatitis, alcohol abuse, and chronic cholangitis. Other factors that can also predispose AAT-deficient individuals to liver disease are male sex and obesity (Bowlus et al., 2005).The underlying cause may be the intrahepatic accumulation of polymerized AAT molecules. The polymers of Z-mutant AAT can be identified in endoplasmic reticulum by the electron microscopy as diastase resistant inclusion bodies reacting positively with PASstaining (Periodic acid-Schiff). Intracellular inclusion bodies in the liver have also been observed with other polymer-forming phenotypes of AAT deficiency (Mmalton (52Phe del), and

**1-Antitrypsin Deficiency Antitrypsin Deficiency**

**Plasma deficiency early onset emphysema** 

**Z m utation Glu Z m utation Glu<sup>342</sup>Lys<sup>342</sup>**

**Polymerization of Polymerization of 1-antitrypsin antitrypsin**

**Figure 3.** Schematic diagram depicting the role of polymers of α1-Antitrypsin (AAT) in the development of liver and lung diseases. A) AAT polymers stained with rabbit polyclonal antibody against human AAT, B) AAT polymers stained

emphysema (Janciauskiene et al., 2010).

8 Acute Phase Proteins

Siiyama (Ser53Phe) (Tuder et al., 2010).

**Intracellular accumulation Intracellular accumulation neonatal hepatitis, liver cirrhosis**

with mouse monoclonal ATZ11 antibody against human Z polymers.

Given the concept of the protease/antiprotease imbalance role in causing emphysema, augmentation of circulating AAT was introduced 30 years ago to treat emphysema patients with severe ZZ deficiency of AAT. Augmentation therapy with human AAT has been performed for over a decade in the United States and in a number of European countries. Worldwide, more than 4,000 patients are currently receiving regular AAT substitution. Most patients receive weekly intravenous application of 3–5 g AAT (60 mg/kg body weight), which is derived from pooled human plasma. Patients with emphysema may be considered for augmentation if their serum concentration is below 0.8 g/L (≤ 11 µmol/L), if their postbronchodilator FEV1 is between 35% and 60% of predicted, or if their annual decline of FEV1 is more than 100 mL (for review see (Mohanka et al., 2012))

Substitution therapy has been studied in a few clinical trials, and the evidence for its efficacy is limited. A last double-blind, placebo-controlled trial EXACTLE (the EXAcerbations and computed tomography (CT) scan as Lung Endpoints), was designed to explore the use of CT densitometry as an outcome measure for the assessment of the effect of AAT augmentation therapy on the progression of emphysema in individuals with inherited AAT deficiency (Stockley et al., 2010). This study showed that the rate of lung density decline was reduced by the intravenous augmentation therapy, and although the exacerbation frequency was unal‐ tered by this treatment, a reduction in severity of exacerbations was observed.

To date fewer than 50 cases of panniculitis associated with various phenotypes of AAT deficiency have been reported (Piliang & Stoller, 2008). The clinical features that distinguish the AAT deficiency-associated panniculitis include higher frequency of ulceration, a vigorous neutrophilic response and histological evidence of both necrosis and elastin breakdown (Smith et al., 1989). In support of AAT deficiency as a contributor to the inflammatory pathogenesis of panniculitis, a few case reports provide evidence that the infusion of purified pooled human AAT induces a rapid clinical resolution of panniculitis (Gross et al., 2009; O'Riordan et al., 1997; Furey et al., 1996).

The putative association between AAT deficiency and vasculitis (Wegener's granulomatosis) is based on the fact that AAT deficiency variants occur more frequently among individuals with multisystem vascultitis (anti-neutrophilic cytoplasmic antibodies (C-ANCA) or antiprotease-3 (PR-3) and glomerulonephritis (Esnault et al., 1993; O'Donoghue et al., 1993; Montanelli et al., 2002). Moreover, since AAT plays an important role in inhibiting PR3, it has been suggested that AAT deficiency could trigger an autoimmune response due to increased extracellular exposure to PR3 (Esnault et al., 1997). Alternatively, although unproven, it is conceivable that circulating Z AAT polymers could prompt a vascular response.

**Acute-phase protein Main biological function**

Fibronectin Wound healing Ferritin Iron binding Angiotensinogen Renin substrate

Complement factors: C3, C4, C9, factor B, C1 inhibitor, C4b-

binding protein, mannose-binding lectin

Coagulation and fibrinolysis factors: fibrinogen, plasminogen, tissue plasminogen activator, urokinase, protein S, vitronectin, plasminogen-activator inhibitor 1

**Proteins whose concentration decrease**

**Table 1.** Diverse functional activities of APPs.

**7. Haptoglobin**

C-reactive protein (CRP) Binding of phosphocholine (opsonin); immunoregulation

Ceruloplasmin Contains copper, has histaminase-and ferroxidase-activity;

Interleukin1-receptor antagonist modulates a variety of interleukin 1 related immune and

Alpha1-Antitrypsin (AAT) Inhibits proteolytic enzymes, immune-modulatory activity

Alpha2-HS glycoprotein Carrier protein, forms soluble complexes with calcium and

Haptoglobin (Hp) is constitutively present in the plasma (normal plasma levels 0.3 to 3.0 mg/ ml) and functions mainly as a scavenger protein for hemoglobin (Hb) that is released from

phosphate

Haptoglobin (Hp) Binds haemoglobin; binds to CD11b/CD18 integrines

Alpha1-acid glycoprotein (AGP) Influences T-cell function; binds steroids

Albumin Regulates the osmotic pressure of blood Transferrin Carrier protein, immunoregulation

Transthyretin Binds to aromatic compounds, carrier of retinol Alpha-fetoprotein Binds various cations, fatty acids and bilirubin

Alpha1-Antichymotrypsin (ACT) Inhibits proteolytic enzymes Alpha2-macroglobulin Inhibits proteolytic enzymes

Thyroxine-binding globulin Binds thyroid hormone

degrade extracellular matrix

Immunoregulatory Properties of Acute Phase Proteins — Specific Focus on α1-Antitrypsin

molecular structure of viruses

invading microbes, chemotaxis

scavenges Fe2+ and free radicals

inflammatory responses

Enhancing phagocytosis of antigens, attracting

Coagulation, degradation of blood clots, trapping

macrophages and neutrophils, lysis membranes of foreign cells, clumping of antigen-bearing agents, altering the

http://dx.doi.org/10.5772/56393

11

Alpha1-Acid Glycoprotein (AGP) Carry lipophilic compounds; immunoregulation Serum amyloid A (SAA) Recruitment of immune cells; activation enzymes that

**Proteins whose plasma concentration increase**

#### **5.2. New perspectives for use of the AAT augmentation therapy**

Administration of exogenous human plasma-derived AAT is used in various animal models to test the value of AAT augmentation. Several studies report that administration of AAT results in a change from the pro-inflammatory to the anti-inflammatory pathways that is necessary for the resolution of inflammation. Cystic Fibrosis (CF) is a condition caused by a known gene defect which predisposes individuals to chronic lung inflammation and infection. To date work has demonstrated that CF neutrophils secrete abnormally high levels of the proinflammatory cytokines, such as IL-8 and TNFα, and proteolytic enzymes specifically elastase which not only causes lung parenchymal damage, but can also perpetuate a vicious cycle of inflammation by inducing expression of the neutrophil chemoattractant, IL-8, from bronchial epithelial cells. Therefore, the interest in the application of treatment with inhaled AAT in CF lung disease is discussed (Siekmeier, 2010)

Based on preclinical and clinical studies, it is suggested that AAT therapy can be successfully used for non-deficient individuals with Type-1 and Type-2 diabetes, acute myocardial infarction, rheumatoid arthritis, inflammatory bowel disease, cystic fibrosis, transplant rejection, graft-versus-host-disease and multiple sclerosis. AAT also appears to be antibacterial and an inhibitor of viral infections, such as influenza and HIV, and is currently evaluated in clinical trials for Type-1 diabetes, cystic fibrosis and graft-versus-host-disease (Blanco et al., 2011; Lewis, 2012). New experimental approaches show that AAT therapy might be an option for arthritis treatment in a combination with doxycycline (Grimstein et al., 2010)

Thus, AAT can be used as potential treatment for a broad spectrum of inflammatory and immune-mediated diseases. Future treatment developments include gene therapy (via injections of viral or non-viral vector systems carrying the SERPINA1-cDNA), strategies to inhibit intra-hepatic AAT polymerization by small chemicals and chaperons, and inhibition of neutrophil elastase by using small molecules. Inhaled application of AAT is currently under development by several companies.

## **6. Immunoregulatory properties of other APPs**

The change in the concentrations of APPs is universally used to monitor the course of the disease, independently of its nature (Parra et al., 2006). However, specific APPs can also modify inflammatory responses. The spectrum of action of various APPs extends to regulation of leukocyte migration, adhesion and production of inflammatory mediators, control of ion channels and mucus secretion, and modulation of other host defence mechanisms (table 1).


**Table 1.** Diverse functional activities of APPs.

## **7. Haptoglobin**

protease-3 (PR-3) and glomerulonephritis (Esnault et al., 1993; O'Donoghue et al., 1993; Montanelli et al., 2002). Moreover, since AAT plays an important role in inhibiting PR3, it has been suggested that AAT deficiency could trigger an autoimmune response due to increased extracellular exposure to PR3 (Esnault et al., 1997). Alternatively, although unproven, it is

Administration of exogenous human plasma-derived AAT is used in various animal models to test the value of AAT augmentation. Several studies report that administration of AAT results in a change from the pro-inflammatory to the anti-inflammatory pathways that is necessary for the resolution of inflammation. Cystic Fibrosis (CF) is a condition caused by a known gene defect which predisposes individuals to chronic lung inflammation and infection. To date work has demonstrated that CF neutrophils secrete abnormally high levels of the proinflammatory cytokines, such as IL-8 and TNFα, and proteolytic enzymes specifically elastase which not only causes lung parenchymal damage, but can also perpetuate a vicious cycle of inflammation by inducing expression of the neutrophil chemoattractant, IL-8, from bronchial epithelial cells. Therefore, the interest in the application of treatment with inhaled AAT in CF

Based on preclinical and clinical studies, it is suggested that AAT therapy can be successfully used for non-deficient individuals with Type-1 and Type-2 diabetes, acute myocardial infarction, rheumatoid arthritis, inflammatory bowel disease, cystic fibrosis, transplant rejection, graft-versus-host-disease and multiple sclerosis. AAT also appears to be antibacterial and an inhibitor of viral infections, such as influenza and HIV, and is currently evaluated in clinical trials for Type-1 diabetes, cystic fibrosis and graft-versus-host-disease (Blanco et al., 2011; Lewis, 2012). New experimental approaches show that AAT therapy might be an option

Thus, AAT can be used as potential treatment for a broad spectrum of inflammatory and immune-mediated diseases. Future treatment developments include gene therapy (via injections of viral or non-viral vector systems carrying the SERPINA1-cDNA), strategies to inhibit intra-hepatic AAT polymerization by small chemicals and chaperons, and inhibition of neutrophil elastase by using small molecules. Inhaled application of AAT is currently under

The change in the concentrations of APPs is universally used to monitor the course of the disease, independently of its nature (Parra et al., 2006). However, specific APPs can also modify inflammatory responses. The spectrum of action of various APPs extends to regulation of leukocyte migration, adhesion and production of inflammatory mediators, control of ion channels and mucus secretion, and modulation of other host defence mechanisms (table 1).

for arthritis treatment in a combination with doxycycline (Grimstein et al., 2010)

conceivable that circulating Z AAT polymers could prompt a vascular response.

**5.2. New perspectives for use of the AAT augmentation therapy**

lung disease is discussed (Siekmeier, 2010)

10 Acute Phase Proteins

development by several companies.

**6. Immunoregulatory properties of other APPs**

Haptoglobin (Hp) is constitutively present in the plasma (normal plasma levels 0.3 to 3.0 mg/ ml) and functions mainly as a scavenger protein for hemoglobin (Hb) that is released from erythrocytes. Hp-Hb complexes are rapidly cleared from the circulation predominantly in the liver (Kupfer cells) expressing the Hp-Hb receptor CD163 (Graversen et al., 2002). Hp not only prevents loss of Hb/iron by renal excretion and protects from iron-driven oxidative tissue damage, but also acts as a bacteriostatic protein. Hence, Hp restricts access of bacteria to iron that is essential for bacterial growth.

pathologies. Hp gene has been studied as a candidate gene for rheumatoid arthritis, systemic lupus erythematosus, primary sclerosing cholangitis, inflammatory bowel disease and

Immunoregulatory Properties of Acute Phase Proteins — Specific Focus on α1-Antitrypsin

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13

In human two allels code for Hp1 and Hp2 proteins resulting in Hp1-1, Hp2-2 homozygous and Hp2-1 heterozygous genotypes. Clinical studies show that Hp2-2 individuals suffering from diabetes mellitus have a higher risk of vascular complications, especially diabetic nephropathy, compared to Hp2-1 and Hp1-1 individuals (Asleh & Levy, 2005). Epidemiolog‐ ical data also show that Hp2-2 genotype is a major determinant of susceptibility to diabetic

Functional differences between Hp1-1 and Hp2-2 on molecular level can be in part explained by the finding that the Hp1-1-Hb complex is endocytosed more rapidly by the CD163 pathway than Hp2-2-Hb. This results in a more effective clearance of Hb and less oxidative stress in Hp1-1 individuals (Asleh et al., 2003). Moreover, Hp2-2 individuals show greater immuno‐ logical reactivity including higher antibody titers after vaccination compared to Hp1-1 and Hp2-1 individuals (Nevo & Sutton, 1968). When compare to Hp2-2-Hb, Hp1-1-Hb is related to a much greater production of anti-inflammatory cytokines, therefore, Hp1-1Hp results in a more Th2 directed balance between Th1 (inflammatory) and Th2 (anti-inflammatory) T helper cells. Recent findings provide evidence that Hp1-1 individuals are better protected against oxidative stress and Hp1-1 seems to have higher immunomodulatory effects than Hp 2-2

Alpha-1-acid glycoprotein (AGP) also known as orosomucoid (ORM) is a heavily glycosylated (45 %) protein (Schmid et al., 1977) APG belongs to the immunocalin family, a lipocalin subfamily. Whereas the lipocalins function as carriers for small hydrophobic compounds, the immunocalins were shown to modulate inflammatory and immune responses (Logdberg & Wester, 2000; Hochepied et al., 2003). Though, the main biologic function of AGP remains unclear. Indeed, APG was found to be a major carrier for neutral and basic drugs in the blood (Kremer et al., 1988). Evidence also exists supporting a role for AGP in the maintenance of normal capillary permeability and selectivity by binding to the capillary vessel wall putatively as part of the glycocalyx, a dynamic endothelial surface layer of glycosaminoglycans, proteo‐ glycans and absorbed plasma proteins (Curry et al., 1989; Fournier et al., 2000; Pries et al., 2000). Moreover, AGP stabilizes the biological activity of plasminogen activator inhibitor-1

AGP is a positive acute phase protein that under normal conditions circulates in human plasma at a concentration of 0.6 -1.2 mg/ml and rises up to 2- to 7-fold during acute phase response (Colombo et al., 2006; Kremer et al., 1988). Besides the liver, a main source for the circulating AGP levels, production of AGP occurs in human microvascular endothelial cells, pneumo‐ cytes, alveolar macrophages, neutrophils, monocytes and B and T lymphocytes (Sirica et al., 1979; Sorensson et al., 1999; Crestani et al., 1998; Martinez Cordero et al., 2008; Fournier et al.,

diabetes mellitus type 2 (Marquez et al., 2012).

cardiovascular disease (Levy et al., 2010).

(Guetta et al., 2007).

(Smolarczyk et al., 2005).

**8. Alpha-1-acid glycoprotein**

Plasma levels of Hp rise rapidly up to 2 to 5 folds during inflammation, specifically under conditions when there is an extensive amount of necrotic tissue in the wound. Like for other APPs, Hp synthesis is induced by various cytokines but also by ciliary neurotrophic factor. While IL-6 is the most efficient Hp inducer, IFN-γ blocks IL-6-induced Hp synthesis and TNFβ attenuates glucocorticoid-dependent expression of Hp (Baumann et al., 1990; Marinkovic & Baumann, 1990; Raynes et al., 1991; Yoshioka et al., 2002; Yu et al., 1999).

Although the liver is the major site of Hp expression, inducible expression of Hp is also found in lung, skin, spleen and kidney. This suggests that tissue levels of Hp are most likely regulated differently and vary from those in the blood (Abdullah et al., 2009). Therefore, locally expressed Hp, for example in the lungs, might be an important component of a local protection system with antioxidant, bacteriostatic and anti-inflammatory effects (Abdullah et al., 2012).

