The main characteristics of hemostatic soluble plasma proteins (HSPPs)
1. Introduction
Coagulation and inflammation are the interdependent processes attributed to the host defence response to injuries. A synchronized activity of both pathways represents an essential prerequisite for restitution of host homeostasis after ultimate disturbances of the latter. Crosstalk between coagulation and inflammation is considered to inherit from primitive coagulation systems similar to invertebrates. For instance,in Horseshoe “crabs” (
Hemostasis in humans is organized as closely interrelated enzyme cascade systems: (i) fibrin clotting system (coagulation); (ii) multilevel system for preventing uncontrolled fibrin formation (anticoagulation); and (iii) system for limiting the amount of cross-linked fibrin (fibrinolysis). Together, coagulation, anticoagulation and fibrinolysis are associated into a self-regulated and highly organized molecular machine that provides either acceleration or reduction of blood hemostasis process (Spronk et al., 2003). Accurateregulation of the coagulation rate is an effective mechanism, preventing circulatory disorders. This regulatory mechanism is known to be a part of the acute phase of the inflammatory reaction which provides rapid restoration of physiological homeostasis.
Hemostasisis closely linked to innate immunity and inflammation through particular regulatory coupling like protein C system (Fig. 1). Cooperation between hemostasis and innate immunity facilitates injury recognition, vessel wall reparation, and preventionof excessive bleeding without causing thrombosis. These processes are mediated by a balance of cellular surface components, cell-derived factors, and soluble plasma proteins (SPPs). After interruption of the vascular integrity, concentrations of some hemostatic SPPs (HSPPs) change in a manner typical of those of acute-phase proteins (APPs). In agreement with definition of APPs (Kushner & Rzewnicki, 1994; Morley & Kushner, 1982), at least several HSPPs including fibrinogen, plasminogen, and PAI-1 should also be classified as APPs, since: (i) the intensity of synthesis of these HSPPs dramatically changes (by more than 50%) during pathological processes (ii) HSPPs synthesis in hepatocytes during the acute-phase response are regulated by inflammatory-associated cytokines; (iii) due to chronic stimulation by inflammatory mediators, HSPPs may persist in circulation and participate in the formation of a “semantically paradoxical chronic acute-phase response” (Gabay & Kushner, 2001). The above-indicated similarities between inflammation and hemostasis determine their unidirectional changes. These changes involve pro-coagulant activities of inflammatory processes, as well as the pro-inflammatory efficacy of the hemostatic molecular machine.
The capacity of inflammatory mediators to regulate hemostasis, as well as some aspects of the coagulation ability to affect inflammatory events have been extensively reviewed (Butenas et al., 2009; Danese et al., 2010; Esmon, 2005; Jennewein et al., 2011; Levi et al., 2004; Medcalf, 2007). Reciprocal regulation of gene expression is the most important mechanism, by which inflammatory and hemostatic pathways interact with each other. The role of cell surface receptors in providing APP-associated signalling has been recently elucidated (Busso et al., 2008; Guitton et al., 2011). The role of the increased HSPPs in the regulation of hemostasis and inflammation pathways under pathophysiological conditions, however, remains less clear. The molecular mechanisms, responsible for the influence of inflammation upon hemostasis and
2. Cooperation of inflammatory and hemostatic pathways during acute phase response
In normal conditions, HSPPs are presented by a soluble fibrous protein, fibrinogen, abundant serine protease zymogens (inactive enzymatic precursors), and minute amounts of active proteases, as well as by non-enzymatic cofactors and protease inhibitors (Table 1).
Precursor conversion | HSPP identification |
The basic function in hemostasis |
Proteolytic activation | Expression during the acute-phase response | ||
Clotting factors | ||||||
Fibrinogen (Fg) → fibrin (Fn) |
a fibrous (structural) protein | substrate for polymerization to fibrin that is important in tissue repair | by thrombin |
increase, 1,5-4,0-fold |
||
Prothrombin (II) → thrombin (IIa) |
trypsin-like serine protease (endopeptidase) |
the conversion of Fg to Fn leading to the formation of a fibrin clot | by prothrombinase | no change or weak increase |
||
Factor V (V) → activated factor V (Va) |
ceruloplasmin-like binding protein | as a cofactor for Xa participates inthrombin activation, not enzymatically active | by thrombin |
ND | ||
Factor VII (VII) → activated factor VIIa (VIIa) |
serine protease (endopeptidase) | the catalytic component of the extrinsic tenase, activatesIX to IXa and X to Xa | by thrombin, IXa, Xa, XIIa |
ND | ||
Factor VIII (VIII) → activated factor VIIIa (VIIIa) |
ceruloplasmin-like binding protein | cofactor for IXa in conversion of X to Xa, receptor for IXa and X, not enzymatically active | bythrombin |