Hp influences almost every immune cell type of the innate as well as the adaptive immune response. As an example, binding of Hp-Hb to CD163 receptor on the monocytes and tissue macrophages (Kristiansen et al., 2001), induces anti-inflammatory and protective genes such as heme oxygenase-1 (HO-1) (Schaer et al., 2006). HO-1 is involved in heme catabolism that ends up with carbon monoxide, bilirubin and ferritin - all are known to exert potent antiinflammatory and cytoprotective effects (Otterbein et al., 2003).

It has also been demonstrated that Hp inhibits respiratory burst in fMLP, arachindonic acid or opsonised zymosan-stimulated neutrophils (Oh et al., 1990). Moreover, Hp exerts inhibitory effects on fMLP-driven chemotaxis, and shows intracellular bactericidal activity against *E. coli.* (Rossbacher et al., 1999). Evidence exists for an intracellular uptake of Hp by peripheral blood neutrophils and monocytes via endocytosis and for the subsequent exocytosis of Hp following exposure to *Candida albicans* or TNF- α (Berkova et al., 1999; Wagner et al., 1996). Hp reduces LPS-induced pro-inflammatory effects by the selective suppression of TNF-α, IL-10 and IL-12 production *in vivo* and *in vivo*. The importance of anti-inflammatory and immune modulatory effects of Hp is confirmed by enhanced sensitivity of Hp knockout mice to LPS shock compared to wild type mice (Arredouani et al., 2005).

In addition to its effects on innate immunity, Hp also dampens adaptive immune response. It is a powerful inhibitor of the proliferative response of lymphocytes to phytohemagglutinin and concanavalin A, and depending on the concentration used Hp significantly inhibits or enhances mitogenesis in B-cells in response to LPS (Baseler & Burrell, 1983). Using highly purified human T lymphocytes Arredouani et al. presented evidence for a specific binding of Hp to resting and anti-CD3 stimulated CD4+ and CD8+ T cells and for a direct anti-proliferative effect of Hp on T lymphocytes (Arredouani et al., 2003).

Further evidence for the high importance of Hp as anti-oxidative and immunoregulatory compound arises from the existence of different Hp subtypes and their influence on different pathologies. Hp gene has been studied as a candidate gene for rheumatoid arthritis, systemic lupus erythematosus, primary sclerosing cholangitis, inflammatory bowel disease and diabetes mellitus type 2 (Marquez et al., 2012).

In human two allels code for Hp1 and Hp2 proteins resulting in Hp1-1, Hp2-2 homozygous and Hp2-1 heterozygous genotypes. Clinical studies show that Hp2-2 individuals suffering from diabetes mellitus have a higher risk of vascular complications, especially diabetic nephropathy, compared to Hp2-1 and Hp1-1 individuals (Asleh & Levy, 2005). Epidemiolog‐ ical data also show that Hp2-2 genotype is a major determinant of susceptibility to diabetic cardiovascular disease (Levy et al., 2010).

Functional differences between Hp1-1 and Hp2-2 on molecular level can be in part explained by the finding that the Hp1-1-Hb complex is endocytosed more rapidly by the CD163 pathway than Hp2-2-Hb. This results in a more effective clearance of Hb and less oxidative stress in Hp1-1 individuals (Asleh et al., 2003). Moreover, Hp2-2 individuals show greater immuno‐ logical reactivity including higher antibody titers after vaccination compared to Hp1-1 and Hp2-1 individuals (Nevo & Sutton, 1968). When compare to Hp2-2-Hb, Hp1-1-Hb is related to a much greater production of anti-inflammatory cytokines, therefore, Hp1-1Hp results in a more Th2 directed balance between Th1 (inflammatory) and Th2 (anti-inflammatory) T helper cells. Recent findings provide evidence that Hp1-1 individuals are better protected against oxidative stress and Hp1-1 seems to have higher immunomodulatory effects than Hp 2-2 (Guetta et al., 2007).

## **8. Alpha-1-acid glycoprotein**

erythrocytes. Hp-Hb complexes are rapidly cleared from the circulation predominantly in the liver (Kupfer cells) expressing the Hp-Hb receptor CD163 (Graversen et al., 2002). Hp not only prevents loss of Hb/iron by renal excretion and protects from iron-driven oxidative tissue damage, but also acts as a bacteriostatic protein. Hence, Hp restricts access of bacteria to iron

Plasma levels of Hp rise rapidly up to 2 to 5 folds during inflammation, specifically under conditions when there is an extensive amount of necrotic tissue in the wound. Like for other APPs, Hp synthesis is induced by various cytokines but also by ciliary neurotrophic factor. While IL-6 is the most efficient Hp inducer, IFN-γ blocks IL-6-induced Hp synthesis and TNFβ attenuates glucocorticoid-dependent expression of Hp (Baumann et al., 1990; Marinkovic &

Although the liver is the major site of Hp expression, inducible expression of Hp is also found in lung, skin, spleen and kidney. This suggests that tissue levels of Hp are most likely regulated differently and vary from those in the blood (Abdullah et al., 2009). Therefore, locally expressed Hp, for example in the lungs, might be an important component of a local protection system

Hp influences almost every immune cell type of the innate as well as the adaptive immune response. As an example, binding of Hp-Hb to CD163 receptor on the monocytes and tissue macrophages (Kristiansen et al., 2001), induces anti-inflammatory and protective genes such as heme oxygenase-1 (HO-1) (Schaer et al., 2006). HO-1 is involved in heme catabolism that ends up with carbon monoxide, bilirubin and ferritin - all are known to exert potent anti-

It has also been demonstrated that Hp inhibits respiratory burst in fMLP, arachindonic acid or opsonised zymosan-stimulated neutrophils (Oh et al., 1990). Moreover, Hp exerts inhibitory effects on fMLP-driven chemotaxis, and shows intracellular bactericidal activity against *E. coli.* (Rossbacher et al., 1999). Evidence exists for an intracellular uptake of Hp by peripheral blood neutrophils and monocytes via endocytosis and for the subsequent exocytosis of Hp following exposure to *Candida albicans* or TNF- α (Berkova et al., 1999; Wagner et al., 1996). Hp reduces LPS-induced pro-inflammatory effects by the selective suppression of TNF-α, IL-10 and IL-12 production *in vivo* and *in vivo*. The importance of anti-inflammatory and immune modulatory effects of Hp is confirmed by enhanced sensitivity of Hp knockout mice to LPS

In addition to its effects on innate immunity, Hp also dampens adaptive immune response. It is a powerful inhibitor of the proliferative response of lymphocytes to phytohemagglutinin and concanavalin A, and depending on the concentration used Hp significantly inhibits or enhances mitogenesis in B-cells in response to LPS (Baseler & Burrell, 1983). Using highly purified human T lymphocytes Arredouani et al. presented evidence for a specific binding of Hp to resting and anti-CD3 stimulated CD4+ and CD8+ T cells and for a direct anti-proliferative

Further evidence for the high importance of Hp as anti-oxidative and immunoregulatory compound arises from the existence of different Hp subtypes and their influence on different

with antioxidant, bacteriostatic and anti-inflammatory effects (Abdullah et al., 2012).

Baumann, 1990; Raynes et al., 1991; Yoshioka et al., 2002; Yu et al., 1999).

inflammatory and cytoprotective effects (Otterbein et al., 2003).

shock compared to wild type mice (Arredouani et al., 2005).

effect of Hp on T lymphocytes (Arredouani et al., 2003).

that is essential for bacterial growth.

12 Acute Phase Proteins

Alpha-1-acid glycoprotein (AGP) also known as orosomucoid (ORM) is a heavily glycosylated (45 %) protein (Schmid et al., 1977) APG belongs to the immunocalin family, a lipocalin subfamily. Whereas the lipocalins function as carriers for small hydrophobic compounds, the immunocalins were shown to modulate inflammatory and immune responses (Logdberg & Wester, 2000; Hochepied et al., 2003). Though, the main biologic function of AGP remains unclear. Indeed, APG was found to be a major carrier for neutral and basic drugs in the blood (Kremer et al., 1988). Evidence also exists supporting a role for AGP in the maintenance of normal capillary permeability and selectivity by binding to the capillary vessel wall putatively as part of the glycocalyx, a dynamic endothelial surface layer of glycosaminoglycans, proteo‐ glycans and absorbed plasma proteins (Curry et al., 1989; Fournier et al., 2000; Pries et al., 2000). Moreover, AGP stabilizes the biological activity of plasminogen activator inhibitor-1 (Smolarczyk et al., 2005).

AGP is a positive acute phase protein that under normal conditions circulates in human plasma at a concentration of 0.6 -1.2 mg/ml and rises up to 2- to 7-fold during acute phase response (Colombo et al., 2006; Kremer et al., 1988). Besides the liver, a main source for the circulating AGP levels, production of AGP occurs in human microvascular endothelial cells, pneumo‐ cytes, alveolar macrophages, neutrophils, monocytes and B and T lymphocytes (Sirica et al., 1979; Sorensson et al., 1999; Crestani et al., 1998; Martinez Cordero et al., 2008; Fournier et al., 1999; Theilgaard-Monch et al., 2005; Rahman et al., 2008; Gahmberg & Andersson, 1978; Dirienzo et al., 1987). Hepatic AGP expression is induced by the IL-1β, IL-6 and TNF-α and inhibited by the growth hormone (Barraud et al., 1996; Mejdoubi et al., 1999).

Taken together AGP seems to exhibit both pro- and anti-inflammatory effects. The resulting net function of AGP most likely depends on contextual factors, e.g. interacting cell type,

Immunoregulatory Properties of Acute Phase Proteins — Specific Focus on α1-Antitrypsin

http://dx.doi.org/10.5772/56393

15

Human C-reactive protein (CRP) belongs to a family of pentraxins (Myles et al., 1990) and is composed of five identical 23 kDa subunits (protomers), linked together non-covalently to form pentameric CRP. The liver is the main source for circulating CRP. Hepatic secretion of CRP is primarily regulated by IL-6 and IL-1 (Weinhold et al., 1997; Zhang et al., 1995). Plasma CRP is a highly dynamic protein with a concentration range from 0.05 to 500 µg/ml (Shine et al., 1981; Pepys & Hirschfield, 2003). Plasma CRP levels rise up to 10.000-fold in response to acute tissue injury or inflammation and decline rapidly due to a relatively short half-life (about 19 hours) (Macintyre et al., 1982; Claus et al., 1976; Vigushin et al., 1993). Cardiovascular disease is correlated to chronic inflammation and serum CRP above 3 µg/ml is a good predictive of

Both *in vitro* and *in vivo* CRP exists in a monomeric form (mCRP) with a molecular weight of 23 kDa, too (Taylor & van den Berg, 2007). The pentameric native form of CRP (nCRP) is usually found in the plasma whereas the momomeric form of CRP (mCRP) is present in tissues and at sites of inflammation (Diehl et al., 2000; Potempa et al., 1987; Rees et al., 1988). pCRP also undergoes conformational rearrangement in the absence of calcium and dissociates into mCRP (Eisenhardt et al., 2009). The modified CRP isoform is generated *in vivo* when pCRP binds to damaged membrane surfaces such as activated platelets, apoptotic microparticles (Haber‐ sberger et al., 2012), liposomes containing lysophosphatidylcholine (Volanakis & Narkates, 1981), and oxidized but not native low-density lipoprotein (Chang et al., 2002). Modified CRP displays an antigenicity that is distinct from pCRP. In fact, modified CRP may exist as

One critical function of modified CRP is binding to C1q and activation of the immune system's complement cascade (Ji et al., 2006). In addition to its roles in the regulation of classical and alternative complement pathways, CRP has been shown to interact with ficolin-2 (Ng et al., 2007). Recent study has shown that infection-induced local inflammatory conditions trigger a strong interaction between CRP and ficolin-2; this elicits complement amplification and enhances antimicrobial activation of the classical and lectin pathways (Zhang et al., 2009).

Despite evidence that modified CRP is more strongly associated with inflammation (Zouki et al., 2001) current CRP diagnostics are unable to distinguish between the common isoforms.

Taken together, physiological function of CRP is not fully understood. The most reproducible observations indicate that CRP contributes to innate immunity against bacterial infections like pneumococci (Horowitz et al., 1987). Experimental data provide evidence that transgenic mice over-expressing human CRP are more resistant to *Pneumococci* sepsis than wild-type mice (Szalai et al., 1995). Indeed, CRP is expressed by respiratory epithelial cells and CRP concen‐

additional stimuli, inflammatory status of the host.

increased risk for the disease (Black et al., 2004).

aggregates of mCRP on cell membrane surfaces (Ji et al., 2007).

**9. C-reactive protein**

The immunomodulatory activities of AGP are specifically directed against exaggerated inflammatory response to tissue damage. For example, AGP in a concentration-dependent manner regulates neutrophil chemotactic migration and superoxide generation (Costello et al., 1984; Hochepied et al., 2003; Laine et al., 1990). AGP also inhibits monocyte chemotaxis and diminishes cellular leakage caused by histamine and bradykinin. Moreover, AGP induces secretion of soluble TNFα receptor and IL-1 receptor antagonist from peripheral blood monocytes. (Tilg et al., 1993; Samak et al., 1982; Muchitsch et al., 1996).

The effects of AGP on lymphocytes are mainly immunosuppressive. AGP significantly suppresses induced synthesis of IL-2 and proliferation of lymphocytes (Chiu et al., 1977; Elg et al., 1997). Notably, different glycan variants of AGP show different degrees of inhibition of lymphocyte proliferation (Bennett & Schmid, 1980). The Con A non-reactive fraction of AGP (AGP-A) inhibits anti-CD3 stimulated lymphocyte proliferation stronger than Con A reactive AGP forms emphasizing the importance of the carbohydrate moiety of AGP (Pos et al., 1990).

*In vivo* AGP has been found to protect mice against TNF-α induced lethal shock but not from LPS-induced lethality or Fas-mediated cell death in lethal hepatitis. These findings imply that the protecting effects of AGP are, most likely, TNF-α -specific (Muchitsch et al., 1998; Van Molle et al., 1999).

Pro-inflammatory and immuno-stimulatory effects of AGP have been described, too. Previous studies demonstrated that AGP activates monocytes to produce IL-1β, IL-6, IL-12, TNF-α and tissue factor (Su & Yeh, 1996; Tilg et al., 1993). Moreover, AGP has found to potentiate the effect of suboptimal concentrations of LPS to induce IL-1β, IL-6 and TNF-α in peritoneal and alveolar macrophages (Boutten et al., 1992). Nakamura and collaborators presented evidence that monocytes stimulated with inflammatory cytokines produce AGP and suggested that high expression of AGP may potentially create a positive feedback loop for further production of IL-1β (Nakamura et al., 1993). The observation that AGP-induced secretion of TNFα can be inhibited by protein tyrosine kinase inhibitors led to the proposal that AGP involves tyrosine kinase signalling pathway (Su et al., 1999). This is in line with the finding that AGP binds to chemokine receptor CCR5 on macrophages and signals via tyrosine kinases (Atemezem et al., 2001). In neutrophil models AGP interacts with lectin-like receptors (Siglecs), Siglec-5 and/or Siglec-14, directly induces an intracellular calcium rise and regulates expression of L-selectin (Gunnarsson et al., 2007).

Recently, it has been reported that AGP up-regulates the expression of the Hb scavenger receptor CD163 on monocytic cells *in vitro* (Komori et al., 2012). *In vivo*, in the phenylhydrazineinduced hemolysis mice model AGP induced CD163 expression with a subsequent increase in Hb clearance and reduced oxidative stress. The effect of AGP on CD163 expression seems to be indirect and mediated by the IL-6 and IL-10, known inducers of CD163. According to Komori and co-workers AGP induces CD163 expression via the TLR4/CD14 pathway (Komori et al., 2012).

Taken together AGP seems to exhibit both pro- and anti-inflammatory effects. The resulting net function of AGP most likely depends on contextual factors, e.g. interacting cell type, additional stimuli, inflammatory status of the host.

## **9. C-reactive protein**

1999; Theilgaard-Monch et al., 2005; Rahman et al., 2008; Gahmberg & Andersson, 1978; Dirienzo et al., 1987). Hepatic AGP expression is induced by the IL-1β, IL-6 and TNF-α and

The immunomodulatory activities of AGP are specifically directed against exaggerated inflammatory response to tissue damage. For example, AGP in a concentration-dependent manner regulates neutrophil chemotactic migration and superoxide generation (Costello et al., 1984; Hochepied et al., 2003; Laine et al., 1990). AGP also inhibits monocyte chemotaxis and diminishes cellular leakage caused by histamine and bradykinin. Moreover, AGP induces secretion of soluble TNFα receptor and IL-1 receptor antagonist from peripheral blood

The effects of AGP on lymphocytes are mainly immunosuppressive. AGP significantly suppresses induced synthesis of IL-2 and proliferation of lymphocytes (Chiu et al., 1977; Elg et al., 1997). Notably, different glycan variants of AGP show different degrees of inhibition of lymphocyte proliferation (Bennett & Schmid, 1980). The Con A non-reactive fraction of AGP (AGP-A) inhibits anti-CD3 stimulated lymphocyte proliferation stronger than Con A reactive AGP forms emphasizing the importance of the carbohydrate moiety of AGP (Pos et al., 1990).