increase | ||
Factor IX (IX) → activated factor IXa (IXa) |
serine protease (endopeptidase) | the enzyme component of the intrinsic tenase, activates X to Xa | by XIa or TF-VIIa/PL-Ca2+ |
ND | ||
Factor X (X) → activated factor Xa (Xa) |
serine protease (endopeptidase) | the enzyme component of the prothrombinase is responsible for rapid thrombin activation | by IXa-VIIIa/PL-Ca2+,
TF-VIIa/PL-Ca2+ |
ND | ||
Factor XI (X) → activated factor XIa(XIa) | serine protease (endopeptidase) | the conversion of IX to IXa within the intrinsic pathway | by surface bound α-XIIa | ND | ||
Factor XII (XII) → activated factor XII (α-XIIa) |
serine protease (endopeptidase) | initiation of the intrinsic coagulation pathway via conversion XI to XIa | by kallikrein |
decrease | ||
Factor α-XIIa (α-XIIa)→factor β-XIIa (β-XIIa) |
serine protease (endopeptidase) | solution phase activation of kallikrein, factor VII and thecomplement cascade | by kallikrein |
decrease | ||
Factor XIII (XIII) → activated factor XIIIa (XIIIa) |
transglutami-nase (transpeptidase) | stabilization of the fibrin clot via cross linking the α and γ-chains of Fn, α2-PI, V, vWF | by thrombin | ND | ||
Anticoagulants | ||||||
Tissue factor pathway inhibitor (TFPI) | Kunitz-type protease inhibitor | reverse inhibition of Xa and IIa, then TF-VIIa independently from Ca2+ | - |
decrease | ||
Antithrombin (AT) |
serpin | Inhibition of VIIa, IXa, Xa and XIa, kallikrein, plasmin, IIa | - | decrease | ||
Protein C (PC)→ Activated protein C (APC) | trypsin-like serine protease (endopeptidase) | inactivation of Va and VIIIa, that inhibits the prothrombinase and tenase and, finally, IIa | by α-throm-bin/throm-bomodulin,by Xa or IIawithout Ca2+ | no change or decrease |
||
Protein S(PS) | binding protein | as cofactor for APC | - | increase | ||
Fibrinolytic proteins | ||||||
Tissue-type plasminogen activator,single chain form (sc-tPA) → active two-chain form (tc-tPA) | serine protease (endopeptidase) | the main endothelium- derived activator of the fibrinolytic system, converts Pg to Pm |
by plasmin |
decrease or weak increase |
||
Glu-plasminogen(Glu-Pg) →plasmin (Pm) | serine protease (endopeptidase) | responsible for the fibrin clot digestion | by t-PA, u-PA, elastase, XIIa | increase, 2-3- fold |
||
Urokinase-type plasminogen activator, single chain(sc-uPA) | serine protease (endopeptidase) | activator of the Pg conversion to Pm | - | increase | ||
Proteinase inhibitors | ||||||
α1-Antitrypsin or alpha 1-proteinase inhibitor (αl -PI) |
serpin | protects tissues from proteolytic enzymes, inhibits IIa and APC | - |
increase | ||
α2-macroglobulin (α2-M) |
broad-range protease inhibitor | inhibits IIa, APC Pm, kallikrein, a remover of plasma enzymes | - |
increase, about 100-fold |
||
α2-antiplasmin (α2 –plasmin inhibitor) (α2 -PI) |
serpin | inhibitor of Pm, forms covalent complexesinterfered with the binding of Pg(Pm) to Fn | - | ND | ||
Thrombin activablefibrinolytic inhibitor (TAFI) → activated form (TAFIa) | carboxypepti-dase | inhibitor of fibrinolysis, removes Pg-binding sites on Fn | by thrombin,plasmin, trypsin |
ND | ||
Plasminogen activator inhibitor of type 1 (PAI-1) |
serpin | the major inhibitor of tPA that regulates the fibrinolysis by attenuation of Pmproduction | - |
increase | ||
Plasminogen activator inhibitor of type2 (PAI-2) | serpin | inhibitor of urokinase as well as tPA | - | ND | ||
Plasminogen activator inhibitor of type3 (PAI-3 or PCI) | serpin | the major inhibitor of APC as well as tPA and urokinase | - | ND |
Physiological anticoagulants are also available to suppress appropriate clotting factors. Some of the clotting factors (like thrombin or factor V) can promote both coagulation and anticoagulation; thus, these factors are called Janus-faced proteins.
A hemostatic response to the activating signal is manifested by a series of transformations of proenzymes to activated enzymes in a cascade-like manner.The formation of thrombin at the final stage of the coagulation cascade is aimed at conversion of soluble fibrinogen into insoluble fibrin, the non-cellular matrix of blood thrombus. The thrombus formation is considerably accelerated due to accumulation of tissue factor (TF) at the sites of vascular endothelial damage. Tissue factor, a membrane-bound glycoprotein, is considered the common physiologic trigger of both hemostasis and inflammation pathways.Under normal conditions, none cells, which contact with the bloodstream, express TF. An injury, as the initial triggering signal, starts up the TF expression and the externalization at the surface ofinflammatory cells (primarily, monocytes) and vascular wall cells (endothelial or smooth muscle cells). Immediately upon exposure to the bloodstream, TF contacts with activated coagulation factor VIIa (VIIa), whose trace amounts (about 1 % of total inactive enzyme precursor, coagulation factor VII) are conventionally present in circulation. The formation of macromolecular complex TF-VIIa is a crucial event that initiates the first stage of the coagulation process, initiation phase. Alongside with that TF initiates local inflammatory reaction.