*In vivo* AGP has been found to protect mice against TNF-α induced lethal shock but not from LPS-induced lethality or Fas-mediated cell death in lethal hepatitis. These findings imply that the protecting effects of AGP are, most likely, TNF-α -specific (Muchitsch et al., 1998; Van Molle

Pro-inflammatory and immuno-stimulatory effects of AGP have been described, too. Previous studies demonstrated that AGP activates monocytes to produce IL-1β, IL-6, IL-12, TNF-α and tissue factor (Su & Yeh, 1996; Tilg et al., 1993). Moreover, AGP has found to potentiate the effect of suboptimal concentrations of LPS to induce IL-1β, IL-6 and TNF-α in peritoneal and alveolar macrophages (Boutten et al., 1992). Nakamura and collaborators presented evidence that monocytes stimulated with inflammatory cytokines produce AGP and suggested that high expression of AGP may potentially create a positive feedback loop for further production of IL-1β (Nakamura et al., 1993). The observation that AGP-induced secretion of TNFα can be inhibited by protein tyrosine kinase inhibitors led to the proposal that AGP involves tyrosine kinase signalling pathway (Su et al., 1999). This is in line with the finding that AGP binds to chemokine receptor CCR5 on macrophages and signals via tyrosine kinases (Atemezem et al., 2001). In neutrophil models AGP interacts with lectin-like receptors (Siglecs), Siglec-5 and/or Siglec-14, directly induces an intracellular calcium rise and regulates expression of L-selectin

Recently, it has been reported that AGP up-regulates the expression of the Hb scavenger receptor CD163 on monocytic cells *in vitro* (Komori et al., 2012). *In vivo*, in the phenylhydrazineinduced hemolysis mice model AGP induced CD163 expression with a subsequent increase in Hb clearance and reduced oxidative stress. The effect of AGP on CD163 expression seems to be indirect and mediated by the IL-6 and IL-10, known inducers of CD163. According to Komori and co-workers AGP induces CD163 expression via the TLR4/CD14 pathway (Komori

inhibited by the growth hormone (Barraud et al., 1996; Mejdoubi et al., 1999).

monocytes. (Tilg et al., 1993; Samak et al., 1982; Muchitsch et al., 1996).

et al., 1999).

14 Acute Phase Proteins

(Gunnarsson et al., 2007).

et al., 2012).

Human C-reactive protein (CRP) belongs to a family of pentraxins (Myles et al., 1990) and is composed of five identical 23 kDa subunits (protomers), linked together non-covalently to form pentameric CRP. The liver is the main source for circulating CRP. Hepatic secretion of CRP is primarily regulated by IL-6 and IL-1 (Weinhold et al., 1997; Zhang et al., 1995). Plasma CRP is a highly dynamic protein with a concentration range from 0.05 to 500 µg/ml (Shine et al., 1981; Pepys & Hirschfield, 2003). Plasma CRP levels rise up to 10.000-fold in response to acute tissue injury or inflammation and decline rapidly due to a relatively short half-life (about 19 hours) (Macintyre et al., 1982; Claus et al., 1976; Vigushin et al., 1993). Cardiovascular disease is correlated to chronic inflammation and serum CRP above 3 µg/ml is a good predictive of increased risk for the disease (Black et al., 2004).

Both *in vitro* and *in vivo* CRP exists in a monomeric form (mCRP) with a molecular weight of 23 kDa, too (Taylor & van den Berg, 2007). The pentameric native form of CRP (nCRP) is usually found in the plasma whereas the momomeric form of CRP (mCRP) is present in tissues and at sites of inflammation (Diehl et al., 2000; Potempa et al., 1987; Rees et al., 1988). pCRP also undergoes conformational rearrangement in the absence of calcium and dissociates into mCRP (Eisenhardt et al., 2009). The modified CRP isoform is generated *in vivo* when pCRP binds to damaged membrane surfaces such as activated platelets, apoptotic microparticles (Haber‐ sberger et al., 2012), liposomes containing lysophosphatidylcholine (Volanakis & Narkates, 1981), and oxidized but not native low-density lipoprotein (Chang et al., 2002). Modified CRP displays an antigenicity that is distinct from pCRP. In fact, modified CRP may exist as aggregates of mCRP on cell membrane surfaces (Ji et al., 2007).

One critical function of modified CRP is binding to C1q and activation of the immune system's complement cascade (Ji et al., 2006). In addition to its roles in the regulation of classical and alternative complement pathways, CRP has been shown to interact with ficolin-2 (Ng et al., 2007). Recent study has shown that infection-induced local inflammatory conditions trigger a strong interaction between CRP and ficolin-2; this elicits complement amplification and enhances antimicrobial activation of the classical and lectin pathways (Zhang et al., 2009).

Despite evidence that modified CRP is more strongly associated with inflammation (Zouki et al., 2001) current CRP diagnostics are unable to distinguish between the common isoforms.

Taken together, physiological function of CRP is not fully understood. The most reproducible observations indicate that CRP contributes to innate immunity against bacterial infections like pneumococci (Horowitz et al., 1987). Experimental data provide evidence that transgenic mice over-expressing human CRP are more resistant to *Pneumococci* sepsis than wild-type mice (Szalai et al., 1995). Indeed, CRP is expressed by respiratory epithelial cells and CRP concen‐ trations in secretions from both inflamed and non-inflamed human respiratory tract are sufficiently high for an antimicrobial effect. This suggests that CRP is involved in the bacterial clearance in the respiratory tract (Gould & Weiser, 2001).

**References**

121-128.

*flammation* , 20, 191-202.

*Cell Biol* , 10, 5967-5976.

[1] Abdullah, M, et al. (2012). Pulmonary haptoglobin and CD163 are functional immu‐

Immunoregulatory Properties of Acute Phase Proteins — Specific Focus on α1-Antitrypsin

http://dx.doi.org/10.5772/56393

17

[2] Abdullah, M, et al. (2009). Expression of the acute phase protein haptoglobin in hu‐ man lung cancer and tumor-free lung tissues. *Pathol Res Pract* , 205, 639-647.

[3] Adam, C, et al. (1996). Inhibition of neutrophil elastase by the alpha1-proteinase in‐

[4] Al-omari, M, et al. (2011). Acute-phase protein alpha1-antitrypsin inhibits neutrophil

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[6] Amelinckx, A, et al. (2011). Neutrophil Chemotaxis Induced by Molecular Modifica‐ tions of Alpha-1 Antitrypsin (AAT) in COPD Subjects with and without AAT Defi‐

[7] Arredouani, M, et al. (2003). Haptoglobin directly affects T cells and suppresses T

[8] Arredouani, M. S, et al. (2005). Haptoglobin dampens endotoxin-induced inflamma‐

[9] Asleh, R, et al. (2005). In vivo and in vitro studies establishing haptoglobin as a major susceptibility gene for diabetic vascular disease. *Vasc Health Risk Manag* , 1, 19-28. [10] Asleh, R, et al. (2003). Genetically determined heterogeneity in hemoglobin scaveng‐ ing and susceptibility to diabetic cardiovascular disease. *Circ Res* , 92, 1193-1200. [11] Atemezem, A, et al. (2001). Human alpha1-acid glycoprotein binds to CCR5 ex‐ pressed on the plasma membrane of human primary macrophages. *Biochem J* , 356,

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[14] Baseler, M. W, et al. (1983). Purification of haptoglobin and its effects on lymphocyte

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Recent findings lend experimental support to the hypothesis that biological activities of CRP might be dependent on both, its molecular form and the property to interact with plasma membrane lipid microdomains (Ji et al., 2009).

For example, CRP and two CRP derived peptides, CRP(174-185) and CRP(201-206), but not peptide CRP(77-82) are capable of diminishing attachment of human neutrophils to LPSstimulated human endothelial cells and consequently limiting leukocyte traffic into inflamed tissues (Zouki et al., 1997). CRP as well as the two peptides rapidly downregulate the expres‐ sion of L-selectin on the neutrophil surface.

In summary, CRP is used as an indicator of disease outcome and to monitor the disease course. CRP is measured objectively and affordably in clinical practice worldwide.

#### **10. Conclusions**

In this chapter we have shown that AAT, only one among many APPs, holds tremendous potential as an anti-inflammatory and immuno-modulatory protein.

Several *in vitro* and *in vivo* studies have been published in which a specific APP switched the pro-inflammatory to the anti-inflammatory pathways necessary for the resolution of inflam‐ mation. Although the physiological roles of APPs are not completely understood, existing findings provide evidence that APPs act on a variety of cells involved in early and late stages of inflammation and that their effects are time, concentration and molecular conformationdependent. It cannot be excluded that these proteins may have more common characteristics and biological effects, however the lack of high quality purified endotoxin or other contami‐ nant free proteins limits current understanding.

The mechanisms of action for the APPs are still being investigated, however, there remain a number of challenges to face in the development of APPs as a true anti-inflammatory thera‐ peutic agents and diagnostic markers.

#### **Author details**

S. Janciauskiene, S. Wrenger and T. Welte

Department of Respiratory Medicine, Hannover Medical School, Hannover, Germany

#### **References**

trations in secretions from both inflamed and non-inflamed human respiratory tract are sufficiently high for an antimicrobial effect. This suggests that CRP is involved in the bacterial

Recent findings lend experimental support to the hypothesis that biological activities of CRP might be dependent on both, its molecular form and the property to interact with plasma

For example, CRP and two CRP derived peptides, CRP(174-185) and CRP(201-206), but not peptide CRP(77-82) are capable of diminishing attachment of human neutrophils to LPSstimulated human endothelial cells and consequently limiting leukocyte traffic into inflamed tissues (Zouki et al., 1997). CRP as well as the two peptides rapidly downregulate the expres‐

In summary, CRP is used as an indicator of disease outcome and to monitor the disease course.

In this chapter we have shown that AAT, only one among many APPs, holds tremendous

Several *in vitro* and *in vivo* studies have been published in which a specific APP switched the pro-inflammatory to the anti-inflammatory pathways necessary for the resolution of inflam‐ mation. Although the physiological roles of APPs are not completely understood, existing findings provide evidence that APPs act on a variety of cells involved in early and late stages of inflammation and that their effects are time, concentration and molecular conformationdependent. It cannot be excluded that these proteins may have more common characteristics and biological effects, however the lack of high quality purified endotoxin or other contami‐

The mechanisms of action for the APPs are still being investigated, however, there remain a number of challenges to face in the development of APPs as a true anti-inflammatory thera‐

Department of Respiratory Medicine, Hannover Medical School, Hannover, Germany

CRP is measured objectively and affordably in clinical practice worldwide.

potential as an anti-inflammatory and immuno-modulatory protein.

clearance in the respiratory tract (Gould & Weiser, 2001).

membrane lipid microdomains (Ji et al., 2009).

sion of L-selectin on the neutrophil surface.

nant free proteins limits current understanding.

peutic agents and diagnostic markers.

S. Janciauskiene, S. Wrenger and T. Welte

**Author details**

**10. Conclusions**

16 Acute Phase Proteins


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[173] Volanakis, J. E, et al. (1981). Interaction of C-reactive protein with artificial phospha‐

[174] Wagner, L, et al. (1996). Haptoglobin phenotyping by newly developed monoclonal antibodies. Demonstration of haptoglobin uptake into peripheral blood neutrophils

[175] Warnock, D. E, et al. (1993). Determination of plasma membrane lipid mass and com‐ position in cultured Chinese hamster ovary cells using high gradient magnetic affini‐

[176] Weinhold, B, et al. (1997). Interleukin-6 is necessary, but not sufficient, for induction of the humanC-reactive protein gene in vivo. *Biochem J* 325 ( Pt 3), 617-621.

[177] Wiemer, A. J, et al. (2010). Calpain inhibition impairs TNF-alpha-mediated neutro‐

[178] Wu, L, et al. (2005). The low-density lipoprotein receptor-related protein-1 associates

[179] Yoon, I. S, et al. (2007). Low-density lipoprotein receptor-related protein promotes amyloid precursor protein trafficking to lipid rafts in the endocytic pathway. *FASEB*

phil adhesion, arrest and oxidative burst. *Mol Immunol* , 47, 894-902.

transiently with lipid rafts. *J Cell Biochem* , 96, 1021-1033.

*J* , 21, 2742-2752.

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*Sci U S A* , 109, 564-569.

28 Acute Phase Proteins

neutrophils. *J Leukoc Biol* , 78, 462-470.

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and monocytes. *J Immunol* , 156, 1989-1996.

ty chromatography. *J Biol Chem* , 268, 10145-10153.


**Chapter 2**

**Inflammation and Acute Phase Proteins in Haemostasis**

Inflammation is a very complex reaction to infection or injury the endeavour being to contain the infection and harm to a limited area. The process is associated with the activation of the coagulation system. To think that inflammation occurs and then leads to activation of the coagulation system is not quite true as there is much cross-talk between the two systems. This is a natural host response to cellular damage or infection however an overshooting of this crosstalk between coagulation and inflammation can lead to an exaggerated prothrombotic state and exacerbate the disease process. A wide range of inflammatory conditions such as infec‐ tions, acute respiratory distress syndrome and SIRS (systemic inflammatory response syn‐ drome) following major surgery e.g.cardiac surgery can lead to an uncontrolled inflammatory response and to profound disturbance of the coagulation system leading to an imbalance in the normal anticoagulated state of blood to that of a procoagulant state. When coagulation is compromised it can contribute to the pathogenesis of the inflammatory condition with deposition of fibrin within the microvasculature directly enhancing the inflammatory reaction. This in turn leads to a modulation of protein manufacture mainly via Liver hepatocytes in the upregulation and downregulation of at least twenty factors directly involved in blood coagulation. This process is controlled via cytokines and leads to the imbalance in what are called the haemostatic acute phase proteins. All of which puts the haemostatic system at an

The haemostatic system maintains blood in a fluid phase under normal physiological condi‐ tions and provides a mechanism to prevent exsanguination upon vascular damage. Morawitz had created the 'classic' theory of blood coagulation in 1905 but it was Macfarlane who first reported the coagulation cascade as a biochemical amplification pathway of pro-enzymeenzyme transformations in 1964 [5]. Davie and Ratnoff later the same year referred to it as a waterfall stepwise sequence of activation [6]. Macfarlane's idea that amplification of the cascade and acceleration of earlier stages of the pathway culminating in the conversion of

> © 2013 Davidson; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Simon J. Davidson

**1. Introduction**

http://dx.doi.org/10.5772/55998

increased thrombotic potential [1, 2, 3, 4].

Additional information is available at the end of the chapter

## **Inflammation and Acute Phase Proteins in Haemostasis**

Simon J. Davidson

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55998

## **1. Introduction**

Inflammation is a very complex reaction to infection or injury the endeavour being to contain the infection and harm to a limited area. The process is associated with the activation of the coagulation system. To think that inflammation occurs and then leads to activation of the coagulation system is not quite true as there is much cross-talk between the two systems. This is a natural host response to cellular damage or infection however an overshooting of this crosstalk between coagulation and inflammation can lead to an exaggerated prothrombotic state and exacerbate the disease process. A wide range of inflammatory conditions such as infec‐ tions, acute respiratory distress syndrome and SIRS (systemic inflammatory response syn‐ drome) following major surgery e.g.cardiac surgery can lead to an uncontrolled inflammatory response and to profound disturbance of the coagulation system leading to an imbalance in the normal anticoagulated state of blood to that of a procoagulant state. When coagulation is compromised it can contribute to the pathogenesis of the inflammatory condition with deposition of fibrin within the microvasculature directly enhancing the inflammatory reaction. This in turn leads to a modulation of protein manufacture mainly via Liver hepatocytes in the upregulation and downregulation of at least twenty factors directly involved in blood coagulation. This process is controlled via cytokines and leads to the imbalance in what are called the haemostatic acute phase proteins. All of which puts the haemostatic system at an increased thrombotic potential [1, 2, 3, 4].

The haemostatic system maintains blood in a fluid phase under normal physiological condi‐ tions and provides a mechanism to prevent exsanguination upon vascular damage. Morawitz had created the 'classic' theory of blood coagulation in 1905 but it was Macfarlane who first reported the coagulation cascade as a biochemical amplification pathway of pro-enzymeenzyme transformations in 1964 [5]. Davie and Ratnoff later the same year referred to it as a waterfall stepwise sequence of activation [6]. Macfarlane's idea that amplification of the cascade and acceleration of earlier stages of the pathway culminating in the conversion of

© 2013 Davidson; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

fibrinogen to fibrin was a major breakthrough in the understanding of how the coagulation factors interacted and forms the basis of what we understand today.

Coagulation activation yields proteases that not only interact with coagulation protein zymogens but also with specific cell receptors to induce signaling pathways that mediate inflammatory responses [9]. Many in vitro observations point to a role of coagulation proteases in upregulating the expression of proinflammatory mediators [10]. The most important mechanism by which coagulation proteases influence inflammation is by binding to protease activated receptors (PARs), of which four types (PAR 1 to 4) have been identified, all belonging to the family of transmembrane domain, G-protein–coupled receptors [11]. Tissue factor is also a potential mediator of intracellular signaling of established inflammatory pathways, func‐ tioning as an intermediate for factor VIIa–induced activation of mitogen-activated protein kinases and calcium signalling [12]. It is tissue factor that binds to factor VII and drives thrombin generation leading to fibrin formation. Tissue factor is an integral membrane protein normally separated from blood by the vascular endothelium. Tissue factor is expressed in the vascular adventitia in astroglial cells. It also appears in tumour cells where it appears related to their metastatic potential. All of this activation of coagulation increases in some procoagu‐ lant factors (fibrinogen and fator VIII) with reduced fibrinolytic response and dampening of

Thrombin plays many parts (Figure 2) and with the anticoagulant protein, activated protein C, can activate specific cell receptors on mononuclear cells and endothelial cells which can

Thrombin

**Figure 2.** Thrombins role in activating some of the components of coagulation and inflammation. Coagulation factors V, VIII,XI and XIII, TM – thrombomodulin, TAFI – thrombin activatable fibrinolytic inhibitor, t-PA – tissue plasminogen

TAFI

Figure 2. Thrombins role in activating some of the components of coagulation and inflammation. Coagulation factors V, VIII,XI and XIII, TM –

Pathogen recognition receptors, Toll receptors (TLR), are essential in the host defence against pathogens. Platelets express these pattern recognition receptors involved in innate immunity. TLR's recognise microbial structures that are conserved among species. TLR1,2,4 6,8 & 9 all appear on platelets and form one of the many bridges between inflammation and coagulation. TLR's are responsible for LPS induced thrombocytopenia[13]. Platelets express TLR – pattern recognition receptors involved in innate immunity. TLR's are able to recognise danger associated molecular patterns (DAMPs). For example fibrinogen that is released during inflammation is a DAMP and further enhances the proinflammatory response through TLR4. It is via TLR and DAMPs that induction of caspase-1 activation which causes the processing of the proinflammatory response through various cytokines[14].

Fibrinogen

thrombomodulin, TAFI – thrombin activatable fibrinolytic inhibitor, t-PA – tissue plasminogen activator

t-PA ICAM-1 P-selectin

**2. Initiation of the pro-inflammatory response** 

Platelet

Activated protein C

TM

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33

the natural anticoagulant potential have a profound effect on mortality.

affect cytokine production and inflammatory cell apoptosis.

Vascular endothelium

VIII V

XI

Leukocyte

Figure 3

XIII

activator, ICAM-1 intracellular adhesion molecule 1

Platelets are the primary haemostatic plug when damage first occurs to a vessel. The multi‐ meric protein von Willebrand factor mediates the adhesion of platelets to the site of vascular damage. The platelets bind to the matrix proteins exposed by the damage to the vessel wall, particularly collagen. These platelets are activated by small amounts of thrombin (~1nM) produced by tissue factor exposure at the site of vascular damage. The tissue factor binds factor VII that is rapidly activated. The tissue factor-VIIa complex subsequently activates factor X. The activated platelet provides binding sites for the coagulation enzymes, localising coagula‐ tion to the site of injury and prolonging activation of coagulation by protecting the enzymes from inhibition and inactivation [7]. Factor X is essential to this 'propagation' phase of coagulation. When bound to the platelet and activated via the VIIIa-IXa complex Xa is protected from inhibition by tissue factor pathway inhibitor and antithrombin.

**Figure 1.** Cell based model coagulation. Reproduced with kind permission of Professor M.Hoffman

Tissue factor pathway inhibitor (TFPI) rapidly blunts this driving force of tissue factor-VIIa complex that initiates coagulation and generates the sudden burst of thrombin [8]. Once trace amounts of thrombin have been formed this is then able to activate factors V, VIII, IX and XI. This positive feedback mechanism ensures prolonged activation of the system with sufficient quantities of thrombin being produced to activate platelets, white cells, endothelial cells, and the protein C anticoagulant pathway and to continue producing thrombin.

Coagulation activation yields proteases that not only interact with coagulation protein zymogens but also with specific cell receptors to induce signaling pathways that mediate inflammatory responses [9]. Many in vitro observations point to a role of coagulation proteases in upregulating the expression of proinflammatory mediators [10]. The most important mechanism by which coagulation proteases influence inflammation is by binding to protease activated receptors (PARs), of which four types (PAR 1 to 4) have been identified, all belonging to the family of transmembrane domain, G-protein–coupled receptors [11]. Tissue factor is also a potential mediator of intracellular signaling of established inflammatory pathways, func‐ tioning as an intermediate for factor VIIa–induced activation of mitogen-activated protein kinases and calcium signalling [12]. It is tissue factor that binds to factor VII and drives thrombin generation leading to fibrin formation. Tissue factor is an integral membrane protein normally separated from blood by the vascular endothelium. Tissue factor is expressed in the vascular adventitia in astroglial cells. It also appears in tumour cells where it appears related to their metastatic potential. All of this activation of coagulation increases in some procoagu‐ lant factors (fibrinogen and fator VIII) with reduced fibrinolytic response and dampening of the natural anticoagulant potential have a profound effect on mortality.

Thrombin plays many parts (Figure 2) and with the anticoagulant protein, activated protein C, can activate specific cell receptors on mononuclear cells and endothelial cells which can affect cytokine production and inflammatory cell apoptosis.

Vascular endothelium

**2. Initiation of the pro-inflammatory response** 

Figure 3

fibrinogen to fibrin was a major breakthrough in the understanding of how the coagulation

Platelets are the primary haemostatic plug when damage first occurs to a vessel. The multi‐ meric protein von Willebrand factor mediates the adhesion of platelets to the site of vascular damage. The platelets bind to the matrix proteins exposed by the damage to the vessel wall, particularly collagen. These platelets are activated by small amounts of thrombin (~1nM) produced by tissue factor exposure at the site of vascular damage. The tissue factor binds factor VII that is rapidly activated. The tissue factor-VIIa complex subsequently activates factor X. The activated platelet provides binding sites for the coagulation enzymes, localising coagula‐ tion to the site of injury and prolonging activation of coagulation by protecting the enzymes from inhibition and inactivation [7]. Factor X is essential to this 'propagation' phase of coagulation. When bound to the platelet and activated via the VIIIa-IXa complex Xa is

factors interacted and forms the basis of what we understand today.

32 Acute Phase Proteins

protected from inhibition by tissue factor pathway inhibitor and antithrombin.

**Figure 1.** Cell based model coagulation. Reproduced with kind permission of Professor M.Hoffman

the protein C anticoagulant pathway and to continue producing thrombin.

Tissue factor pathway inhibitor (TFPI) rapidly blunts this driving force of tissue factor-VIIa complex that initiates coagulation and generates the sudden burst of thrombin [8]. Once trace amounts of thrombin have been formed this is then able to activate factors V, VIII, IX and XI. This positive feedback mechanism ensures prolonged activation of the system with sufficient quantities of thrombin being produced to activate platelets, white cells, endothelial cells, and

Figure 2. Thrombins role in activating some of the components of coagulation and inflammation. Coagulation factors V, VIII,XI and XIII, TM – thrombomodulin, TAFI – thrombin activatable fibrinolytic inhibitor, t-PA – tissue plasminogen activator **Figure 2.** Thrombins role in activating some of the components of coagulation and inflammation. Coagulation factors V, VIII,XI and XIII, TM – thrombomodulin, TAFI – thrombin activatable fibrinolytic inhibitor, t-PA – tissue plasminogen activator, ICAM-1 intracellular adhesion molecule 1

Pathogen recognition receptors, Toll receptors (TLR), are essential in the host defence against pathogens. Platelets express these pattern recognition receptors involved in innate immunity. TLR's recognise microbial structures that are conserved among species. TLR1,2,4 6,8 & 9 all appear on platelets and form one of the many bridges between inflammation and coagulation. TLR's are responsible for LPS induced thrombocytopenia[13]. Platelets express TLR – pattern recognition receptors involved in innate immunity. TLR's are able to recognise danger associated molecular patterns (DAMPs). For example fibrinogen that is released during inflammation is a DAMP and further enhances the proinflammatory response through TLR4. It is via TLR and DAMPs that induction of caspase-1 activation which causes the processing of the proinflammatory response through various cytokines[14].

## **2. Initiation of the pro-inflammatory response**

Pathogen recognition receptors, Toll receptors (TLR), are essential in the host defence against pathogens. Platelets express these pattern recognition receptors involved in innate immunity. TLR's recognise microbial structures that are conserved among species. TLR1,2,4 6,8 & 9 all appear on platelets and form one of the many bridges between inflammation and coagulation. TLR's are responsible for LPS induced thrombocytopenia [13]. Platelets express TLR – pattern recognition receptors involved in innate immunity. TLR's are able to recognise danger associated molecular patterns (DAMPs). For example fibrinogen that is released during inflammation is a DAMP and further enhances the proinflammatory response through TLR4. It is via TLR and DAMPs that induction of caspase-1 activation which causes the processing of the proinflammatory response through various cytokines [14]. Figure 3

involved in regulating the coagulation response during inflammation are IL-6, TNF-alpha,

Acute phase proteins are released as mediators of the inflammatory cascade as a chemical and cellular response to injury. They increase rapidly in plasma in response to a inflammatory insult. Some acute phase proteins increase transiently (C-reactive protein) while others have

The inflammatory response to surgery, atherosclerosis, infection and cardiovascular disease has a profound effect upon the haemostatic system, including fibrinolysis. The cytokines interleukin 1β (IL-1β) and interleukin 6 (IL-6) modulate the production and suppression of

The effect is to make the endothelium and whole coagulation system more procoagulant.

Inflammation

t-PA Tissue factor

An acute-phase protein has been defined as one whose plasma concentration increases (positive acute-phase proteins) or decreases (negative acute-phase proteins) by at least 25 percent during inflammatory disorders [16]. The changes in the concentrations of acutephase proteins are due largely to changes in their production by hepatocytes. Although

vWf PAI-1 PAF P-selectin Procoagulant Antifibrinolytic Pro-inflammatory

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35

IL-8, MCP-1 and IL-1 [15].

Figure 4

a more sustained elevation (Haptoglobin) [16].

Anticoagulant Anti-inflammatory Profibrinolytic

many of the coagulation enzymes formed by the Liver.

TM EPCR Prostoglandin

**3. Coagulation acute phase proteins**

**Figure 4.** Inflammatory drive to a procoagulant state following endothelial stimulation.

**Figure 3.** Cross-talk between coagulation and innate immune pathways in response to DAMPs (danger associated molecular patterns); DAMPS from invading pathogens or damaged host cellsare recognised by pathogen recognition receptors (PRRs) on antigen presenting cells, neutrophils, monocytes, macropahges, endothelial cells and platelets. This results in tissue factor exposure sustained by cytokines and chemokines with proinflammatory and opsonic prop‐ erties and associated with increaased expressionof leukocyte adhesion molecules. In parallel DAMP-induced comple‐ ment activation via any of the complement activation pathways leads to generation of the complement factors C3a and C5a and the membrane attack complex C5b-9. C5a feeds back to promote further tissue factor expresssion and C5b-9 also supports generation of thrombin. Green lines indicate increase in response, red lines indicate supression. Reproduced with kind permission of Dr EM Conway.

Cytokines are small molecules that have relatively short half-lives of between minutes and hours but play a pivotal role in the inflammatory response. The most important cytokines involved in regulating the coagulation response during inflammation are IL-6, TNF-alpha, IL-8, MCP-1 and IL-1 [15].

Acute phase proteins are released as mediators of the inflammatory cascade as a chemical and cellular response to injury. They increase rapidly in plasma in response to a inflammatory insult. Some acute phase proteins increase transiently (C-reactive protein) while others have a more sustained elevation (Haptoglobin) [16].

The inflammatory response to surgery, atherosclerosis, infection and cardiovascular disease has a profound effect upon the haemostatic system, including fibrinolysis. The cytokines interleukin 1β (IL-1β) and interleukin 6 (IL-6) modulate the production and suppression of many of the coagulation enzymes formed by the Liver.

The effect is to make the endothelium and whole coagulation system more procoagulant. Figure 4

**Figure 4.** Inflammatory drive to a procoagulant state following endothelial stimulation.

#### **3. Coagulation acute phase proteins**

**2. Initiation of the pro-inflammatory response**

34 Acute Phase Proteins

of the proinflammatory response through various cytokines [14]. Figure 3

Pathogen recognition receptors, Toll receptors (TLR), are essential in the host defence against pathogens. Platelets express these pattern recognition receptors involved in innate immunity. TLR's recognise microbial structures that are conserved among species. TLR1,2,4 6,8 & 9 all appear on platelets and form one of the many bridges between inflammation and coagulation. TLR's are responsible for LPS induced thrombocytopenia [13]. Platelets express TLR – pattern recognition receptors involved in innate immunity. TLR's are able to recognise danger associated molecular patterns (DAMPs). For example fibrinogen that is released during inflammation is a DAMP and further enhances the proinflammatory response through TLR4. It is via TLR and DAMPs that induction of caspase-1 activation which causes the processing

**Figure 3.** Cross-talk between coagulation and innate immune pathways in response to DAMPs (danger associated molecular patterns); DAMPS from invading pathogens or damaged host cellsare recognised by pathogen recognition receptors (PRRs) on antigen presenting cells, neutrophils, monocytes, macropahges, endothelial cells and platelets. This results in tissue factor exposure sustained by cytokines and chemokines with proinflammatory and opsonic prop‐ erties and associated with increaased expressionof leukocyte adhesion molecules. In parallel DAMP-induced comple‐ ment activation via any of the complement activation pathways leads to generation of the complement factors C3a and C5a and the membrane attack complex C5b-9. C5a feeds back to promote further tissue factor expresssion and C5b-9 also supports generation of thrombin. Green lines indicate increase in response, red lines indicate supression.

Cytokines are small molecules that have relatively short half-lives of between minutes and hours but play a pivotal role in the inflammatory response. The most important cytokines

Reproduced with kind permission of Dr EM Conway.

An acute-phase protein has been defined as one whose plasma concentration increases (positive acute-phase proteins) or decreases (negative acute-phase proteins) by at least 25 percent during inflammatory disorders [16]. The changes in the concentrations of acutephase proteins are due largely to changes in their production by hepatocytes. Although the mechanism by which the liver processes the stimulation to increase and decrease protein production may be different in different forms of inflammatory insult e.g. sepsis and chronic inflammation [17].

During an inflammatory reaction fibrinogen can increase in the order of 2-3 fold and this will significantly increase blood viscosity and cause some degree of red cell aggregation as well as transform vascular pathologies such as atherosclerotic plaques. Fibrinogen by means of increasing the production of endothelin-1, is also capable of directly inducing

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37

**Figure 5.** Fibrinogen and fibrin modulation of inflammation. Fibrin(ogen) modulates the inflammatory response by affecting leukocyte migration but also by induction of cytokine/chemokine expression mostly via Mac-1 signalling. NO-nitric oxide, Mac-1 Macrophage antigen 1, PI3K Phosphoinositide-3-kiniase, TNF tumor necrosis factor. Repro‐

There is good evidence that fibrinogen has a function in regulation of the inflammatory response as is seen in the increased concentration of fibrinogen associated with atherosclerosis and cardiovascular risk. Fibrin(ogen) also participates in activation of vascular cells and regulation of the inflammatory response via the ability to bind to and activate a number of immune cells. The cellular ligand binding of fibrinogen is totally distinct to any coagulation

duced with kind permission of Prof. K Zacharowski

vasoconstriction [18].


**Table 1.** Acute phase proteins that directly affect haemostasis

## **4. Coagulation proteins whose plasma concentrations increase during inflammation**

#### **4.1. Fibrinogen**

Fibrinogen is a soluble glycoprotein synthesised in the Liver with a normal plasma concen‐ tration of 2 – 4 g/L and half life of 4 days. When the coagulation cascade is activated fibrinogen is the final substrate in the formation of a clot being converted to its insoluble fibrin form. Thrombin cleaves fibrinogen releasing fibrinopeptide A and B forming fibrin monomers which have exposed polymerisation sites on the fibrin molecule. Thrombin activates factor XIII which cross-links these fibrin fibrils increasing clot strength and rendering them more resistant to proteolysis [18]. Fibrin creation is required with platelets (stimulating platelet aggregation by binding to the glycoprotein IIb/IIIa platelet membrane receptor) to repair any breach in the vascular integrity and prevent haemorrhage. This process is not left unchecked and is regulated via the fibrinolytic system (plasmin production) to prevent excess fibrin accumula‐ tion at the site of damage despite the procoagulant signalling drive. The local production of plasmin is regulated via two plasminogen activators, tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) which under normal physiological conditions keeps this fibrin matrix production and its lysis tightly controlled [19].