2.1 Initiation phase of the coagulation process
Tissue factor-VIIa complexes, newly appeared on the boundary between blood flow and the vessel wall, begin to bind plasma-derived coagulation factor VII to produce additionally factor VIIa. Thus, TF acts as a cofactor in the factor VII/VIIa autoactivation process (Fig. 2). Membrane-bound TF/VIIa complexes also interact with small amounts of coagulation factor X (X) and coagulation factor IX (IX). Activated factors X (Xa) and IX (IXa) start up prothrombin (coagulation factor II) conversion to thrombin (IIa). This first cycle of restricted thrombin production is limited by two plasma-derived inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT). The former one neutralizes factor Xa when forms a quaternary structure with TF-VIIa-Xa. As well, AT upon binding to heparan sulphate/heparin, rapidly inactivates free factors Xa, IXa, and thrombin, initially accumulated at the site of vascular injury. So, TF-VIIa itself is incapable to generate substantial amounts of thrombin during the initiation phase. However, the TF-dependent cycle of thrombin production can overcome inhibition by TFPI and AT, when TF is maintained at a sufficiently high level (Tanaka et al., 2009). Blood-borne forms of TF (soluble sTF or TF-positive microparticles) shed from disrupted cell membranes of different origin presumably can be an additional driver of the increased TF-initiated thrombin production (Sommeijer et al., 2006). Apparently, cell-exposed and blood-borne TF can promote transduction of inflammatory signals
2.2. Propagation phase of the coagulation process
Trace amounts of thrombin promote formation of a IXa-VIIIa-Ca2+-phospholipid assembly (tenase complex) ora Xa-Va-Ca2+-phospholipid assembly (prothrombinase complex)
inflammation and coagulation. Furthermore, the formation of thrombi in the microvasculature provides a mechanical barrier that blocks spreading of inflammatory/coagulation mediators into the circulation. Such limited clotting restricts thrombus propagation and prevents from acute local inflammation turning into systemic complications. An anti-clotting molecular process induced by several physiological anticoagulants and fibrinolytic agents is designated for regulation of an adequate clot size and formation of an effective thrombus. Two hemostatic pathways, anticoagulation and fibrinolysis, both are responsible for coagulation termination (Fig. 2).
2.3 Termination phaseof the coagulation process
After bleeding is arrested, and the injured vessel is repaired, coagulation attenuation begins to dominate over its propagation due to accumulation of inhibitors of blood clotting. Natural clotting inhibitors are orchestrated through successive induction of three major anticoagulant-dependent pathways: tissue factor pathway inhibitor-, heparin/antithrombin- and the protein C-dependent pathways. A tissue factor pathway inhibitor (TFPI), as was mentioned above, is a most effective inhibitor of coagulation at the initiation phase. This inhibitor specifically blocks the TF-VIIa-Xa complex after trace factor Xa has been formed (Spronk et al., 2003). Antithrombin (AT) is a direct protease inhibitor, which attenuates accumulation of coagulation factors IXa, Xa, and IIa during the propagation phase. Heparin-like glycosaminoglycans accelerate the rate of inactivation of these clotting factors by AT. The protein C system provides multi-directional attenuation of thrombin procoagulant activity and this terminates coagulation. Thrombomodulin (TM), a endothelial cell membrane-associated protein, is capable of to tackling excessive thrombin, thus changing its specific enzyme activity. Within the thrombin-thrombomodulin complex, thrombin looses its affinity to fibrinogen or cellular PARs. Instead this, it possesses an ability to convert precursor protein C (PC) into an anticoagulant enzyme, activated protein C (APC). Activated protein C interrupts thrombin propagation
In a complementary mode with respect to anticoagulation, the surplus clots are dissolved by proteases of the fibrinolytic system. Activation of intravascular fibrinolysis is controlled through enhancing synthesis and secretion of tissue plasminogen activator (tPA) by endothelial cells during fibrin clotting. Tissue plasminogen activator is released into the clot and binds in the clot volume with fibrin(ogen) and plasminogen (Pg). The formation of ternary Fn-tPA-Pg complex extremely effectively accelerates Pg converting into the serine protease plasmin (Pm). Plasmin cleaves fibrin into soluble fragments, the so called fibrin degradation products (FDPs). The rate and extent of local delivery of tPA during the clot formation is important for enhancing the process of fibrinolysis (Schrauwen et al., 1994). To avoid excessive clot digestion, which can affect bleeding, the activity of fibrinolytic system is down-regulated by numerous plasma- and cell-derived inhibitors (Meltzer et al., 2010). These are (i) plasminogen activator inhibitor of type 1 (PAI-1) that highly specifically inactivates tPA, (ii) α2-antiplasmin, primary Pm inhibitor that prevents Pm-dependent non-specific proteolysis due to effective neutralization of Pm, and (iii) thrombin activablefibrinolytic inhibitor (TAFI) that down-regulates the cofactor activity of fibrin during activation of plasminogen and, thereby, suppresses fibrinolysis.