During an inflammatory reaction fibrinogen can increase in the order of 2-3 fold and this will significantly increase blood viscosity and cause some degree of red cell aggregation as well as transform vascular pathologies such as atherosclerotic plaques. Fibrinogen by means of increasing the production of endothelin-1, is also capable of directly inducing vasoconstriction [18].

the mechanism by which the liver processes the stimulation to increase and decrease protein production may be different in different forms of inflammatory insult e.g. sepsis

**4. Coagulation proteins whose plasma concentrations increase during**

Fibrinogen is a soluble glycoprotein synthesised in the Liver with a normal plasma concen‐ tration of 2 – 4 g/L and half life of 4 days. When the coagulation cascade is activated fibrinogen is the final substrate in the formation of a clot being converted to its insoluble fibrin form. Thrombin cleaves fibrinogen releasing fibrinopeptide A and B forming fibrin monomers which have exposed polymerisation sites on the fibrin molecule. Thrombin activates factor XIII which cross-links these fibrin fibrils increasing clot strength and rendering them more resistant to proteolysis [18]. Fibrin creation is required with platelets (stimulating platelet aggregation by binding to the glycoprotein IIb/IIIa platelet membrane receptor) to repair any breach in the vascular integrity and prevent haemorrhage. This process is not left unchecked and is regulated via the fibrinolytic system (plasmin production) to prevent excess fibrin accumula‐ tion at the site of damage despite the procoagulant signalling drive. The local production of plasmin is regulated via two plasminogen activators, tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) which under normal physiological conditions keeps

**inflammation**

Histidine rich glycoprotein Thrombomodulin

Endothelial protein C receptor

Protein C (? No change/decrease)

Thrombin activatable fibrinolytic inhibitor TAFI?

Factor XII Antithrombin

**Proteins whose plasma concentrations decrease with**

and chronic inflammation [17].

**inflammation**

36 Acute Phase Proteins

Fibrinogen Factor VIII Protein S

protein Urokinase α 1 Antitrypsin α 2 Macrogloulin von Willebrand factor C1-esterase inhibitor C-reactive protein Thrombopoietin

**inflammation**

**4.1. Fibrinogen**

**Proteins whose plasma concentrations increase with**

Plasminogen activator inhibitor PAI-1 C4b-binding

Thrombin activatable fibrinolytic inhibitor (TAFI)?

**Table 1.** Acute phase proteins that directly affect haemostasis

this fibrin matrix production and its lysis tightly controlled [19].

**Figure 5.** Fibrinogen and fibrin modulation of inflammation. Fibrin(ogen) modulates the inflammatory response by affecting leukocyte migration but also by induction of cytokine/chemokine expression mostly via Mac-1 signalling. NO-nitric oxide, Mac-1 Macrophage antigen 1, PI3K Phosphoinositide-3-kiniase, TNF tumor necrosis factor. Repro‐ duced with kind permission of Prof. K Zacharowski

There is good evidence that fibrinogen has a function in regulation of the inflammatory response as is seen in the increased concentration of fibrinogen associated with atherosclerosis and cardiovascular risk. Fibrin(ogen) also participates in activation of vascular cells and regulation of the inflammatory response via the ability to bind to and activate a number of immune cells. The cellular ligand binding of fibrinogen is totally distinct to any coagulation function. Fibrin(ogen) can bind to the integrin receptor Mac-1 which is found on many myeloid cells including monocytes and neutrophils and also T cells. The Mac-1 integrin is involved in phagocytosis, adhesion, migration through the endothelium as well as apoptosis and degra‐ nulation (Figure 5). Binding of fibrinogen to Mac-1 also induces production of cytokines IL-1β, IL-6 and TNF-α potentiating the inflammatory response, as many acute phase proteins do [20].

Protein S has been shown to increase during inflammation. This may in part be due to counterbalancing the procoagulant drive of the coagulation system in these circumstances by providing more anticoagulant effect via the protein C pathway. It may also have to do with other non-anticoagulant actions it has via its binding with C4BP (see below). Certainly inhibition of protein S in in-vivo models of bacteraemia have shown to provoke the cytokine response and what was a non-lethal injected dose of *E.coli* in baboons resulted in a lethal

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The complement binding protein C4BP is a co-factor to a serine protease in the degredation of C4 in the classical complement pathway and providing it has been suggested protection from inflammation at a cellular level. The C4BP molecule has a central region with seven α chains and one β chain emanating from it each with distinct ligand binding regions. It is on the β chain side arm that the protein S binding region resides and in normal human plasma ~80% of C4BP is found as this β chain form. C4BP is an acute phase protein and its levels can increase to 400% of normal [25]. However this is mainly of the C4BPα form so the Protein S-C4BP complex concentration is not generally affected. As so much protein S is normally bound to C4BP and the concentration of the free anticoagulant active co-factor protein S level quite tightly controlled is there a physiological role for the PS-C4BP complex? It has been suggested that protein S forms one of the bridges between coagulation and inflammation. As protein S has a high affinity to bind to negatively charged phospholipids it can bring C4BP into close proximity to these sites allowing controlled complement activation at areas of vascular damage where coagulation is activated. This has been linked to apoptosis and the complement system allowing rapid clearance of apoptotic cells from the site of damage by macrophages but more probably limiting or inhibiting further complement activation via necrotic cells [27, 28].

The fibrinolytic system is the opposite of the coagulation system. It limits clot formation to the site of injury and breaks down existing clot by an enzyme cascade. It is also, like haemostasis, linked to inflammation with acute phase proteins that increase and others that are decreased. Plasminogen provides the fibrinolytic potential and when converted to its active form plasmin is able to bind to fibrin and begin its degradation. Plasminogen is activated by two activators, tissue plasminogen activator (t-PA) and urokinase tissue plasmingen activator (u-PA). These in turn are inhibited by plasminogen activator inhibitor 1 and 2 (PAI-1, PAI-2) and plasmin is inhibited by α-2-antiplasmin. Of these inhibitors PAI-1 is a positive acute phase protein and

(TNF-α) suppresses finbrinolysis by down regulating t-PA expression in endothelial cells while production PAI-1 then in turn PAI-1 inhibits TNF-α is inhibited by PAI-1. It has also

reaction [26].

**4.4. C4b-binding protein**

**4.5. Plasminogen activator inhibitor PAI-1**

increases during inflammation. The cytokine tissue necrosis factor

been shown that by inhibiting TNF-α plasma levels of PAI-1 decrease [19].

When fibrin is laid down in the microvasculature bed this enhances local and systemic inflammation via expression of proinflammatory mediators. Fibrin(ogen) increases mRNA concentration and induces synthesis of the proinflammatory cytokines IL-6 and TNF-α in monocytes and macrophages which is induced by chemokine expression, macrophage chemotactic protein (MCP-1) and macrophage inflammatory protein (MIP-1 and 2). The Toll receptor TLR 4 facilitates this fibrin(gen) chemokine expression signalling [20].

Breakdown products of fibrin, D-dimers stimulate monocytes to release the following IL-1, IL-6 and PAI-1 and fibrin degradation products also induce C-reactive protein production.

#### **4.2. Factor VIII**

Factor VIII is a procoagulant whose deficiency leads to classical haemophilia and a bleeding diathesis [21]. Factor VIII once activated by small amounts of thrombin becomes a co-factor for activated factor IXa in the formation of the tenase complex whereby these catalysts convert factor X to its activated form Xa. A number of publications have shown raised factor VIII to be linked with venous thromboembolism and increased thrombin generation [22, 23]. This increase in factor VIII has been linked with an increase in basal inflammatory reaction. Factor VIII increased production is mediated by the cytokine IL-6, however it is debateable as to how much a persistently raised factor VIII is responsible to inflammation by some [24]. Post operatively particularly after cardiac surgery factor VIII can be raised by as much as 2-3 fold.

#### **4.3. Protein S**

Protein S is a co-factor for protein C and produced mainly in the hepatocyes but also endothelial cells and megakaryocytes. Once protein C has been activated by the thrombin thrombomo‐ dulin complex, which is augmented by the endothelial protein C receptor, activated protein C dissociates from the endothelial protein C receptor and binds with protein S. This complex is then able to inhibit factors Va and VIIIa, protein S enhancing the reactive cleaving of specific sites by APC on factors V and VIII by anything up to 20-fold. At any one time only approxi‐ mately 40% of circulating protein S is free and able to participate in this reaction. The remaining 60% of circulating protein S is bound to the complement regulator protein C4B-binding protein and lacks the co-factor functionality in this reaction. Consequently the protein S-C4BP complex limits the functionality of activated protein C in its anticoagulant role. Therefore protein S plays an important role in the regulation of thrombin generation although it being a risk factor for venous thrombosis in deficient patients remains unclear and the odds risks have varied between 0 – 11.5 fold in protein S deficient cases.

Protein S has an accelerating role in APC mediated PAI-1 inhibition thereby promoting clot lysis and a possible inhibition of activation of thrombin activatable fibrinolytic inhibitor [25].

Protein S has been shown to increase during inflammation. This may in part be due to counterbalancing the procoagulant drive of the coagulation system in these circumstances by providing more anticoagulant effect via the protein C pathway. It may also have to do with other non-anticoagulant actions it has via its binding with C4BP (see below). Certainly inhibition of protein S in in-vivo models of bacteraemia have shown to provoke the cytokine response and what was a non-lethal injected dose of *E.coli* in baboons resulted in a lethal reaction [26].

#### **4.4. C4b-binding protein**

function. Fibrin(ogen) can bind to the integrin receptor Mac-1 which is found on many myeloid cells including monocytes and neutrophils and also T cells. The Mac-1 integrin is involved in phagocytosis, adhesion, migration through the endothelium as well as apoptosis and degra‐ nulation (Figure 5). Binding of fibrinogen to Mac-1 also induces production of cytokines IL-1β, IL-6 and TNF-α potentiating the inflammatory response, as many acute phase proteins do [20].

When fibrin is laid down in the microvasculature bed this enhances local and systemic inflammation via expression of proinflammatory mediators. Fibrin(ogen) increases mRNA concentration and induces synthesis of the proinflammatory cytokines IL-6 and TNF-α in monocytes and macrophages which is induced by chemokine expression, macrophage chemotactic protein (MCP-1) and macrophage inflammatory protein (MIP-1 and 2). The Toll

Breakdown products of fibrin, D-dimers stimulate monocytes to release the following IL-1, IL-6 and PAI-1 and fibrin degradation products also induce C-reactive protein production.

Factor VIII is a procoagulant whose deficiency leads to classical haemophilia and a bleeding diathesis [21]. Factor VIII once activated by small amounts of thrombin becomes a co-factor for activated factor IXa in the formation of the tenase complex whereby these catalysts convert factor X to its activated form Xa. A number of publications have shown raised factor VIII to be linked with venous thromboembolism and increased thrombin generation [22, 23]. This increase in factor VIII has been linked with an increase in basal inflammatory reaction. Factor VIII increased production is mediated by the cytokine IL-6, however it is debateable as to how much a persistently raised factor VIII is responsible to inflammation by some [24]. Post operatively particularly after cardiac surgery factor VIII can be raised by as much as 2-3 fold.

Protein S is a co-factor for protein C and produced mainly in the hepatocyes but also endothelial cells and megakaryocytes. Once protein C has been activated by the thrombin thrombomo‐ dulin complex, which is augmented by the endothelial protein C receptor, activated protein C dissociates from the endothelial protein C receptor and binds with protein S. This complex is then able to inhibit factors Va and VIIIa, protein S enhancing the reactive cleaving of specific sites by APC on factors V and VIII by anything up to 20-fold. At any one time only approxi‐ mately 40% of circulating protein S is free and able to participate in this reaction. The remaining 60% of circulating protein S is bound to the complement regulator protein C4B-binding protein and lacks the co-factor functionality in this reaction. Consequently the protein S-C4BP complex limits the functionality of activated protein C in its anticoagulant role. Therefore protein S plays an important role in the regulation of thrombin generation although it being a risk factor for venous thrombosis in deficient patients remains unclear and the odds risks have varied

Protein S has an accelerating role in APC mediated PAI-1 inhibition thereby promoting clot lysis and a possible inhibition of activation of thrombin activatable fibrinolytic inhibitor [25].

receptor TLR 4 facilitates this fibrin(gen) chemokine expression signalling [20].

**4.2. Factor VIII**

38 Acute Phase Proteins

**4.3. Protein S**

between 0 – 11.5 fold in protein S deficient cases.

The complement binding protein C4BP is a co-factor to a serine protease in the degredation of C4 in the classical complement pathway and providing it has been suggested protection from inflammation at a cellular level. The C4BP molecule has a central region with seven α chains and one β chain emanating from it each with distinct ligand binding regions. It is on the β chain side arm that the protein S binding region resides and in normal human plasma ~80% of C4BP is found as this β chain form. C4BP is an acute phase protein and its levels can increase to 400% of normal [25]. However this is mainly of the C4BPα form so the Protein S-C4BP complex concentration is not generally affected. As so much protein S is normally bound to C4BP and the concentration of the free anticoagulant active co-factor protein S level quite tightly controlled is there a physiological role for the PS-C4BP complex? It has been suggested that protein S forms one of the bridges between coagulation and inflammation. As protein S has a high affinity to bind to negatively charged phospholipids it can bring C4BP into close proximity to these sites allowing controlled complement activation at areas of vascular damage where coagulation is activated. This has been linked to apoptosis and the complement system allowing rapid clearance of apoptotic cells from the site of damage by macrophages but more probably limiting or inhibiting further complement activation via necrotic cells [27, 28].

#### **4.5. Plasminogen activator inhibitor PAI-1**

The fibrinolytic system is the opposite of the coagulation system. It limits clot formation to the site of injury and breaks down existing clot by an enzyme cascade. It is also, like haemostasis, linked to inflammation with acute phase proteins that increase and others that are decreased. Plasminogen provides the fibrinolytic potential and when converted to its active form plasmin is able to bind to fibrin and begin its degradation. Plasminogen is activated by two activators, tissue plasminogen activator (t-PA) and urokinase tissue plasmingen activator (u-PA). These in turn are inhibited by plasminogen activator inhibitor 1 and 2 (PAI-1, PAI-2) and plasmin is inhibited by α-2-antiplasmin. Of these inhibitors PAI-1 is a positive acute phase protein and increases during inflammation. The cytokine tissue necrosis factor

(TNF-α) suppresses finbrinolysis by down regulating t-PA expression in endothelial cells while production PAI-1 then in turn PAI-1 inhibits TNF-α is inhibited by PAI-1. It has also been shown that by inhibiting TNF-α plasma levels of PAI-1 decrease [19].

The increase in PAI-1 levels is again a driver of the haemostatic system towards a more prothrombotic state. PAI-2 will not be discussed here but for interested readers they are directed to the following publication [29].

**4.9. C1-esterase inhibitor**

inhibiting role [36].

ADAMTS-13.

of cleavage of the ultralarge VWF multimers.

of exhaustion of metalloprotease activity [37].

**4.10. von Willebrand factor**

C1-inhibitor (C1INH) is a serpin and major inhibitor of the contact coagulation system that involves factor XII, kallikrein and kininogen. It is also an important regulator of complement activation inhibiting the first component of complement C1. Another important biological role of C1INH is vascular permeability regulation [35]. This is well illustrated in patients who suffer from hereditary angioedema where there is a deficiency of the C1INH activity. As it now seems factor XII plays a substantial part in the formation of thrombin during sepsis and inflammation where neutrophil extracellular traps are present that release polyphosphate that in turn activate factor XII it would appear normal for C1INH to act as a positive acute phase protein in limiting this activation as well as its complement inhibitory role. A rapid appearance of C1INH-factor XIIa complexes is reported during sepsis with a sharp fall in the C1INH activity wherein the inhibitory function of α-2Macroglobin becomes more important in its kallikrein

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The von Willebrand factor (VWF) is a multimeric protein that mediates the adhesion of platelets to the exposed subendothelium at the site of vascular injury. It also serves as a carrier protein for the labile coagulation factor VIII. VWF is synthesised in and stored in the Weibal-Palade bodies of endothelial cells and megakaryocytes/platelets. Endothelial cells release VWF when activated or stimulated. These VWF molecules are ultralarge and hyperactive, proficient in binding to platelets via the glycoprotein Ib-X-V complex without any external activation. Normally the ultralarge VWF multimers are cleaved into smaller less active multimers before being released into plasma. Cleavage is performed by ADAMTS-13 (a disintegrin and metal‐ loprotease with thrombospondin motif). A deficiency of ADAMTS-13 leads to a thrombotic thrombocytopenic purpura a thrombotic microangiopathy. Increased plasma levels of VWF have been reported in a number of disease states including coronary artery disease, autoim‐ mune disease, trauma and infections. A common underlying process in all of these is inflam‐ mation. There appears to be a complex interaction between the common inflammatory cytokines IL-6, IL-1β and TNF-α the release and cleavage of the VWF multimers by

Release of VWF multimers from endothelial cells occurs in a dose dependent fashion when stimulated with IL-8 and TNF-α and IL-6 inhibits cleavage of the ultrlarge multimers. This demonstrates that inflammatory cytokines interfere with the equilibrium of release and rate

This increases prothrombotic risk in this setting as most of the positive acute phase proteins have a propensity to do creating a procoagulant environment. It is of interest to note that IL-8 resides in the Weibel-Palade bodies of the platelet the same place as VWF and is involved in the platelet leukocyte aggregation and it is TNF-α that activates endothelial cells releasing IL-8. The inhibition by IL-6 of ultralarge multimer cleavage by ADAMTS-13 may be by way of synthetic and secretion inhibition. In overwhelming sepsis there is also probably an element

#### **4.6. Urokinase**

Urokinase plasminogen activator (u-PA) is a multifunctional serine protease. It has been shown to act as both a signalling ligand and a proteolytic enzyme. As an acute phase protein its increase has been found in a number of metastatic carcinomas and this has led to a link in its role of tumor growth and cellular expansion in conditions such as cardiac fibrosis and atherosclerosis [30]. As well as displaying involvement in tissue remodelling u-PA also appears to regulate macrophage activation and function. The macrophages not only synthesis and release u-PA but also have receptors for the u-PA protease on their membranes. The excreted u-PA induces TNF-α synthesis and secretion. The u-PA molecule has also been shown to initiate inflammation via the release of IL-6 and IL-1β from monocytes and lymphocytes [31]. The u-PA and its receptor uPAR also mediate immune complex induced inflammation in the lung. This is achieved via generation of u-PA at the site of cellular inflammation with subsequent activation of its receptor uPAR. This then sets off cellular signalling with C5a/C5aR on the alveolar macrophages, recruitment of polymorphonuclear leukocytes and adequate TNF-α production [32].

#### **4.7. α 1 Antitrypsin**

The α-1-antitrypsin glycoprotein is a proteinase inhibitor synthesised and secreted in the hepatocytes of the Liver. It is one of two physiological inhibtors of activated protein C. It also has a profound anti-inflammatory effect in the lungs with concentrations during a inflamma‐ tory response reaching levels found in the plasma. The range of antiprotease activity seen in the lungs includes neutrophil elastase and plasminogen acitvators. If there is a deficiency of α-1-antitrypsin (as with the genetically abnormal SERPINA1 gene mutations) then neutrophil elastase provoked by infection and inflammation will unchecked breakdown elastin and destroy alveolar walls leading to emphysema [33].

#### **4.8. Alpha-2-macroglobulin**

Alpha-2-Macroglobulin (α-2M) is an ancient serine protease binding host or foreign peptides and particles, thereby serving as humoral defense barrier against pathogens in the plasma and tissues. In humans α-2M, interacts and captures virtually any proteinase whether self or foreign, suggesting a function as a unique "panproteinase inhibitor." In adult humans it provides somewhere between 10-25% of the overall anti-thrombin activity in plasma. It is also the primary inhibitor of thrombin in neonates and infants under 1 year of age until the Liver matures and begins producing sufficient Antithrombin to take over the role. At times of inflammation when antithrombin levels are low α-2M can become a 'back-up' thrombin inhibitor [34].

#### **4.9. C1-esterase inhibitor**

The increase in PAI-1 levels is again a driver of the haemostatic system towards a more prothrombotic state. PAI-2 will not be discussed here but for interested readers they are

Urokinase plasminogen activator (u-PA) is a multifunctional serine protease. It has been shown to act as both a signalling ligand and a proteolytic enzyme. As an acute phase protein its increase has been found in a number of metastatic carcinomas and this has led to a link in its role of tumor growth and cellular expansion in conditions such as cardiac fibrosis and atherosclerosis [30]. As well as displaying involvement in tissue remodelling u-PA also appears to regulate macrophage activation and function. The macrophages not only synthesis and release u-PA but also have receptors for the u-PA protease on their membranes. The excreted u-PA induces TNF-α synthesis and secretion. The u-PA molecule has also been shown to initiate inflammation via the release of IL-6 and IL-1β from monocytes and lymphocytes [31]. The u-PA and its receptor uPAR also mediate immune complex induced inflammation in the lung. This is achieved via generation of u-PA at the site of cellular inflammation with subsequent activation of its receptor uPAR. This then sets off cellular signalling with C5a/C5aR on the alveolar macrophages, recruitment of polymorphonuclear leukocytes and adequate

The α-1-antitrypsin glycoprotein is a proteinase inhibitor synthesised and secreted in the hepatocytes of the Liver. It is one of two physiological inhibtors of activated protein C. It also has a profound anti-inflammatory effect in the lungs with concentrations during a inflamma‐ tory response reaching levels found in the plasma. The range of antiprotease activity seen in the lungs includes neutrophil elastase and plasminogen acitvators. If there is a deficiency of α-1-antitrypsin (as with the genetically abnormal SERPINA1 gene mutations) then neutrophil elastase provoked by infection and inflammation will unchecked breakdown elastin and

Alpha-2-Macroglobulin (α-2M) is an ancient serine protease binding host or foreign peptides and particles, thereby serving as humoral defense barrier against pathogens in the plasma and tissues. In humans α-2M, interacts and captures virtually any proteinase whether self or foreign, suggesting a function as a unique "panproteinase inhibitor." In adult humans it provides somewhere between 10-25% of the overall anti-thrombin activity in plasma. It is also the primary inhibitor of thrombin in neonates and infants under 1 year of age until the Liver matures and begins producing sufficient Antithrombin to take over the role. At times of inflammation when antithrombin levels are low α-2M can become a 'back-up' thrombin

directed to the following publication [29].

**4.6. Urokinase**

40 Acute Phase Proteins

TNF-α production [32].

**4.8. Alpha-2-macroglobulin**

inhibitor [34].

destroy alveolar walls leading to emphysema [33].

**4.7. α 1 Antitrypsin**

C1-inhibitor (C1INH) is a serpin and major inhibitor of the contact coagulation system that involves factor XII, kallikrein and kininogen. It is also an important regulator of complement activation inhibiting the first component of complement C1. Another important biological role of C1INH is vascular permeability regulation [35]. This is well illustrated in patients who suffer from hereditary angioedema where there is a deficiency of the C1INH activity. As it now seems factor XII plays a substantial part in the formation of thrombin during sepsis and inflammation where neutrophil extracellular traps are present that release polyphosphate that in turn activate factor XII it would appear normal for C1INH to act as a positive acute phase protein in limiting this activation as well as its complement inhibitory role. A rapid appearance of C1INH-factor XIIa complexes is reported during sepsis with a sharp fall in the C1INH activity wherein the inhibitory function of α-2Macroglobin becomes more important in its kallikrein inhibiting role [36].

#### **4.10. von Willebrand factor**

The von Willebrand factor (VWF) is a multimeric protein that mediates the adhesion of platelets to the exposed subendothelium at the site of vascular injury. It also serves as a carrier protein for the labile coagulation factor VIII. VWF is synthesised in and stored in the Weibal-Palade bodies of endothelial cells and megakaryocytes/platelets. Endothelial cells release VWF when activated or stimulated. These VWF molecules are ultralarge and hyperactive, proficient in binding to platelets via the glycoprotein Ib-X-V complex without any external activation. Normally the ultralarge VWF multimers are cleaved into smaller less active multimers before being released into plasma. Cleavage is performed by ADAMTS-13 (a disintegrin and metal‐ loprotease with thrombospondin motif). A deficiency of ADAMTS-13 leads to a thrombotic thrombocytopenic purpura a thrombotic microangiopathy. Increased plasma levels of VWF have been reported in a number of disease states including coronary artery disease, autoim‐ mune disease, trauma and infections. A common underlying process in all of these is inflam‐ mation. There appears to be a complex interaction between the common inflammatory cytokines IL-6, IL-1β and TNF-α the release and cleavage of the VWF multimers by ADAMTS-13.

Release of VWF multimers from endothelial cells occurs in a dose dependent fashion when stimulated with IL-8 and TNF-α and IL-6 inhibits cleavage of the ultrlarge multimers. This demonstrates that inflammatory cytokines interfere with the equilibrium of release and rate of cleavage of the ultralarge VWF multimers.

This increases prothrombotic risk in this setting as most of the positive acute phase proteins have a propensity to do creating a procoagulant environment. It is of interest to note that IL-8 resides in the Weibel-Palade bodies of the platelet the same place as VWF and is involved in the platelet leukocyte aggregation and it is TNF-α that activates endothelial cells releasing IL-8. The inhibition by IL-6 of ultralarge multimer cleavage by ADAMTS-13 may be by way of synthetic and secretion inhibition. In overwhelming sepsis there is also probably an element of exhaustion of metalloprotease activity [37].

#### **4.11. C-reactive protein**

C-reactive protein (CRP) is a pentameric molecule that increases several hundred fold following an inflammatory stimulatory response this being primarily due to IL-6 stimulation of production of CRP in the hepatocytes of the Liver. CRP amplifies the host defence mecha‐ nism by activation of complement via C1 and stimulation of macrophages. CRP also upregu‐ lates tissue factor expression on monocytes [38] and induces release of PAI-1 thereby downregulating fibrinolysis [39] promoting a procoagulant state. The pentameric CRP is thought to be directly proinflammatory at high concentration like those of sepsis or major surgery but more subtle inflammatory reactions take place when monomeric CRP is released from the pentameric form. This appears to be driven by activated platelets revealing new lipid messenger sites; lysophosphatidycholine (LPC) that bind and dissociate the pentameric form of CRP to the monmeric form [40].

endothelial and platelet activation. AT also appears to directly act as an anti-inflammatory agent by binding with leukocytes receptors blocking their communication with endothelial cells and limiting their adhesion and migration [45]. In animaI models it has also been shown that AT induces the release of prostacyclin from endothelial cells. The prostacyclin is a platelet inhibitor and abrogates neutrophils adhering to endothelial cells both of which contribute to

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In addition AT can also modulate cellular receptor expression by downregulating the expres‐

AT is also able to decrease expression of tissue factor and IL-6 expression on monocytes and

Histindine rich glycoprotein (HRGP) is a single polypeptide chain protein that is synthesised in the Liver found in plasma and on the surface of leukocytes, monocytes and the α-granules of platelets. HRGP has been shown to bind to a number of molecules including, haem, Zn2+, plasminogen, fibrinogen, IgG, complement and factor XIIa (not the inactive zymogen) [49]. HRGP exerts some degree of innate immune response by exhibiting antimicrobial activity to some organisms and removal of necrotic cells by binding to free decondensed DNA (polyphos‐

HRGP behaves as a negative acute protein during inflammation [50]. This is of interest as fibrinogen one of its primary ligands is increased. Possibly more importantly is the fact that HRGP strongly inhibits polyphosphate induced factor XII autoactivation via increased Zn2+ levels in response to local platelet activation, showing it to be a significant modulator of factor XII activation during sepsis and inflammation [51]. Because of its negative acute phase nature this will allow increased activation of factor XII in these circumstances potentiating the pro-

Thrombomodulin (TM) is present on endothelial cells of the entire vasculature and is a transmembrane protein with epidermal growth factor (EGF) repeating molecules that stick out of the plasma membrane and provide binding and activation sites for a number of molecules. There is a high affinity binding site for thrombin on EGF domain 5-6 from which protein C is activated at EGF domains 4-6. Thrombin activatable fibrinolytic inhibitor is activated by thrombin at EGF sites 3-6 [52]. When thrombin binds and complexes with thrombomodulin on the cell membrane its ability to activate protein C increases by greater than 1000-fold. Activated protein C anti-inflammatory effects are discussed later in this chapter, see below. Once thrombin has bound to TM it becomes quickly neutralised by its natural serine proteases inhibitors antithrombin, heparin co-factor II and protein C inhibitor and therefore activation

The effect of inflammation on TM is to decrease its presence on the endothelial cell surface by its cellular internalisation by endocytosis via TNF-α. This creates a site where coagulation can

sion of CD11b/CD18 on leucocytes which bind factor X aiding its activation [47].

endothelial cells reducing the inflammatory drive [48].

the inflammatory response [46].

**5.2. Histidine rich glycoprotein**

inflammatory and pro-coagulant condition.

**5.3. Thrombomodulin**

of protein C also ceases.

phate).

During sepsis when disseminated intravascular coagulation (DIC) occurs this has been found experimentally to co-inside with formation of a calcium-dependent complex between CRP and very low density lipoprotein (VLDL). The VLDL molecular makeup is different in septic patients to normal controls due to a deficiency of phosphatidylethanolamine. This CRP-VLDL lipid raft increases the procoagulant effect through an increase in prothrombinase activity. The CRP-VLDL complex exists in vivo and it has been postulated that is has a pathogenic role in disseminating the intravascular coagulation [41].

## **5. Coagulation proteins whose plasma concentrations decrease during inflammation**

#### **5.1. Antithrombin**

Antithrombin (AT) is one of the major natural anticoagulats inhibiting thrombin, factor Xa, IXa and factor VIIa bound to tissue factor. Inhibition of factors Xa, IXa and the VIIa-tissue factor are accelerated via the endothelial cell heparin like proteoglycans. The importance of this anticoagulant pathway is highlighted when AT levels are low, either congenitally or acquired, the risk of thrombosis is significantly increased.

AT has been shown in-vitro using HUVEC cells and IL-6 to act as a negative acute phase protein [42]. Other mechanisms that can reduce AT function during an inflammatory response include increased consumption from activation of haemostasis and increased degradation by proteo‐ lytic enzymes (elastase released from activated neutrophils). Furthermore inflammatory cytokines can induce a reduction in the production of glycosaminoglycans such as heparin and chondroitin sulphate on the endothelial cell which may contribute to the impaired function of AT due to GAGs acting as physiological heparin-like cofactors promoting the anticoagulant anti-thrombin activity of AT [43, 44].

AT can indirectly act as an anti-inflammatory molecule by directly inhibiting thrombin reducing its inflammatory properties of haemostatic and complement activation, leukocyte, endothelial and platelet activation. AT also appears to directly act as an anti-inflammatory agent by binding with leukocytes receptors blocking their communication with endothelial cells and limiting their adhesion and migration [45]. In animaI models it has also been shown that AT induces the release of prostacyclin from endothelial cells. The prostacyclin is a platelet inhibitor and abrogates neutrophils adhering to endothelial cells both of which contribute to the inflammatory response [46].

In addition AT can also modulate cellular receptor expression by downregulating the expres‐ sion of CD11b/CD18 on leucocytes which bind factor X aiding its activation [47].

AT is also able to decrease expression of tissue factor and IL-6 expression on monocytes and endothelial cells reducing the inflammatory drive [48].

#### **5.2. Histidine rich glycoprotein**

**4.11. C-reactive protein**

42 Acute Phase Proteins

of CRP to the monmeric form [40].

**inflammation**

**5.1. Antithrombin**

disseminating the intravascular coagulation [41].

the risk of thrombosis is significantly increased.

anti-thrombin activity of AT [43, 44].

C-reactive protein (CRP) is a pentameric molecule that increases several hundred fold following an inflammatory stimulatory response this being primarily due to IL-6 stimulation of production of CRP in the hepatocytes of the Liver. CRP amplifies the host defence mecha‐ nism by activation of complement via C1 and stimulation of macrophages. CRP also upregu‐ lates tissue factor expression on monocytes [38] and induces release of PAI-1 thereby downregulating fibrinolysis [39] promoting a procoagulant state. The pentameric CRP is thought to be directly proinflammatory at high concentration like those of sepsis or major surgery but more subtle inflammatory reactions take place when monomeric CRP is released from the pentameric form. This appears to be driven by activated platelets revealing new lipid messenger sites; lysophosphatidycholine (LPC) that bind and dissociate the pentameric form

During sepsis when disseminated intravascular coagulation (DIC) occurs this has been found experimentally to co-inside with formation of a calcium-dependent complex between CRP and very low density lipoprotein (VLDL). The VLDL molecular makeup is different in septic patients to normal controls due to a deficiency of phosphatidylethanolamine. This CRP-VLDL lipid raft increases the procoagulant effect through an increase in prothrombinase activity. The CRP-VLDL complex exists in vivo and it has been postulated that is has a pathogenic role in

**5. Coagulation proteins whose plasma concentrations decrease during**

Antithrombin (AT) is one of the major natural anticoagulats inhibiting thrombin, factor Xa, IXa and factor VIIa bound to tissue factor. Inhibition of factors Xa, IXa and the VIIa-tissue factor are accelerated via the endothelial cell heparin like proteoglycans. The importance of this anticoagulant pathway is highlighted when AT levels are low, either congenitally or acquired,

AT has been shown in-vitro using HUVEC cells and IL-6 to act as a negative acute phase protein [42]. Other mechanisms that can reduce AT function during an inflammatory response include increased consumption from activation of haemostasis and increased degradation by proteo‐ lytic enzymes (elastase released from activated neutrophils). Furthermore inflammatory cytokines can induce a reduction in the production of glycosaminoglycans such as heparin and chondroitin sulphate on the endothelial cell which may contribute to the impaired function of AT due to GAGs acting as physiological heparin-like cofactors promoting the anticoagulant

AT can indirectly act as an anti-inflammatory molecule by directly inhibiting thrombin reducing its inflammatory properties of haemostatic and complement activation, leukocyte, Histindine rich glycoprotein (HRGP) is a single polypeptide chain protein that is synthesised in the Liver found in plasma and on the surface of leukocytes, monocytes and the α-granules of platelets. HRGP has been shown to bind to a number of molecules including, haem, Zn2+, plasminogen, fibrinogen, IgG, complement and factor XIIa (not the inactive zymogen) [49]. HRGP exerts some degree of innate immune response by exhibiting antimicrobial activity to some organisms and removal of necrotic cells by binding to free decondensed DNA (polyphos‐ phate).