The activities of both coagulation and fibrinolytic cascades are normally latent but have the potential to be accelerated in an extremely acute manner during inflammation. The coagulation and fibrinolysis pathways have to follow each other, retaining a delicate physiological balance preventing thrombosis and bleeding. Activation of the coagulation cascade leads to increases in the plasma levels of coagulation factors VIIa, Xa, and thrombin, which are pro-inflammatory factors contributing to the acute-phase response (Fig. 2). In addition, fibrin deposition and fibrin degradation products, FDPs, enhance inflammation.Coagulation factors elicit inflammation
A failure of the control of these processes causes incorrect inflammation termination or even its propagation. Such an inconsistency leads to deregulation of hemostasis, which, in turn, might force the further leap of inflammatory responses. Under pathological conditions, cytokines are released by immune regulatory cells in sites of the local inflammatory response. This process may be acute but limited in time (reverting to the normal homeostatic state) or persistent (resulting in chronic activation of coagulation and fibrinolysis). Initially acting within the frame of the adaptive defence system, inflammation and hemostasis might develop from a local response to a systemic host reaction. Escalation of inflammation can induce endothelial dysfunction subsequently activating the coagulation cascade, and
Under these conditions mutual activation of coagulation and fibrinolysis might follow to potentially exhausting consumptive coagulopathy and disseminated intravascular coagulation. A detrimental inflammatory response resulting from coupling of procoagulant and pro-inflammatory stimuli might cause thrombophilia, and, furthermore, provoke the thrombotic events. In such a way, inflammation/coagulation interaction drastically increases a risk of thrombus formation implicated in the pathogenesis of several diseases in humans. On the one hand, these are thrombophilias, atherosclerosis, and other cardio-vascular pathologies, as well as intercurrent illnesses (like trauma and cancer) or surgery. On the other hand, these are acute/chronic inflammatory diseases, including sepsis, inflammatory bowel disease, and lung and heart inflammation (Fig. 3).
Abbreviations: IIa, VIIa, IXa, Xa, and XIa indicate activated coagulation factors; Va and VIIIa – non enzymatic cofactors; Fn, fibrin; TF, tissue factor; sTF, blood-borne (soluble) forms of tissue factor; PARs, protease-activated receptors; IL, interleukin; CRP, C-reactive protein; TNF−α, tumour necrosis factor alpha; tPA, tissue activator of plasminogen; APC, activated protein C; AT, antithrombin; PAI-1, plasminogen activator inhibitor of type 1; TAFI, thrombin activable fibrinolytic inhibitor.
3. Pro-inflammatory environment sustains coagulation
Recently, a clear association of high APP levels with a human procoagulant phenotype and impaired fibrinolysis were found in some studies. Indeed, a certain relationship is believed between the plasma levels of C-reactive protein (CRP) and some HSPPs. C-reactive protein is at present used as a sensitive biomarker of acute and chronic inflammation. This is the only APP that correctly displays the severity of vascular pathologies: from an endothelium-derived focal inflammatory response to a hard coronary lesion (Calabrò et al., 2009, Willerson & Ridker, 2004).CRP levels are now detected using a high-sensitivity assay (hsCRP); these indices are found to be an accurate predictor of cardiovascular disease (CVD) (de Ferranti & Rifai, 2007).
The procoagulant function of the C-reactive protein is still debated (MacCallum, 2005), but there is some evidence proving that CRP is associated with the metabolism of HSPP. In
The studies that elucidate the inhibitory role of cytokines in fibrinolysis are not limited to that of CRP.A multifunctional cytokine, IL-1, was shown to stimulate up-regulation of specific mRNA expression of urokinase-type plasminogen activator(u-PA) (Wojta et al., 1994). IL-1 also increased accumulation of PAI-1 in cardiac microvascular endothelial cells (Okada et al., 1998) and also controlled expression of PAI-1 and u-PA in human astrocytes (Kasza et al., 2002). Production of PAI-1 protein in human adult cardiac myocytes was found to be increased up to two times by interleukin-1α and tumour necrosis factor-α (TNF-α) and up to five times by transforming growth factor-ß (TGF- ß ) and oncostatin M. However, t-PA production in human cardiac myocytesdid not change after cytokine treatment (Macfelda et al., 2002). By contrast, IL-1 and tissue necrosis factor alpha inhibited t-PA in HUVEC (Bevilacqua et al., 1986).
During severe inflammation, the function offibrinolysis can be impaired. The same is true with respect to anticoagulant pathways. It was recently documented that an increase in serum CRP level in dogs was accompanied by lowering of the AT concentration (Cheng et al., 2009). Under inflammation conditions, the AT plasma level can decreased due to impaired synthesis (like other negative APPs) or due to protein degradation by elastase produced by activated neutrophils (Viles-Gonzalez et al., 2005).In addition, AT might be consumed proportionally to inhibition of the target proteases or removed from circulation after binding to fluid-phase complement attack complexes within the complement cascade (Esmon, 2005). Another natural anticoagulant, TFPI, seems to be incapable of regulating an enhanced thrombin amount during severe inflammation, since a low endogenous concentration of the anticoagulant does not increase (Bastarache et al., 2008). It would be noted that TFPI concentrations do not follow the development of disseminated intravascular coagulation and cannot prevent hypercoagulation (Wiinberg et al., 2008).TFPI is expressed primarily in the microvessels; thus, it might only nominally participate in hemostasis balancing in the larger vessels. Apparently, this pathway only slightly contributes to the coagulation/inflammation cross-over (DelGiudice & White, 2009). The PC anticoagulant system is more extensively present in the vascular network (Viles-Gonzalez et al., 2005). This system plays a pivotal role in hemostasis, shutting down coagulation and promoting fibrinolysis. These events might fail because of the presence of some vulnerable components. Down-regulation of membrane TM and EPCR by endotoxin, IL-1b, and TNF-α has been noted elsewhere (Esmon, 2005). The disappearance of TM from the endothelial cell surface impairs the process of activation of protein C. Not only the amount of APC but also its anticoagulant activity might be decreased under pathological conditions. Protein S, when forming an inactive complex with complement protein C4b (C4BP), thereby looses its ability to promote APC(Dahlback, 1991). Additionally, soluble forms of TM and EPCR can appear during inflammation in the blood flow. They may bind APC without potentiation of its activity or, moreover, even might inhibit APC anticoagulant function. sEPCRs have been found to block binding of protein C and APC to phospholipids and to alter the active site of APC (Liaw et al., 2000).