HRGP behaves as a negative acute protein during inflammation [50]. This is of interest as fibrinogen one of its primary ligands is increased. Possibly more importantly is the fact that HRGP strongly inhibits polyphosphate induced factor XII autoactivation via increased Zn2+ levels in response to local platelet activation, showing it to be a significant modulator of factor XII activation during sepsis and inflammation [51]. Because of its negative acute phase nature this will allow increased activation of factor XII in these circumstances potentiating the proinflammatory and pro-coagulant condition.

#### **5.3. Thrombomodulin**

Thrombomodulin (TM) is present on endothelial cells of the entire vasculature and is a transmembrane protein with epidermal growth factor (EGF) repeating molecules that stick out of the plasma membrane and provide binding and activation sites for a number of molecules. There is a high affinity binding site for thrombin on EGF domain 5-6 from which protein C is activated at EGF domains 4-6. Thrombin activatable fibrinolytic inhibitor is activated by thrombin at EGF sites 3-6 [52]. When thrombin binds and complexes with thrombomodulin on the cell membrane its ability to activate protein C increases by greater than 1000-fold. Activated protein C anti-inflammatory effects are discussed later in this chapter, see below. Once thrombin has bound to TM it becomes quickly neutralised by its natural serine proteases inhibitors antithrombin, heparin co-factor II and protein C inhibitor and therefore activation of protein C also ceases.

The effect of inflammation on TM is to decrease its presence on the endothelial cell surface by its cellular internalisation by endocytosis via TNF-α. This creates a site where coagulation can take place as the anticoagulant barrier has been removed and it may also stimulated further inflammatory response [43]. CRP has also been shown in experimental conditions using human coronary artery endothelial cells treated with CRP in a dose and time dependent manner to reduce messenger RNA levels of TM [53]. TM also provides anti-inflammatory protection from complement activation by enhancing inactivation of C3b and by promoting activation of thrombin-activatable fibrinolysis inhibitor that inactivates complement anaphy‐ latoxins C5a and C3a [54]. Others have shown that thrombin-activatable fibrinolysis inhibitor activation via TM is attenuated by platelet factor 4 released from activated platelets [55].

**6. Other major components of the acute phase response that affect**

The protein C pathway is known to be an important anticoagulant system with patients deficient in protein C being at risk of thrombosis or in its homozygous form purpura fulminans. Activated protein C inhibits factors V and VIII this being supported by the activation of protein C by thrombin bound to thrombomodulin on the endothelial cell surface. As well as acting as an anticoagulant activated protein C is also able to inhibit PAI-1. The anticoagulant and antifibrinolytic aspects of the protein C pathway have been elucidated and well described although there still appears much to learn from the interaction of protein C during the

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It is unclear from studies of the acute phase proteins whether protein C acts as a positive or negative acute phase protein. Most studies show it to have no change in concentration during an inflammatory response or its plasma concentration to decrease. A decrease in protein C could be attributed to consumption as well as a cytokines limiting the natural anticoagulant response. Activated protein C also confers a cytoprotective, anti-inflammatory, anti-apoptosis

Activated protein C signals its anti-inflammatory effects mainly via PAR-1 pathways whereby following Gi signalling and sphingosine-1-phosphate production there is improvement in

Transcriptional profiling studies using cell cultures of human umbilical vein endothelial cells (HUVECs) have demonstrated that recombinant human activated protein C can regulate endothelial cell gene expression linked to inflammation and cell survival [63]. The activated protein C suppresses NFκB a cell nuclear transcription factor, and by reducing its expression

Cyotkines are a superfamily of molecules involved in cell signalling many of which are increased greater than 1000-fold during an inflammatory insult. Cytokines such as interleukin (IL)-6, but also platelet-derived growth factor and monocyte chemoattractant protein (MCP)-1 are capable of stimulating tissue factor expression in mononuclear cells. Tissue factor being

IL-6 is a multifunctional cytokine that is induced in many disease states such as sepsis, endotoxaemia and in-vitro after administration of tumor necrosis factor (TNF). Several studies have suggested IL-6 to be a potential mediator of endotoxin induced coagulation activation. This has been validated by treatment of chimpanzees with a monoclonal anti-IL6 antibody that ablated the activation of coagulation [65]. Cytokines are pivotal in providing a means of cross

and endothelial barrier stabilisation effect when active [61].

and function this in turn causes inhibition of cytokine signaling [64].

the initiator of thrombin generation and fibrin formation.

talk between inflammation and coagulation [66].

**haemostasis**

**6.1. Protein C**

inflammatory response.

**6.2. Cytokines**

endothelial cell barrier function [62].

#### **5.4. Endothelial protein C receptor**

The thrombomodulin-thrombin conversion of protein C to its activated form is facilitated by the endothelial protein C receptor (EPCR). The EPCR is another endothelial cell transmem‐ brane protein located in close proximity to the thrombomodulin molecule and with high affinity binding for protein C. EPCR as well as binding protein C also binds its activated form and via membrane lipid rafts they complex with PAR-1 [56]. The EPCR-activated protein C molecule activates PAR-1 in a different way to thrombin which allows it to signal through a potent Gi protein pathway by which anti-inflammatory pathways are stimulated within the endothelial cell [57]. C-reactive protein has also been shown, as in the case of thrombomodulin, under experimental cell culture conditions to down regulate EPCR.

#### **5.5. Thrombin activatable fibrinolytic inhibitor**

Thrombin activatable fibrinolytic inhibitor (TAFI) is a basic carboxypeptidase identical to the plasma procarboxypeptidases B, U and R. TAFI is activated by thrombin and plasmin although the most efficient activator is the thrombomodulin-thrombin cellular membrane bound complex. TAFI inhibits fibrinolysis by cleaving lysine residues from fibrin which restricts the binding of tissue plasminogen activator to these sites and enhancing the plasminogen con‐ version to plasmin potentiating further fibrin breakdown [58]. TAFI certainly modulates the balance between coagulation and fibrinolysis but it appears its linking role with inflammation still needs to be fully elucidated. For instance TAFI has been shown to be a positive acute phase protein in mice [59] but in the same year Boffa et al interestingly showed that IL-6 administered to cultured HepG2 cells resulted in a 60% decrease in TAFI mRNA [58]. Subsequently TAFI has been shown to be raised in experimental endotoxemia [60]*.* Its anti-inflammatory proper‐ ties are also downregulated by platelet factor 4 which is release from activated platelets and endothelial cells during activating stimuli and inflammatory insults which prevents TAFI activation by binding to the thrombin-thrombomodulin complex thereby preventing TAFI's inactivation of the complement anaphylatoxins C5a and C3a [55].

## **6. Other major components of the acute phase response that affect haemostasis**

#### **6.1. Protein C**

take place as the anticoagulant barrier has been removed and it may also stimulated further inflammatory response [43]. CRP has also been shown in experimental conditions using human coronary artery endothelial cells treated with CRP in a dose and time dependent manner to reduce messenger RNA levels of TM [53]. TM also provides anti-inflammatory protection from complement activation by enhancing inactivation of C3b and by promoting activation of thrombin-activatable fibrinolysis inhibitor that inactivates complement anaphy‐ latoxins C5a and C3a [54]. Others have shown that thrombin-activatable fibrinolysis inhibitor activation via TM is attenuated by platelet factor 4 released from activated platelets [55].

The thrombomodulin-thrombin conversion of protein C to its activated form is facilitated by the endothelial protein C receptor (EPCR). The EPCR is another endothelial cell transmem‐ brane protein located in close proximity to the thrombomodulin molecule and with high affinity binding for protein C. EPCR as well as binding protein C also binds its activated form and via membrane lipid rafts they complex with PAR-1 [56]. The EPCR-activated protein C molecule activates PAR-1 in a different way to thrombin which allows it to signal through a potent Gi protein pathway by which anti-inflammatory pathways are stimulated within the endothelial cell [57]. C-reactive protein has also been shown, as in the case of thrombomodulin,

Thrombin activatable fibrinolytic inhibitor (TAFI) is a basic carboxypeptidase identical to the plasma procarboxypeptidases B, U and R. TAFI is activated by thrombin and plasmin although the most efficient activator is the thrombomodulin-thrombin cellular membrane bound complex. TAFI inhibits fibrinolysis by cleaving lysine residues from fibrin which restricts the binding of tissue plasminogen activator to these sites and enhancing the plasminogen con‐ version to plasmin potentiating further fibrin breakdown [58]. TAFI certainly modulates the balance between coagulation and fibrinolysis but it appears its linking role with inflammation still needs to be fully elucidated. For instance TAFI has been shown to be a positive acute phase protein in mice [59] but in the same year Boffa et al interestingly showed that IL-6 administered to cultured HepG2 cells resulted in a 60% decrease in TAFI mRNA [58]. Subsequently TAFI has been shown to be raised in experimental endotoxemia [60]*.* Its anti-inflammatory proper‐ ties are also downregulated by platelet factor 4 which is release from activated platelets and endothelial cells during activating stimuli and inflammatory insults which prevents TAFI activation by binding to the thrombin-thrombomodulin complex thereby preventing TAFI's

under experimental cell culture conditions to down regulate EPCR.

inactivation of the complement anaphylatoxins C5a and C3a [55].

**5.5. Thrombin activatable fibrinolytic inhibitor**

**5.4. Endothelial protein C receptor**

44 Acute Phase Proteins

The protein C pathway is known to be an important anticoagulant system with patients deficient in protein C being at risk of thrombosis or in its homozygous form purpura fulminans. Activated protein C inhibits factors V and VIII this being supported by the activation of protein C by thrombin bound to thrombomodulin on the endothelial cell surface. As well as acting as an anticoagulant activated protein C is also able to inhibit PAI-1. The anticoagulant and antifibrinolytic aspects of the protein C pathway have been elucidated and well described although there still appears much to learn from the interaction of protein C during the inflammatory response.

It is unclear from studies of the acute phase proteins whether protein C acts as a positive or negative acute phase protein. Most studies show it to have no change in concentration during an inflammatory response or its plasma concentration to decrease. A decrease in protein C could be attributed to consumption as well as a cytokines limiting the natural anticoagulant response. Activated protein C also confers a cytoprotective, anti-inflammatory, anti-apoptosis and endothelial barrier stabilisation effect when active [61].

Activated protein C signals its anti-inflammatory effects mainly via PAR-1 pathways whereby following Gi signalling and sphingosine-1-phosphate production there is improvement in endothelial cell barrier function [62].

Transcriptional profiling studies using cell cultures of human umbilical vein endothelial cells (HUVECs) have demonstrated that recombinant human activated protein C can regulate endothelial cell gene expression linked to inflammation and cell survival [63]. The activated protein C suppresses NFκB a cell nuclear transcription factor, and by reducing its expression and function this in turn causes inhibition of cytokine signaling [64].

#### **6.2. Cytokines**

Cyotkines are a superfamily of molecules involved in cell signalling many of which are increased greater than 1000-fold during an inflammatory insult. Cytokines such as interleukin (IL)-6, but also platelet-derived growth factor and monocyte chemoattractant protein (MCP)-1 are capable of stimulating tissue factor expression in mononuclear cells. Tissue factor being the initiator of thrombin generation and fibrin formation.

IL-6 is a multifunctional cytokine that is induced in many disease states such as sepsis, endotoxaemia and in-vitro after administration of tumor necrosis factor (TNF). Several studies have suggested IL-6 to be a potential mediator of endotoxin induced coagulation activation. This has been validated by treatment of chimpanzees with a monoclonal anti-IL6 antibody that ablated the activation of coagulation [65]. Cytokines are pivotal in providing a means of cross talk between inflammation and coagulation [66].

Inflammation and coagulation can not be considered as two separate processes because there are several interlocking points making them a unique defensive host reaction. The endotheli‐ um is one of the major links between the two since damaged endothelium during inflammation represents a surface where proteins involved in both coagulation and fibrinolysis and the development of inflammation are expressed. Cytokines down regulate the surface receptor thrombomodulin and the activation of protein C but at the same time increase the expression of tissue factor. Platelets adhere to these sites of vascular damage and when activated also release several cytokine mediators of inflammation, adhesion molecules and growth factors including IL-1β, CD40 ligand, vitronectin and RANTES [13], [67].

chromatin which are laced with histones that have antimicrobial properties. It also appears that this may be an 'overshooting' of the hosts defence mechanism with further platelet

Inflammation and Acute Phase Proteins in Haemostasis

http://dx.doi.org/10.5772/55998

47

activation, thrombosis and endothelial cell injury [71], [72], [73].

Figure 6. Inflammation and platelet activation. Platelet derived growth factor – PDGF, platetlet factor 4 – PF4.

**Figure 6.** Inflammation and platelet activation. Platelet derived growth factor – PDGF, platetlet factor 4 – PF4.

Endothelial cells in the vasculature are actively involed in haemostasis providing an anticoa‐ gulant surface preventing activation of the coagulation system. Endothelial cells produce elements with proinflammatory, procoagulant and antifibrinolytic properties as well as those with the opposite anti-inflammatory, anticoagulant and profibrinolytic properties. When endothelial cells are activated or damaged they release into the local surroundings procoagu‐ lant components such as von Willebrand factor ( a platelet binding factor and a carrier protein for factor VIII) and thromboxane A2 (a platelet activator) and plasminogen activator inhibitor (PAI-1 a potent inhibitor of tissue plasmingen activator). The opposite is true of components that provide an anticoagulant surface in the milieu of the blood vascular barrier. Thrombo‐ modulin, a thrombin binding transmembrane protein that switches thrombin from a procoa‐ gulant enzyme to one that activates protein C, a nautral anticoagulant that inhibits the activity of factors V and VIII, in internalised within the endothelial cell. Thrombomodulin is also cleaved from the endothelium by activated Neutrophils. Tissue factor is expressed on the cell surface along with adhesion molecules that mediate the interaction of neutrophils and platelets these included vascular cell adhesion molecule (VCAM-1), P and E selectin and intracellular

included vascular cell adhesion molecule (VCAM-1), P and E selectin and intracellular

directly interact with microbes and bacteria.

[73].

**6.4. Endothelium** 

**6.4. Endothelium**

**6.5. Complement** 

are inhibited by TAFI.

The concept that platelets play a key role in the host defence and inflammatory response has taken longer to realise mainly due to their role in primary haemostasis. This new role of platelets as immune effector cells is enhanced by the finding that platelets

Platelet activation along with procoagulant events and fibrin formation seem crucial for the containment and killing of bacteria. Platelets have recently been shown to induce the formation of neutrophil extracellulat traps (NETs). NETs are lattice arrangements of decondensed nuclear chromatin which are laced with histones that have antimicrobial properties. It also appears that this may be an 'overshooting' of the hosts defence mechanism with further platelet activation, thrombosis and endothelial cell injury[71],[ 72],

Endothelial cells in the vasculature are actively involed in haemostasis providing an anticoagulant surface preventing activation of the coagulation system. Endothelial cells produce elements with proinflammatory, procoagulant and antifibrinolytic properties as well as those with the opposite anti-inflammatory, anticoagulant and profibrinolytic properties. When endothelial cells are activated or damaged they release into the local surroundings procoagulant components such as von Willebrand factor ( a platelet binding factor and a carrier protein for factor VIII) and thromboxane A2 (a platelet activator) and plasminogen activator inhibitor (PAI-1 a potent inhibitor of tissue plasmingen activator). The opposite is true of components that provide an anticoagulant surface in the milieu of the blood vascular barrier. Thrombomodulin, a thrombin binding transmembrane protein that switches thrombin from a procoagulant enzyme to one that activates protein C, a nautral anticoagulant that inhibits the activity of factors V and VIII, in internalised within the endothelial cell. Thrombomodulin is also cleaved from the endothelium by activated Neutrophils. Tissue factor is expressed on the cell surface along with adhesion molecules that mediate the interaction of neutrophils and platelets these

cell adhesion molecule (ICAM-1) all of which promote the inflammatory response. So the endothelium serves as an interface for the inflammatory response leading to local activation of the coagulation system, vasodilatation and pro-inflammatory state[3], [57].