Tissue factor, in addition to its procoagulant function, has been recently identified as a key secondary inflammatory mediator that markedly accelerates the feedback intensification of coagulation and inflammation pathways. Its concentration in circulation dramatically increases when the endothelium is disrupted and the blood begins to contact with extra vascular cells. In addition, inflammatory mediators many times increase the tissue factor protein amount and activity through stimulation of expression of this protein and through increasing the number of TF-positive microparticles as a consequence of paracrine and autocrine activity of the inflammatory cells (Esmon, 2005). It should be noted that TNF-α and IL-1ß are produced by lymphocytes and macrophages during vascular inflammation, and these events can also enhance the expression of the TF. The TF expression can be stimulated by several inflammatory mediators, namely TNF-α, IL-1, IL-6, activated complement, and immune complexes (DelGiudice & White, 2009).Activated T cells increase both TF expression and activity
Coagulation factor Xa was found to increase induction of endothelial TF and E-selectin by all the pro-inflammatory cytokines (e.g. TNF, IL-1ß, and CD40L). TF, in turn, initiates a new wave of factor Xa production after the formation of the TF-VIIa complex and activation of zymogens of factors IX and X. Binding of TF-VIIa to PAR-2 also results in up-regulation of the inflammatory responses in macrophages and neutrophils (Cunningham et al., 1999). A synergistic pattern of activity of factor Xa and inflammatory cytokines, resulting in both re-activation of coagulation cascade and augmentation of inflammatory mediators, is a good illustration of the apparent positive feedback mechanism, by which enhanced coagulation maintains pro-inflammatory environment and
4. Hypercoagulation and impaired fibrinolysis perpetuate inflammation
The above-mentioned facts proved the capacity of the inflammatory factors to regulate coagulation and fibrinolysis. A converse molecular mechanism, by which hemostasis stimulates inflammation is at present less obvious but undergoes increasing investigations. Fibrinogen, the precursor of fibrin, is considered a rapid and sensitive marker of both coagulation and the acute-phase response, while its synthesis is enhanced during early inflammatory reactions. Fibrinogen contributes to coagulation being a terminal substrate in plasma clotting, which is cleaved specifically by thrombin. By splitting fibrinopeptide A and fibrinopeptide B from fibrinogen thrombin forms fibrin-monomers are spontaneously polymerized producing fibrin.Fibrin, in turn, provides plasma clotting, platelet aggregation and wound healing or thrombus formation. In addition, fibrin(ogen) participates in activation of vascular cells and regulation of the inflammatory response. Pro-inflammatory effects of fibrin(ogen) manifest itself after the abnormal fibrin deposition; this affects the vascular bed and enhances primarily local and, then, systemic inflammation through expression of the pro-inflammatory mediators. In fact, fibrin(ogen) increases the mRNA levels and induces synthesis of inflammatory cytokines IL-6 and TNF-alpha in human peripheral blood mononuclear cells (PBMCs) (Jensen et al.,2007), of IL-8 in HUVEC (Qi et al., 1997), and of transcription factor NF-kB in mononuclear phagocytes (Sitrin et al., 1998).Fg can manifest a pro-inflammatory action independently of its clotting function due to the existence of a high-affinity integrin binding site or multiple low-affinity binding sites, which interact with inflammatory competent cells. In particular, induction of cytokines IL-1β, IL-6, TNF-α has been found to be associated with fibrin binding to integrin receptors Mac-1 (CD11b/CD18) in monocytes (Trezzini et al., 1991). Cytokine secretion is suggested to be directly triggered by the process of Fg polymerization to Fn. The activity of thrombin that increases in Fg-deficient mice after LPS administration does not correlate with the levels of inflammatory mediators produced by bone marrow-derived macrophages and duration of their action. Both thrombin and Fg acting separately or in combination exert no effect on the cytokine production. It was concluded that up-regulation of secretion results in conformation changes of the Fg molecular structure during its conversion into Fn (Cruz-Topete et al., 2006). Some recently obtained data supported this conclusion. Molecular determinants of fibrin(ogen)-mediated pro-inflammatory activity were found to be localized in a γ-chain. These determinants can enhance the inflammatory cell recruitment and activation
The involvement of fibrinogen in coagulation, as well as that of fibrin in the fibrinolytic process, is accompanied by generation of the various fibrin(ogen) degradation products, FDPs. These small and large fragments can exert an independent regulatory effect on the inflammatory process. In particular, fibrinopeptides A and B, the products of Fg conversion into Fn, are suggested to show a pro-inflammatory action on leucocytes functioning as chemoattractants. In contrast, the peptide Bβ15-42 which is generated by plasmin cleavage of fibrin, can mediate powerful anti-inflammatory effects. FDPs, which are formed after Fg(Fn) digestion by plasmin, also seem to modulate inflammation (Jennewein et al., 2011). Fg, Fn and FDPs were shown to induce intensification of CRP production in vascular smooth muscle cells.Herein, FDPs have the most prominent pro-inflammatory potency, as compared to that of fibrin(ogen) (Guo et al., 2009).