Complement is part of the innate immune system and the effector of antibody mediated immunity. The biological functions of complement include the defence against infections and the clearance of immune complexes and apoptotic cells. The complement cascade is made up of approximately 30 proteins circulating in plasma and expressed on cellular surfaces. The complement cascade is activated via three pathways: classical, lectin and alternative. The classical pathway is initiated by the binding of C1q to antigen– antibody complexes. The lectin pathway is initiated via the binding of mannose-binding lectin or ficolins to sugars found at the bacterial cell wall. Both of these pathways lead to the formation of a C3 convertase. The alternative pathway is stimulated by spontaneous hydrolysis of internal thioester bonds within C3. C3a and C5a are anaphylatoxins and inflammatory mediators which

In addition to the inflammatory cytokines like interleukin 1 and TNF, infection per se can trigger the release of neutrophil extracellular traps (NETs). Which in turn release cytokines and microparticles exacerbating the acute phase haemostatic response [68].

Cytokines also upregulate the complement system activated C5b9 complexes can assemble clot promoting membrane phospholipids, TNF-α downregulates thrombomodulin and vascular heparin sulphates promoting a procoagulant environment. The net effect of this is to further lessen the inhibitory mechanisms that control thrombin generation.

#### **6.3. Platelets**

Platelet numbers increase following surgery, trauma and sepsis following a inflammatory response. Both IL-6 and thrombopoetin stimulate the production of platelets [69]. This reactive thrombocytosis can last up to13 days post surgery and may responsible for PAF adverse events in liver reperfusion and thrombotic complications post cardiac surgery. It appears that thrombopoietin is an acute phase reactant but not uniquely responsible for the rise in the platelet count during a reactive thrombocytosis but is probably aided and abetted by IL-6 [70].

Platelets release CD40 ligand which induces tissue factor expression and increases inflamma‐ tory cytokines IL-6 and IL-8. (Esmon CT). The CD40 ligand is a transmembrane protein related to TNF-α which was originally identified on stimulated CD4<sup>+</sup> T cells. The interaction of CD40 on T and B cells is integral to the development and function of the humoral immune response. It is now known that CD40 ligand is found on many cells including macrophages, endothelial cells and platelets. Upon activation platelets express CD40L within seconds. As wtih TNF-α and IL-1 the CD40L on platelets induces endothelial cells to exude cytokines and up regulate the expression of adhesion molecules. This all increases the general recruitment of leukocytes to the site of injury. Platelets therefore directly initiate the inflammatory response at the vessel wall [14]. Figure 6

The concept that platelets play a key role in the host defence and inflammatory response has taken longer to realise mainly due to their role in primary haemostasis. This new role of platelets as immune effector cells is enhanced by the finding that platelets directly interact with microbes and bacteria.

Platelet activation along with procoagulant events and fibrin formation seem crucial for the containment and killing of bacteria. Platelets have recently been shown to induce the formation of neutrophil extracellulat traps (NETs). NETs are lattice arrangements of decondensed nuclear chromatin which are laced with histones that have antimicrobial properties. It also appears that this may be an 'overshooting' of the hosts defence mechanism with further platelet activation, thrombosis and endothelial cell injury [71], [72], [73].

Figure 6. Inflammation and platelet activation. Platelet derived growth factor – PDGF, platetlet factor 4 – PF4. **Figure 6.** Inflammation and platelet activation. Platelet derived growth factor – PDGF, platetlet factor 4 – PF4.

#### The concept that platelets play a key role in the host defence and inflammatory response has taken longer to realise mainly due to their role in primary haemostasis. This new role of platelets as immune effector cells is enhanced by the finding that platelets **6.4. Endothelium**

**6.5. Complement** 

are inhibited by TAFI.

directly interact with microbes and bacteria.

Inflammation and coagulation can not be considered as two separate processes because there are several interlocking points making them a unique defensive host reaction. The endotheli‐ um is one of the major links between the two since damaged endothelium during inflammation represents a surface where proteins involved in both coagulation and fibrinolysis and the development of inflammation are expressed. Cytokines down regulate the surface receptor thrombomodulin and the activation of protein C but at the same time increase the expression of tissue factor. Platelets adhere to these sites of vascular damage and when activated also release several cytokine mediators of inflammation, adhesion molecules and growth factors

In addition to the inflammatory cytokines like interleukin 1 and TNF, infection per se can trigger the release of neutrophil extracellular traps (NETs). Which in turn release cytokines

Cytokines also upregulate the complement system activated C5b9 complexes can assemble clot promoting membrane phospholipids, TNF-α downregulates thrombomodulin and vascular heparin sulphates promoting a procoagulant environment. The net effect of this is to

Platelet numbers increase following surgery, trauma and sepsis following a inflammatory response. Both IL-6 and thrombopoetin stimulate the production of platelets [69]. This reactive thrombocytosis can last up to13 days post surgery and may responsible for PAF adverse events in liver reperfusion and thrombotic complications post cardiac surgery. It appears that thrombopoietin is an acute phase reactant but not uniquely responsible for the rise in the platelet count during a reactive thrombocytosis but is probably aided and abetted by IL-6 [70]. Platelets release CD40 ligand which induces tissue factor expression and increases inflamma‐ tory cytokines IL-6 and IL-8. (Esmon CT). The CD40 ligand is a transmembrane protein related to TNF-α which was originally identified on stimulated CD4<sup>+</sup> T cells. The interaction of CD40 on T and B cells is integral to the development and function of the humoral immune response. It is now known that CD40 ligand is found on many cells including macrophages, endothelial cells and platelets. Upon activation platelets express CD40L within seconds. As wtih TNF-α and IL-1 the CD40L on platelets induces endothelial cells to exude cytokines and up regulate the expression of adhesion molecules. This all increases the general recruitment of leukocytes to the site of injury. Platelets therefore directly initiate the inflammatory response at the vessel

The concept that platelets play a key role in the host defence and inflammatory response has taken longer to realise mainly due to their role in primary haemostasis. This new role of platelets as immune effector cells is enhanced by the finding that platelets directly interact with

Platelet activation along with procoagulant events and fibrin formation seem crucial for the containment and killing of bacteria. Platelets have recently been shown to induce the formation of neutrophil extracellulat traps (NETs). NETs are lattice arrangements of decondensed nuclear

including IL-1β, CD40 ligand, vitronectin and RANTES [13], [67].

and microparticles exacerbating the acute phase haemostatic response [68].

further lessen the inhibitory mechanisms that control thrombin generation.

**6.3. Platelets**

46 Acute Phase Proteins

wall [14]. Figure 6

microbes and bacteria.

Platelet activation along with procoagulant events and fibrin formation seem crucial for the containment and killing of bacteria. Platelets have recently been shown to induce the formation of neutrophil extracellulat traps (NETs). NETs are lattice arrangements of decondensed nuclear chromatin which are laced with histones that have antimicrobial properties. It also appears that this may be an 'overshooting' of the hosts defence mechanism with further platelet activation, thrombosis and endothelial cell injury[71],[ 72], [73]. **6.4. Endothelium**  Endothelial cells in the vasculature are actively involed in haemostasis providing an anticoagulant surface preventing activation of the coagulation system. Endothelial cells produce elements with proinflammatory, procoagulant and antifibrinolytic properties as well as those with the opposite anti-inflammatory, anticoagulant and profibrinolytic properties. When endothelial cells are activated or damaged they release into the local surroundings procoagulant components such as von Willebrand factor ( a platelet binding factor and a carrier protein for factor VIII) and thromboxane A2 (a platelet activator) and plasminogen activator inhibitor (PAI-1 a potent inhibitor of tissue plasmingen activator). The opposite is true of components that provide an anticoagulant surface in the milieu of the blood vascular barrier. Thrombomodulin, a thrombin binding transmembrane protein that switches thrombin from a procoagulant enzyme to one that activates protein C, a nautral anticoagulant that inhibits the activity of factors V and VIII, in internalised within the endothelial cell. Thrombomodulin is also cleaved from the endothelium by activated Neutrophils. Tissue factor is expressed on the cell surface along with adhesion molecules that mediate the interaction of neutrophils and platelets these included vascular cell adhesion molecule (VCAM-1), P and E selectin and intracellular Endothelial cells in the vasculature are actively involed in haemostasis providing an anticoa‐ gulant surface preventing activation of the coagulation system. Endothelial cells produce elements with proinflammatory, procoagulant and antifibrinolytic properties as well as those with the opposite anti-inflammatory, anticoagulant and profibrinolytic properties. When endothelial cells are activated or damaged they release into the local surroundings procoagu‐ lant components such as von Willebrand factor ( a platelet binding factor and a carrier protein for factor VIII) and thromboxane A2 (a platelet activator) and plasminogen activator inhibitor (PAI-1 a potent inhibitor of tissue plasmingen activator). The opposite is true of components that provide an anticoagulant surface in the milieu of the blood vascular barrier. Thrombo‐ modulin, a thrombin binding transmembrane protein that switches thrombin from a procoa‐ gulant enzyme to one that activates protein C, a nautral anticoagulant that inhibits the activity of factors V and VIII, in internalised within the endothelial cell. Thrombomodulin is also cleaved from the endothelium by activated Neutrophils. Tissue factor is expressed on the cell surface along with adhesion molecules that mediate the interaction of neutrophils and platelets these included vascular cell adhesion molecule (VCAM-1), P and E selectin and intracellular

cell adhesion molecule (ICAM-1) all of which promote the inflammatory response. So the endothelium serves as an interface for the inflammatory response leading to local activation of the coagulation system, vasodilatation and pro-inflammatory state[3], [57].

Complement is part of the innate immune system and the effector of antibody mediated immunity. The biological functions of complement include the defence against infections and the clearance of immune complexes and apoptotic cells. The complement cascade is made up of approximately 30 proteins circulating in plasma and expressed on cellular surfaces. The complement cascade is activated via three pathways: classical, lectin and alternative. The classical pathway is initiated by the binding of C1q to antigen– antibody complexes. The lectin pathway is initiated via the binding of mannose-binding lectin or ficolins to sugars found at the bacterial cell wall. Both of these pathways lead to the formation of a C3 convertase. The alternative pathway is stimulated by spontaneous hydrolysis of internal thioester bonds within C3. C3a and C5a are anaphylatoxins and inflammatory mediators which cell adhesion molecule (ICAM-1) all of which promote the inflammatory response. So the endothelium serves as an interface for the inflammatory response leading to local activation of the coagulation system, vasodilatation and pro-inflammatory state [3], [57].

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#### **6.5. Complement**

Complement is part of the innate immune system and the effector of antibody mediated immunity. The biological functions of complement include the defence against infections and the clearance of immune complexes and apoptotic cells. The complement cascade is made up of approximately 30 proteins circulating in plasma and expressed on cellular surfaces. The complement cascade is activated via three pathways: classical, lectin and alternative. The classical pathway is initiated by the binding of C1q to antigen–antibody complexes. The lectin pathway is initiated via the binding of mannose-binding lectin or ficolins to sugars found at the bacterial cell wall. Both of these pathways lead to the formation of a C3 convertase. The alternative pathway is stimulated by spontaneous hydrolysis of internal thioester bonds within C3. C3a and C5a are anaphylatoxins and inflammatory mediators which are inhibited by TAFI.

Complement and coagulation are again two systems that cannot be viewed separately during an inflammatory response. Complement contributes significantly to the prothrombotic state during inflammation. The direct procoagulant activities of complement involve the activation ofplateletsviaC3aandtheC5b-9membraneattackcomplexandtheupregulationoftissuefactor and PAI-1 expression on various cell types by C5b. Thrombin has also recently been identified as an activator of C5 linked to the coexistence of a C5 convertase enzyme [74], [75], [76].

ItisofinteresttonotethecomplementfactorC4B-bindingproteinisapositiveacutephaseprotein that can increase greater than 400% during inflammatory states. The C4B-bp in normal circum‐ stances binds the natural anticoagulant protein S. Protein S acts as a co-factor with activated protein C in the inactivation of factors V and VIII. It was thought that an increase in C4B-bp may increase the binding of circulating protein S, approximately 60% of protein S is normally bound leaving the other 45% free protein S to aid in the V and VIII inactivation. However this in‐ crease is restricted to the C4BPα+ form, which does not bind to PS. Therefore, the blood levels of the active free form of PS remain stable even during an acute phase response. The normal binding of protein S to C4B-bp probably allows this complex to bind via protein S high affinity fornegativelychargedphospholipidsdepositingitatareaofcellulardamageandlimitingfurther apoptosis through complement activation as it will block C4b (see Protein S).

## **Author details**

Simon J. Davidson

Department of Haematology, Royal Brompton Hospital, London, UK

#### **References**

cell adhesion molecule (ICAM-1) all of which promote the inflammatory response. So the endothelium serves as an interface for the inflammatory response leading to local activation

Complement is part of the innate immune system and the effector of antibody mediated immunity. The biological functions of complement include the defence against infections and the clearance of immune complexes and apoptotic cells. The complement cascade is made up of approximately 30 proteins circulating in plasma and expressed on cellular surfaces. The complement cascade is activated via three pathways: classical, lectin and alternative. The classical pathway is initiated by the binding of C1q to antigen–antibody complexes. The lectin pathway is initiated via the binding of mannose-binding lectin or ficolins to sugars found at the bacterial cell wall. Both of these pathways lead to the formation of a C3 convertase. The alternative pathway is stimulated by spontaneous hydrolysis of internal thioester bonds within C3. C3a and C5a are anaphylatoxins and inflammatory mediators which are inhibited by TAFI.

Complement and coagulation are again two systems that cannot be viewed separately during an inflammatory response. Complement contributes significantly to the prothrombotic state during inflammation. The direct procoagulant activities of complement involve the activation ofplateletsviaC3aandtheC5b-9membraneattackcomplexandtheupregulationoftissuefactor and PAI-1 expression on various cell types by C5b. Thrombin has also recently been identified

ItisofinteresttonotethecomplementfactorC4B-bindingproteinisapositiveacutephaseprotein that can increase greater than 400% during inflammatory states. The C4B-bp in normal circum‐ stances binds the natural anticoagulant protein S. Protein S acts as a co-factor with activated protein C in the inactivation of factors V and VIII. It was thought that an increase in C4B-bp may increase the binding of circulating protein S, approximately 60% of protein S is normally bound leaving the other 45% free protein S to aid in the V and VIII inactivation. However this in‐ crease is restricted to the C4BPα+ form, which does not bind to PS. Therefore, the blood levels of the active free form of PS remain stable even during an acute phase response. The normal binding of protein S to C4B-bp probably allows this complex to bind via protein S high affinity fornegativelychargedphospholipidsdepositingitatareaofcellulardamageandlimitingfurther

as an activator of C5 linked to the coexistence of a C5 convertase enzyme [74], [75], [76].

apoptosis through complement activation as it will block C4b (see Protein S).

Department of Haematology, Royal Brompton Hospital, London, UK

of the coagulation system, vasodilatation and pro-inflammatory state [3], [57].

**6.5. Complement**

48 Acute Phase Proteins

**Author details**

Simon J. Davidson


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**Chapter 3**

**The Role of Haptoglobin and Its Genetic Polymorphism**

Acute phase response is a stereotyped innate nonspecific reaction of the body proceeding specific immune reactions. It´s a systemic homeostatic reaction of the organism to local and or systemic disturbances caused by infections, tissue injury, trauma, immunologic disorders and neoplasias (Ron D *et al* 1990, Trautwein C *et al* 1994, Gruys E *et al* 2005). Proinflammatory cytokines are released at the place of tissue injury, diffuses locally and systemically to the vascular system and activates receptors on different target cells resulting in the activation of hypothalamic-pituitary-adrenal axis (HPAA), results in the production of growth hormone secretion and induces changes in the concentration of several plasma proteins (Ron D *et al* 1990,

These acute phase proteins (APPs) can be positive (higher levels in plasma) or negative (lower levels in plasma). The alteration on mRNA in hepatocytes is due to simultaneous influence of systemic cytokines (IL1, IL6 and TNFα), glucocorticoids and catecholamines (Bowman BH

Haptoglobin together with fibrinogen, α-globulins with antiprotease-activity and lipopoly‐ saccharide binding protein belong to the group of positive APPs that increase 3-fold in

Haptoglobin (Hp) is an acute phase α2 plasma glycoprotein that is a component of innate immunity, which also may influence acquired immunity. Through both types of immunity,

and reproduction in any medium, provided the original work is properly cited.

© 2013 Bicho et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

**in Cancer: A Review**

Maria Clara Bicho, Alda Pereira da Silva,

Additional information is available at the end of the chapter

**1.1. The acute phase response (APR) and haptoglobin (Hp)**

Rui Medeiros and Manuel Bicho

Trautwein C *et al* 1994, Gruys E *et al* 2005).

1993, Ron D *et al* 1990, Trautwein C *et al* 1994).

mammals (Trautwein C *et al* 1994, Gruys E *et al* 2005).

http://dx.doi.org/10.5772/56695

**1. Introduction**