It is interesting that plasmin(ogen) also generates degradation products during activation of fibrinolysis. These are either the first three, or the first four kringle domains (K1-3, K1-4) or only kringle domain 5 (K5). Angiostatin, a proteolytic fragment that contains K1-4, acts as a powerful anti-inflammatory modulator. In particular, angiostatin demonstrated a lowering adhesiveness of leukocytesto extracellular matrix proteins and the endothelium. Interaction of the angiostatin kringle domain K4 with integrin receptor Mac-1 down-regulates transcriptional factor NF-kB, whereby attenuates NF-kB-related expression of neutrophil-derived tissue factor (Chavakis et al., 2005).The kringle domain K5 has been found to restrict the neutrophil chemotactic activity (Perri et al., 2007). Obviously, impaired fibrinolysis looses this anti-inflammatory action.
The formation of fibrin deposition is a direct consequence of increased thrombin production. A pro-inflammatory action of thrombin is realized by two interdependent ways: (i) by direct promotion of hypercoagulation accompanied by the pro-inflammatory effects described above; (ii) by stimulation of vascular and blood-borne cells and their further involvement in the inflammatory response. Being a powerful signal molecule, thrombin interacts specifically with PAR-1, PAR-2, or PAR-3 and activates the signaling pathways in endothelial cells, platelets, mononuclear cells, and fibroblasts. Thrombin-induced intracellular pathways up-regulate the expression of several cytokines and growth factors, as well as the secretion of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (Levi & Poll, 2008). Thrombin is a key protease-agonist, which controls the platelet involvement in the formation of thrombi by stimulation of platelet aggregation, granule secretion, and additional recruitment in the inflammatory process. In an
5. Phenotypic variability of hemostasis during acute and chronic inflammation
Abnormal exposure of the procoagulant and pro-inflammatory agents contributes to sustaining of both local and systemic procoagulant/inflammatory potentials. Prolonged activation of inflammatory cells promotes the production of large amounts of inflammatory mediators by downstream-cells affecting not only
5.1. Transcriptional regulation of HSPP production during inflammation
Unlike CRP (type-1 acute phase protein) up-regulated by synergistic action of IL-6 and IL-1beta, most hemostatic proteases (type-2 acute phase proteins) require IL-6 alone for maximal induction of their synthesis (Trautwein et al., 1994). IL-6 is a key effector that effectively promotes the coagulation pathway, not only by up-regulation of expression ofsome procoagulant factors (such as TF, fibrinogen, and factor VIII) but also by down-regulation of synthesis of some anticoagulants (such as antithrombin and protein S) (Hou et al., 2008). Cytokine IL-6 is suggested to act as a common inductor for several vascular acute-phase proteins (CRP, α- and β- chains of Fg,Pg, α2-macroglobulin, and PAI-1). Under transcriptional control by the cytokine IL-6, their circulating levels increase
Not only IL-6, but a number of cytokines, alone or in a combination, may also influence HSPP synthesis. TNF-α and other inflammatory agents are known to markedly suppress fibrinolysis, mainly
5.2. Alterations of the HSPP plasma levels caused by post-transcriptionaland post-translational events
Marked alterations in the plasma HSPP levelsfollowing an acute-phase stimulus are determined not only by transcriptional regulation but also by post-transcriptional and post-translational mechanisms. The latter were found for such classic APPs, as serum amyloid A (SAA), complement factors B, and C3 (Jiang et al., 1995). It was reported that the α2-macroglobulin gene transcription ratemight increase (up to nine-fold)during the acute inflammatory response, while its protein plasma concentrations can rise much more significantly (to 100-fold) (Hattory et al., 1990). The other study demonstrated that aortic endothelial cells decreased production and secretion of t-PA after incubation with CRP without any alteration of the tPA mRNA level, thus underlying a suggestion that CRP-mediated tPA inhibition is a posttranscriptional event (Singh et al., 2005).In contrast, post-transcriptional regulation should not play a substantial role in monocyte-derived production of fibrinogen α-chain or α-1 protease inhibitor (α -PI) proteins (Jiang et al., 1995).
Despite the HSPPs are largely regulated by transcriptional control, they still strongly require the post-transcriptional regulation (including co-translational and post-translational modification) to confer their optimal functionality. They should form the disulfide bonds to get native conformation as well as should be carboxylated, hydroxylated, phosphorylated, sulfated or glycosylated to achieve a specific function. In particular, the main coagulation factors II, VII, IX, X and protein C (all are the vitamin K-dependent proteins) are processed through further post-translational modification to become biologically active. Prior to secretion into the blood they should be modified by a vitamin K-dependent gamma-glutamyl carboxylase, getting in such a way, an amount of negatively charged γ-carboxyglutamic acid (Gla) residues. Gla-residues have a chelating activity oriented to Ca2+-cations (Table 2). They are orchestrated in the "Gla domain" to participate in the Ca2+-dependent binding of parent protein to cell membrane or macromolecular complexes.
Similarly to most secretory proteins, HSPPs are enriched by disulfide bonds (Table 2). Before secretion, they undergo oxidative maturation that leads to binding of the appropriate pairs of cystein residues. The disulfide bonds are formed in the rough endoplasmic reticulum, since this process requires an oxidative environment. These functional groups are well-known as playing an important role in protein folding (by stabilizing the tertiary and quaternary structure). Furthermore, disulfide bonds can be responsible for intra- and intermolecular reorganization or even proteins aggregation. In the few last years, a number of studies on functional disulfides have highlighted their two important functions, namely catalytic and allosteric (Chen & Hogg, 2006; Manukyan et al., 2008; Popescu et al., 2010).
In addition to carboxylation and formation of disulfide bonds, a series of post-translational modifications occurs to attach N- or O-linked glycans to secreted proteins (Table 2). Several N-linked glycosylation sites are well-known to be an attributive feature of HSPPs, which are glycoproteins. N-glycosylation has been recently discovered to be a crucial event in the regulation of the glycoprotein structure and function.
There are available data, suggesting that glycosylation is higher in proteins synthesized during the acute-phase responses.
Protein | Percent carbo-hydrate (w/w) |
number of modified residue | |||
Glycosylation |
Disulfide bond | Hydroxylation, phosphorylationor sulfation |
Carboxylation | ||
fII | 3 N-linked | 10 (+2 predicted) |
none | 10 Gla residues | |
fV | ~25% | 5 N-linked (+21 predicted) |
6 (+1 predicted) |
1 phosphothreonine 1 phosphoserine 7 sulfotyrosine |
none |
fVII | 13% | 2 O-linked 2 N-linked |
12 | one β-hydroxyaspartate | 10 Gla residues |
fVIII | 1 N-linked (+21predicted) |
7 (+1 predicted) |
6 sulfotyrosine | none | |
fIX | 17% | 4 O-linked 2 N-linked |
11 | one β-hydroxyaspartate 2 phosphoserine 1 sulfotyrosine |
12 Gla residues |
fX | 15 % | 2 O-linked 2 N-linked |
12 | one β-hydroxyaspartate | 11 Gla residues |
fXI | 5% | 5 N-linked | 18 | 2 phosphorilated | none |
fXII | 17% | 7 O-linked 2 N-linked |
20 | none | none |
Glu-Pg | ~2% | 2 O-linked 1 N-linked |
24 | 1 phosphoserine | none |
tPA | 1 O-linked 3 N-linked |
17 | none | none | |
TFPI | 2 N-linked 3 O-linked |
9 | none | none | |
AT | 9% | 4 N-linked | 3 | 1 phosphoserine | none |
PC | 23 % | 4 N-linked | 12 | one β-hydroxyaspartate | 9 Gla residues |
α2 -PI | 14% | 4 N-linked | 1 | 1 sulfotyrosine | none |
PAI-1 | 2 N-linked (+1 predicted) |
none | none | none | |
TAFI | 5 N-linked | 3 | none | none |
in different glycoforms of APP in circulation most likely result from alterations in oligosaccharide branching, increased sialylation, and decreased galactosylation (Gabay & Kushner, 1999). The replacement of individual N-glycans by other ones exerts very specific and diverse effects on the protein structure and/or function. Human hemostatic proteins, coagulation factor IX and protein C, which both are the vitamin K-dependent proteins synthesized and secreted by hepatocytes, vary extensively in their glycosylation levels. Coagulant factor IX has two N-glycosylation sites and is characterised by significantly more heterogeneity of N-glycan structures than anticoagulant protein C.PC has four N-glycosylation sites, Q97, Q248, Q313, and Q329; the latter has an unusual consensus sequence, Asn-X-Cys. Desialylation of PC and factor IX was shown to result in a two-threefold increase in the anticoagulant activity of protein C and in a loss of the coagulant activity of factor IX (Gil et al., 2009; Grinnell et al., 1991). Alterations in the glycosylation pattern have been suggested to be specific in certain diseases (An et al., 2009; Ohtsubo&Marth,2006). Nevertheless, it remains unclear whether inflammation signals control processing of coagulation proenzymes or not. Well-documentedinflammation impact on glycosylation of classic APPs allows researchers to suggest such a control. The most important mechanism, through which the inflammatory environment is able to alter the enzyme activity and/or substrate specificity in local environments or in a systemic disease are modifications of the glycan moiety or heterogeneity.Experiments with glycoprotein deglycosylation showed that the removal of distinct glycan or total deglycosylation usually leads to remarkable reduction of the protein binding and enzymatic activity. However, at least two examples have been recently elucidated (Skropeta, 2009), wherethe enzyme activity increased upon deglycosylation of HSPPs. In particular, removal of the two of four existing glycosylation sites in the human protein C molecule resulted in a two–threefold increase in the anticoagulant activity of APC due to an enhanced affinity of thrombin, the natural activator of PC. Interestingly, two fibrinolytic proteins, tPA and its specific substrate, Pg, interact more or less effectively depending on the peculiarities of attached glycans. Indeed, tPA can exist as two glycoforms, type I with three N-glycosylated sites and type II with two N-glycans. Plasminogen also exists in two glycoforms; type 1 has both N- and O-linked glycans, while type 2 has only an O-linked glycan. The combination of type II tPA with type 2 plasminogen induced a twofold more intense conversion of plasminogen to plasmin compared to interaction of more heavily glycosylated type I tPA with type 1 Pg. Changes in the microheterogenity and unique structure of glycans are now known to be ensued from folding of the glycoprotein early form during post-translation processing in the secretory pathway. Glycosylation is an enzymatic process regulated by distinct glycosyltransferases in the endoplasmic reticulum, which modulate unfolded glycoproteins prior to trafficking to the Golgi apparatus. Unexpectedly, one experiment demonstrated that an alterated O-glycosylation pathway affects the N-glycosylated coagulation proteins in NAcT-1-deficient mice. In particular, the deficiency of a polypeptide GalNAc transferase (ppGalNAcT) contributed to shifting of O-glycan repertoire by other glycosyltransferases, as well as affected blood coagulation resulting in prolongation of the activated partial thromboplastin time, APTT, and bleeding time. These abnormalities were accompanied by mild or moderate decreases in the circulating levels of factors V, VII, VIII, IX, X, and XII, whereas the level of von Willebrand factor tendedto raise(Tenno et al., 2007). The reported results might be interpreted as a consequence of pleuotropic effects of O-glycosylation that contribute to regulation of HSPP expression and/or turnover (primarily secretion and clearance). Additionally, alterations in the degree of branching and of levels of sialylation, fucosylation, and mannosylation can dramatically change the glycoprotein turnover. Although our information about glycan-mediated pathophysiological mechanisms is still very limited, their impact on the enzyme secretion, stability, and activity and on molecular trafficking and clearance allows researchers to suggest that glycosylation plays a special role in the phenotypical variability of hemostatic and inflammatory proteins in circulation. Apparently, the acute-phase response generates a characteristic protein profile by alteration of synthesis, secretion, and clearance of protein reflected in their final concentrations. The actual level of plasma proteins under pathological conditions is also determined by changes in their stability, post-secretion proteolysis, functional activity, and accessibility for interaction.
Marked alterations in the plasma protein levels are probably paralleled by modifications of their disulphide bonds. The role of disulfides in regulation of the functional activity of HSPPs was subjected to intense research. A direct influence of inflammatory conditions on the structure or functions of plasma proteins is an intriguing question. Recently, we demonstrated that the concentration of DTNBA-active polypeptides produced in the course of the reaction of plasma and serum proteins with 5,5'-dithiobis(2-nitrobenzoic acid), was noticeable increased in patients with stable
Although HSPPs are synthesised and secreted principally in hepatocytes (Ruminy et al., 2001) other cell types can be additionally involved. For example, vascular endothelial cells represent an almost exclusive source of such a fibrinolytic component, as tPA produced by the endothelium in both physiological and pathophysiological states. Another fibrinolytic component, PAI-1, has additional sites of synthesis, such as vascular endothelial cells, leukocytes, adipocytes, and platelets, but this occurs predominantly after their activation at inflammatory
It is obvious that the plasma levels of naturally active (e.g., tPA) or
Probably, the recovery of the tPA plasma pool in proportion to excessive consumption by PAI-1 is rapid but transient, since the augmented local tPA secretion is limited by the rate of its synthesis. Because of the fact that the tPA-PAI-1 complex is usually cleared at a lower rate than free tPA, this can lead to the appearance of a disproportion between the antigen and activity values. Notably, activated protein C is suggested to compete with tPA for PAI-1 complexation. The importance of APC-PAI-1
In a study with patients suffering from chronic cardiac failure (CCF) and stable
Some evidence do exist that the plasma levels of PC are associated with the systemic inflammatory response to trauma, infection, resuscitated cardiac arrest, non-stable
6. Conclusion
Recent advances in our understanding of the nature of critical factors, linking hypercoagulation with both acute and chronic inflammation are rather promising. Nevertheless, we only can propose some speculations predicting the balance disruption between procoagulant and anticoagulant components under conditions of abnormal hemostasis, as well as consequences of their ratio abnormality on inflammation duration. The problem is complicated by the existence of poorly predictable mechanisms of most urgent thrombotic events that are happened rather “now” and “here”. A transient deficiency or acute inactivation of common hemostatic soluble plasma proteins, affecting hemostasis and inflammation by a mutual regulatory mechanism, was suggested as a key pathogenic factor of such life-threatening complications. Post-translational HSPPs modifications reviewed here could be considered as crucial phenomenon impacted by the inflammatory process. Apparently, inflammation-associated variations in the structure and function of hemostatic proteins can influence their catalytic efficiency and measurable plasma levels.These changes should be taken into account in indication of pathological hemostasis. The recent knowledge on regulatory crosstalk between hemostatic system components and the inflammatory system allows discovering new therapeutic targets to be developed. This new approach could not only change the traditional paradigm of clotting factor substitution therapy, but also anti-inflammatory therapies. Activated protein C is expected to be an attractive therapeutic target with prominent anticoagulant, profibrinolytic, and anti-inflammatory properties, which can simultaneously regulate both inflammation and coagulation. Nevertheless, the results of several clinical trials with recombinant APC or modified rAPC were found to be rather disappointing. Indeed, the peculiarities of the protein structure, attributed to regulatory components with pleiotropic action such as APC, may play a pivotal role in providing clinical benefit of designed protein variants. Hemostasis is a thorough “molecular machine”, which can not readily be improved. To understand and to reconstruct perturbed functions of this machinery should be a prominent goal for both basic and clinical research studies.
Acknowledgments
The author is thankful to Professor Stanislaw A. Kudinov, Dept. of Enzyme Chemistry and Biochemistry (Palladin Institute of Biochemistry of the NAS of Ukraine) for initiating the idea of this review. The author would like also thank Professor Francisco Veas for advice and for critically reading the manuscript. We acknowledge the financial support from the Anisimov Property Management LTD (Ukraine) and from Sergey Davydov, businessman.
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